Cristian Ilea
Director Marketing & Product Management Romer Labs.
Mycotoxins are toxic secondary metabolites produced by fungi such as Aspergillus, Fusarium, and Penicillium, which commonly contaminate agricultural commodities. These toxins – including aflatoxins, deoxynivalenol (DON), zearalenone, fumonisins, ochratoxin A, and T-2/HT-2 toxins – pose both acute and chronic health risks, ranging from hepatotoxicity and carcinogenicity to immune suppression and reproductive toxicity.
According to the Food and Agriculture Organization (FAO), up to 25% of global food crops may be significantly contaminated with mycotoxins, although occurrence rates in specific commodities and regions can be much higher. Given the strict regulatory limits imposed by authorities such as the European Commission – particularly under Regulation (EU) 2023/915 – robust mycotoxin testing is essential to ensure food and feed safety, maintain market access, and achieve regulatory compliance.
However, mycotoxin testing is inherently complex due to the heterogeneous distribution of toxins, diversity of sample matrices, and environmental conditions that influence fungal growth. This article examines five critical pitfalls in mycotoxin testing – improper sampling, inadequate storage and transportation, poor sample preparation, matrix effects, and test execution errors -and provides evidence-based strategies to address them.
Pitfall 1: Improper Sampling
The Challenge of Uneven Distribution
A primary source of error in mycotoxin testing stems from the uneven distribution of toxins within bulk materials. Unlike uniformly distributed nutrients such as protein or starch, mycotoxins often occur in highly localized “hot spots.” A single grab sample is thus insufficient to represent an entire lot. For example, a feed mill once approved a corn shipment based on preliminary tests showing low aflatoxin and fumonisin levels. However, after incorporation into broiler feed, poor flock performance prompted further investigation, revealing high fumonisin concentrations – ultimately traced to non-representative sampling.
To illustrate the scale of this issue: you typically analyse a 10–50 g test portion, yet this must accurately reflect a batch that may weigh up to 50 metric tons. Failure to capture the full variability of the batch can result in either false negatives (missing contamination) or false positives (overestimating contamination).
Solutions for Effective Sampling
Standardized, multi-point sampling protocols are essential. Regulatory frameworks such as the USDA’s Federal Grain Inspection Service (FGIS) Mycotoxin Handbook and the European Union’s Commission Regulation (EU) No. 2023/2782 outline sampling plans tailored to different commodities and lot sizes. Best practices include:
- Determine Sample Size: Use regulatory guidelines to calculate the number of incremental samples based on batch size and expected variability.
- Collect Incremental Samples: Obtain small, equally sized samples from different points—during transfer (e.g., loading spouts) or at varying depths in silos or storage piles.
- Create an Aggregate Sample: Combine the incremental samples into a homogeneous aggregate, and then reduce it (e.g., via riffle splitting) to obtain the final analytical portion.
For instance, the USDA recommends sampling flat-bottom trucks at multiple evenly spaced points across length, width, and depth using a probe. Additionally, tools such as the FAO Mycotoxin Sampling Tool (an Excel-based decision aid) can help design statistically sound plans for various commodities and risk levels.
By following such protocols, testers can ensure that samples are representative, minimizing the risk of inaccurate results. Studies have shown that sampling error can account for more than 80% of total uncertainty in mycotoxin testing (Whitaker & Dickens, J. AOAC Int., 2004), underscoring the critical importance of this step.
Pitfall 2: Inadequate Storage and Transportation
The Impact of Storage Conditions
Even after accurate sampling, mycotoxin levels may rise during storage and transport if environmental conditions are suboptimal. A grain trader, for example, conducted lateral flow tests on incoming shipments and found low toxin levels. However, follow-up analyses by external labs revealed elevated contamination in stored lots. The root cause: poor storage practices.
Key environmental risk factors include:
- Moisture Content: Grain moisture >14% fosters fungal growth.
- Temperature: Warm conditions (25–35°C) accelerate both mold growth and toxin production.
- Spore Load: Pre-existing spores of Fusarium or Aspergillus can begin producing mycotoxins within days.
- Poor Aeration: Localized “hot spots” due to compaction or poor airflow can create ideal microenvironments for fungal proliferation.
These factors can cause rapid increases in mycotoxin levels, sometimes within days, rendering initial test results obsolete.
Strategies to Mitigate Storage Issues
To minimize post-harvest mycotoxin production:
- Maintain Dry Conditions: Keep grain moisture below 14%, ideally below 13% for long-term storage.
- Control Temperature: Maintain storage temperatures under 25°C where possible.
- Monitor and Aerate: Use sensors to monitor grain temperature and moisture and implement regular aeration to prevent hot spots.
- Perform Spot Checks: Perform periodic testing during storage to detect changes in mycotoxin levels early.
- Combine Testing Methods: Use rapid screening tools (e.g., lateral flow devices) and confirmatory techniques (e.g., LC-MS/MS) for high-value or borderline lots.
Continuous monitoring and documentation of storage conditions can help identify trends and enable rapid responses to rising contamination levels.
Pitfall 3: Inadequate Sample Preparation
The Role of Grinding and Particle Size
Sample preparation – particularly grinding – directly affects extraction efficiency and test result reliability. In one documented case, a feed mill consistently underestimated mycotoxin levels with lateral flow tests compared to external LC-MS/MS results. The discrepancy was traced to inconsistent grinding, resulting in heterogeneous particle sizes and incomplete toxin extraction.
Many mycotoxins – such as aflatoxins and fumonisins – are concentrated near the outer layers of kernels. Uniform grinding increases the exposed surface area and improves solvent penetration during extraction. Coarse or inconsistent particles may trap toxins, leading to underestimation or false negatives.
Best Practices for Sample Preparation
To optimize sample preparation:
- Grind to the Right Particle Size: Follow kit manufacturer guidelines, typically requiring 95% of the ground sample to pass through a 20-mesh (0.84 mm) sieve.
- Use Proper Equipment: Employ well-maintained grinders with sharp blades and calibrate regularly.
- Verify Consistency: Implement QC checks such as sieve analysis or duplicate testing.
- Train Operators: Ensure staff are trained in standardized grinding procedures, especially during high-volume periods.
These steps enhance extraction efficiency and improve the reliability of test results.
Pitfall 4: Matrix Effects
Interference from Complex Matrices
Matrix effects occur when components like fats, proteins, and oils in a sample interfere with test performance, particularly in rapid assays like lateral flow devices. In one instance, a poultry integrator’s on-site tests yielded inconsistent results compared to LC-MS/MS analysis, due to matrix interference in finished feed.
Finished feeds are especially challenging due to their diverse formulation. Additives, binders, and varying moisture or fat contents can affect extraction efficiency and assay performance, leading to false negatives or inconsistent results.
Shifting Focus to Raw Material Testing
Rather than relying solely on testing finished feed, which provides insights only after production, testing raw materials enables proactive risk management. By screening incoming grains, producers can:
- Mitigate Risk: Reject or divert contaminated lots before processing.
- Apply Corrective Measures: Adjust formulations or add mycotoxin binders or deactivators to further mitigate risks.
- Simplify Validation Efforts: Raw materials are often validated matrices in rapid test kits, unlike complex finished feeds where the variability in formulations make universal protocols difficult.
While finished product testing may still be required for regulatory or customer purposes, raw material testing provides earlier and often more reliable insights.
Pitfall 5: Test Execution Errors
Errors Under Pressure
Even the most accurate test method can yield unreliable results if improperly executed. During peak harvest season, for instance, rushed personnel at a feed intake site skipped incubation steps and improperly timed reactions – resulting in false negatives for aflatoxin. The contaminated corn was later rejected by a downstream processor.
Small changes, such as altering incubation times or centrifugation steps, improper reagent volumes, using expired kits or cross-contamination between samples can significantly compromise test accuracy, especially under high-pressure conditions.
Ensuring Test Accuracy
To reduce user error:
- Adhere to Protocols: Follow manufacturer guidelines precisely and avoid improvisation unless validated with the supplier.
- Validate Changes: Work with test kit manufacturers to confirm that any protocol adjustments maintain accuracy.
- Train and Re-train: Offer periodic training and use visual SOPs or checklists.
- Use Controls: Include positive and negative controls regularly.
- Use Test Kits with Simple Workflows: Opt for rapid test kits with streamlined procedures, such as automatic timing or result reading, to reduce operator error.
Conclusion
Reliable mycotoxin testing is critical for food and feed safety – but numerous pitfalls can compromise accuracy, from sampling and storage to test execution. By implementing standardized sampling protocols, optimizing storage and preparation, accounting for matrix effects, and rigorously adhering to test procedures, stakeholders can significantly reduce error and improve compliance.
Resources such as the USDA FGIS Mycotoxin Handbook, EU regulations, the FAO Mycotoxin Sampling Tool, and validated rapid test kits (e.g., AgraStrip® Pro WATEX®) offer practical, effective solutions. Moreover, integrating digital data management tools and traceable workflows can further enhance audit readiness and decision-making.
By proactively addressing these common pitfalls, food and feed producers can better protect both consumers and animals – and ensure safer products in an increasingly complex and regulated global market.