Key insights
- OECD 301B test assesses the ready biodegradability of organic chemicals in an aerobic aquatic environment.
- It is also known as CO2 evolution test or Modified strum test
- This test measures the amount of carbon dioxide released when microorganisms degrade a test substance.
- It is a widely accepted method among environmental regulators, including REACH
- To pass the test, The test product must achieve ≥ 60% Theoretical CO2 within a defined 10-day time window.
Summary
The OECD 301B test is a globally accepted method used to assess whether an organic substance is readily biodegradable under aerobic conditions. This method measures carbon dioxide evolution from microbial activity over 28 days. The test assesses environment risk, supports regulatory submissions and evaluates chemicals.
What is OECD 301B biodegradability testing?
OECD 301B test is one of the six tests of OECD guideline 301 designed to assess biodegradability. This test is also referred to as CO2 evolution test and evaluates whether microorganisms can rapidly and completely break down an organic substance under standard laboratory conditions. It is widely accepted by environmental regulators and frequently used during chemical registration.
Why does OECD 301B matter?
Environmental regulators often expect solid proof that chemicals do not persist in ecosystems. OECD 301B test determines whether a substance can undergo rapid biodegradation after release into natural environments.
The test becomes important as:
- It assesses environmental risk
- Supports REACH and other regulatory submissions
- Helps manufacturers demonstrate sustainability claims
- Provides globally accepted data on biodegradation claims
Why is it called the Modified Strum test?
OECD 301B test is commonly known as the Modified Strum test as it is designed from original carbon dioxide evolution method developed by R.N. Strum used to evaluate biodegradation through CO2 production.
Principle of OECD 301B
OECD 301B principle is based on CO2 evolution. The test method checks ready biodegradability by measuring the amount of carbon dioxide (CO2) released when microorganisms break down the test substance under aerobic conditions. The CO2 produced is compared with the theoretical CO2 of the substance.
Understanding theoretical CO2
Theoretical CO2 (ThCO2) is defined as the maximum amount of carbon dioxide that is generated if all carbon in the test product is completely mineralized by microorganisms. It is used as the reference value against which CO2 produced is compared.
Which products can be tested?
The OECD 301B test is applicable to:
- Surfactants
- Detergents
- Pharmaceuticals
- Industrial chemicals
- Personal care products
OECD 310B test conditions
- Inoculum – Activated sludge from STPs, Natural environment or soil or surface water act as sources of microorganisms used as inoculum. The inoculum is a mixture of bacteria, protozoa and fungi that can break down organic matter
- Test substance concentration – The concentration should range from 10 to 20 mg per litre of dissolved organic carbon (DOC) or Total Organic Carbon (TOC)
- Test duration – 28 days
- Temperature – 22 ± 2 °C
- Aerobic conditions are maintained throughout the test procedure
OECD 301B test procedure
- A mineral medium is prepared and inoculated with microorganisms.
- The test substance is added as the primary carbon source
- The test system is then continuously aerated using CO2-free air.
- The system is incubated for 28 days at specified test conditions
- The evolved CO2 is captured, measured at predefined intervals
- The CO2 evolution is calculated as a cumulative against theoretical CO2
How are OECD 301B results calculated?
Results are expressed as the ThCO2 evolved. To pass the test, test substance must achieve at least 60% of ThCO2 within a 10-day window during the 28 day test period.
Understanding the 10-day window
The 10-day window begins when the test substance reaches 10% biodegradation. From that point, it must achieve 60% biodegradation (based on ThOD or ThCO₂) or 70% DOC removal within the next 10 days and before the end of the 28-day test.
These levels are considered evidence of near-complete biodegradation because the remaining 30-40% of the substance is typically converted into microbial biomass or other natural biological products rather than remaining as the original chemical.
Common reasons why substances fail OECD 301B
Failure in OECD 301B can be due to several reasons:
- Slow microbial adaptation
- Complex molecular structures
- Poor bioavailability
- Low water solubility
- Volatility issues that make accurate assessment difficult
OECD 301B vs other tests
| Parameter | OECD 301B – CO2 evolution test | OECD 301D – Closed bottle test | OECD 301F |
| Purpose | To verify whether a chemical is readily biodegradable by measuring CO2 released | To assess ready biodegradability by measuring oxygen consumption | To determine ready biodegradability by measuring oxygen uptake of microorganisms during biodegradation |
| Scope of application | Non-volatile chemicals, highly soluble, poorly soluble or adsorbing materials (Surfactants, industrial chemicals, oils, greases etc) | Volatile, poorly soluble, and adsorbing substances. Suitable for compounds unsuitable for aeration methods | Suitable for poorly soluble, adsorptive and complex organic substances |
| Test microorganisms | Mixed microbial population obtained from activated sewage sludge, treatment plants or environmental waters | Diluted secondary effluent or surface water (very low concentration of inoculum) | Mixed microbial population obtained from activated sludge, sewage effluent. |
| Results interpretation | ≥ 60% of theoretical CO2 production within a defined 10-day window over 28 days. | ≥ 60% of theoretical oxygen consumption within the 10-day window during the 28-day period. | ≥ 60% of theoretical oxygen demand (ThOD) within the 10-day window during the 28-day test period |
Limitations of OECD 301B
OECD 301B:
- May underestimate degradation of poorly bioavailable substances
- Is not ideal for highly volatile chemicals
- Test results do not directly predict degradation rates in specific ecosystems
Need OECD 301B testing?
Looking to validate your product in accordance with OECD 301B! At Microbe Investigation Switzerland (MIS), WE provide OECD-compliant biodegradability testing services by performing the test with standard protocols and generating reliable, reproducible results.
To discuss your testing needs further, contact our experts now.
FAQs
1. Why is OECD 301B called the CO2 evolution test?
It is called CO2 evolution test because biodegradation by measuring the carbon dioxide produced by microorganisms during degradation
2. What is ThCO2?
ThCO2 is the maximum amount of carbon dioxide that can be produced if the test substance undergoes complete mineralization
3. How long does the OECD 301B test take?
OEC 301B usually takes 28 days to complete
4. Is OECD 301B accepted under REACH
Yes, OECD 301B is accepted under REACH
5. Can poorly soluble substances be tested using OECD 301B?
Yes, the method can be applied to poorly soluble or absorptive substances with suitable testing conditions
6. What happens if a substance fails OECD 301B?
Failure usually indicates the test substance could not achieve the passing criteria. However, re-testing or additional testing can be done to evaluate biodegradability
7. What microorganisms are used in OECD 301B?
The inoculum is a mixture of bacteria, protozoa and fungi that can break down organic matter. Activated sludge from STPs, Natural environment or soil or surface water act as sources of microorganisms used as inoculum.
