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OECD 301B Ready Biodegradability Test (CO₂ Evolution / Modified Sturm Test)
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Summary
OECD 301B is a ready biodegradability test that uses the aerobic biodegradation method to measure the carbon dioxide evolved during microbial degradation. from. It is widely accepted under REACH and EPA regulation to support wether a chemical meets the criteria for being classified as “readily biodegradable”.
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Quick understanding of the test
OECD 301B - Method for the quantitative determination of the biodegradability of organic materials in an aqueous aerobic environment
The test helps determine the biodegradability of organic chemicals in an aerobic aquatic environment measured through CO2 evolution during degradation of microbes
Application
- The inoculum is composed of diverse microbial populations sourced activated sludge, soil, or surface water.
The test requires the preparation of test solution with a mineral medium, inoculation with microbial population, and aeration with CO2-free air. The released CO2 is trapped in a hydroxide solution and then quantified through a titration process or by an inorganic carbon analyzer.
- The test checks if the chemical will break down quickly in the environment, therefore decreasing the risk of long-term ecological damage caused by it.
- The biodegradability data is mandatory under REACH and EPA regulations. If the chemical complies with the OECD 301B test, then it means regulatory requirements.
Turnaround Time
Passing criteria
To pass, at least 60% of Theoretical CO2 must be achieved in a 10-day test window within the 28-day test period.
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What is the OECD 301B Biodegradation Test?
The OECD 301B (Modified Sturm Test), also known as the CO2 Evolution Test, is a widely recognized method for the quantitative determination of the biodegradability of organic materials in an aqueous aerobic environment. The biodegradability properties of several chemicals have a great impact on the environment, especially on aquatic systems. The 301 B test is one of six tests in the OECD series that have been widely adopted for assessing ready biodegradability. Its wide applicability is due to its virtue of simplicity and reliability.
The 301B test measures the release of carbon dioxide during the microbial degradation of a chemical. Conceptually, in the microbial degradation of an organic chemical, microorganisms respire, and the organism consumes O2 and produces CO2 as a byproduct. The production of CO2 is directly used as a measure to quantify biodegradation. The 301B test is specifically applicable for water-soluble, non-volatile chemicals.
When is OECD 301B required?
OECD 301B is commonly required for following p
- Register substances under REACH
- Support environmental fate assessments
- Demonstrate ready biodegradability for regulatory dossiers
- Compare biodegradation performance across formulations
Chemicals applicable for OECD 301B testing
This test is applicable for a broad range of organic substances, including those that are soluble, poorly soluble, or adsorptive but non-volatile. They include :
- Surfactants
- Detergents
- Pesticides
- Pharmaceuticals
- Industrial chemicals
- Personal care products
OECD 301B test method overview
CO₂ evolution principle
OECD 301B test checks ready biodegradability by measuring how much carbon dioxide (CO₂) is released when microorganisms break down the test substance under aerobic conditions. The CO₂ produced is compared against the theoretical CO₂ (ThCO₂) of the substance.
Inoculum and test conditions
- Inoculum: Microorganisms are sources from activated sludge from sewage treatment plants. It could also be sourced from the natural environment, such as soil or surface water. This inoculum is a mixture of microbes such as bacteria, protozoa, and fungi that can break down organic matter.
- Test substance concentration: Test substance concentration should be within the range of 10 to 20 mg per liter of Dissolved Organic Carbon (DOC) or Total Organic Carbon (TOC).
- Test duration : 28 days
- Temperature: 22 ± 2 °C
- Aerobic conditions throughout the study
OEC D 301B test procedure
- A mineral medium is prepared and adjusted to the required pH. An inoculum obtained from activated sludge is added to the medium under aerobic conditions.
- The test substance is introduced as the sole source of carbon to mineral medium containing microorganisms. A reference substance and appropriate control vessels are prepared in parallel.
- The test mixtures are continuously aerated with CO₂-free air. The carbon dioxide produced during biodegradation is trapped in absorption bottles containing an alkaline solution. The system is incubated for 28 days at 22 ± 2°C in the dark or under diffuse light.
- At regular intervals, the amount of trapped CO₂ is measured. Blank corrections are applied, and the cumulative CO₂ produced is calculated.
- The percentage biodegradation is determined by comparing the measured CO₂ with the theoretical CO₂ (ThCO₂) of the test substance.
Interpretation of OECD 301B test results
The substance is said to be readily biodegradable if at least 60% of ThCO₂ is achieved within a 10-day window during the 28-day test period.
OECD 301B vs OECD 301D
Parameter | OECD 301B – CO₂ Evolution Test | OECD 301D – Closed Bottle Test |
Purpose | To evaluate whether a chemical is readily biodegradable by measuring CO₂ released | To assess ready biodegradability by measuring oxygen consumption |
Scope of Products | Non-volatile chemicals,highly soluble, poorly soluble, or adsorbing materials (surfactants, industrial chemicals, oils, greases, etc) | Volatile, poorly soluble, and adsorbing substances since the bottles are sealed. Suitable for compounds unsuitable for aeration methods. |
Test Organisms (Inoculum) | Mixed microbial populations, typically from sources like activated sludge of sewage treatment plants or environmental waters. | Diluted secondary effluent or surface water (very low inoculum concentration) |
Results Interpretation | ≥ 60 % of theoretical CO₂ production within a defined 10-day window ending by day 28. | ≥ 60 % of theoretical oxygen consumption within the 10-day window during the 28-day period. |
Importance of OECD 301B test
Biodegradability testing using OECD standards plays an important role in environmental risk assessment and ensuring compliance with regulatory bodies. The standard’s primary importance lies in its ability to:
- Assess biodegradability
It determines the rate and extent of the degradation of chemicals. Thus, this test helps determine if the chemical is biodegradable and non-toxic to the environment or if it is persistent.
- Regulatory compliance
Many regulatory bodies, such as the European Union’s REACH regulation, demand biodegradability data for chemicals registration and risk evaluation. The test is widely accepted for these aspects.
- Wastewater and environmental impact
The biodegradable nature of chemicals is important for the management of industrial wastewater and control of long-term environmental pollution.
Why choose Our Lab for OECD 301B Testing
At Microbe Investigations Switzerland, we provide reliable OECD 301B testing services to assist you in evaluating the biodegradability of your products, ensuring adherence to international regulations and lowering environmental impact. Our microbiology team delivers accurate results, support your product development and compliance needs.
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Frequently Asked Questions
DR. Martinoz Scholtz
The OECD 301B test measures the ready biodegradability of a chemical in aerobic aqueous conditions by quantifying the CO₂ evolved from microbial breakdown of the substance versus the theoretical maximum CO₂ expected.
The test duration is 28 days.
A substance passes if it reaches ≥ 60 % of theoretical CO₂ (ThCO₂) evolution within a defined 10-day window during the 28-day test period.
Biodegradation is the percentage of CO₂ evolved from the test substance relative to the theoretical maximum CO₂ the substance could produce based on its organic carbon content.
OECD 301B measures biodegradation by tracking the amount of carbon dioxide (CO₂) produced as the substance breaks down in an aerated system. OECD 301D measures biodegradation by monitoring the amount of dissolved oxygen consumed in a sealed bottle.
Yes, OECD 301B is one of the accepted ready biodegradability tests under the REACH.
OECD 301B is used for a broad range of organic substances, including those that are soluble, poorly soluble, or adsorptive but non-volatile.
OECD 301B can be applied to poorly soluble or insoluble substances, if they can be dispersed and their organic carbon must be known to calculate theoretical CO₂.
A substance is readily biodegradable if it achieves ≥ 60 % CO₂ evolution within the specified 10-day window during the 28-day test.
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