When it comes to disinfection, not all microorganisms are created equal. Each microorganism has varying resistance to chemical disinfectants due to its cellular structure, discussed in a previous blog.

Among the toughest to eliminate are bacterial spores. Just like us, bacteria strive to survive in this world. When put under stress and unfavourable conditions, these bacteria strains form into spores and remain dormant for long periods to protect themselves from dying, only to “wake up” when conditions improve. Common examples for bacterial spores are the Bacillus and Clostridium species.

This is why demonstrating sporicidal activity is considered one of the highest benchmarks for disinfectant efficacy performance.

Hydrogen peroxide (H₂O₂) is widely recognized as a high-level disinfectant, especially valued for being strongly oxidizing, residue free (breaks down into water and oxygen) and environmentally friendly, making it preferred by end users in the disinfectant market with high expectations for strong efficacy.

But in reality, the sporicidal performance of H₂O₂ is highly dependent on its concentration, formulation, and most importantly, contact time. Some studies suggest that low concentrations (e.g., 6-7%) can achieve strong efficacy quickly. However, standardized testing tells a more nuanced story. How so?

EN 17126 standard for sporicidal activity

First, let us see what we can interpret directly from the standard method. EN 17126 is a quantitative suspension test designed to evaluate sporicidal activity under defined laboratory conditions. To pass this test, the product must demonstrate a reduction of at least 4 log₁₀ against the challenged spore.

A critical component of this method is the spore susceptibility (resistance) verification, typically performed using reference disinfectants such as:

• Glutaraldehyde (GA)
• Peracetic acid (PAA)

This verification is essential in ensuring the test spores maintain appropriate resistance. Without this verification, partially germinated spores (i.e., converted back to vegetative cells or spores not fully formed) may be present and are easier to kill, leading to false positive outcomes.

Susceptibility criteria for Bacillus spores in EN 17126

 Interpretation:

• Bacillus subtilis shows higher resistance to glutaraldehyde
• Bacillus cereus shows higher resistance to oxidizing agents such as peracetic acid

Internal experimental evaluation of H₂O₂

To determine the concentration of hydrogen peroxide required to achieve passing results, we conducted an internal evaluation in accordance with EN 17126. Given that H₂O₂ is an oxidizing agent, Bacillus cereus was selected as the test organism, as it represents a more stringent challenge for evaluating the efficacy of hydrogen peroxide–based disinfectants.

The testing was performed using Bacillus cereus spores under clean conditions (low organic load, 0.3 g/L BSA). The table below summarizes our results.

Key findings

• 6% H₂O₂ required 180 minutes to achieve ≥4 log₁₀ reduction
• 10% H₂O₂ failed to meet the ≥4 log₁₀ requirement even at 60 minutes
• 15% H₂O₂ successfully achieved ≥4 log₁₀ reduction within 60 minutes

*All solutions were prepared by diluting 50% H₂O₂ with distilled water, and concentrations were verified via titration.

*No stabilizers or formulation enhancers were added.

*Important note: In practical formulations, additives (e.g., surfactants, acids, stabilizers) can significantly enhance efficacy, potentially allowing lower H₂O₂ concentrations to achieve sporicidal claims.

Why your product may pass in one lab but fail in another?

This is one of the most common and misunderstood question in disinfectant testing. And one manufacturers struggle with constantly. You might ask yourself or us, “My product has shown effectiveness previously, but now, with no changes to the formulation, it is suddenly failing miserably under the same conditions. Why is that?”

The answer often comes down to one critical factor: Spore Quality

According to EN 17126, spore suspension prepared must pass the aforementioned susceptibility verification before their initial use. After this, the verified spores may be stored at 4–8°C for a defined period and be used periodically when needed. However, during storage, some spores may germinate into vegetative cells, reducing the overall resistance of the spore suspension. This phenomenon can lead to disinfectants appearing more effective than they truly are.

Independent Validation Matters

While the methodology for efficacy tests is standardized according to the EN standards, each laboratory has its own unique day-to-day operating practices. Nevertheless, reproducibility of the results should still be expected. This is why organizations such as VAH in Germany often require two independent laboratory reports for quality assurance.

Need Support with Sporicidal Testing?

At TECOLAB, we specialize in high-quality, standard-compliant efficacy testing to help you validate your disinfectant claims with confidence. Drop us an email now to have your products tested for sporicidal activity claims as a high level disinfectant.