OECD 301B
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OECD 301B
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. It is 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.
OECD 301B test conditions
Testing requires specific conditions for an accurate measurement of biodegradation. The conditions appear as follows:
- The inoculum must include a high density of varied microbial populations that can break down the test substance.
- The concentration of the test substance should lie between 10 to 20 mg per liter of Dissolved Organic Carbon (DOC) or Total Organic Carbon (TOC).
- The test is carried out in aerobic conditions. The testing temperature is maintained between 22°C ± 2°C to ensure sufficient microbial action. The test is conducted in diffuse light or in the dark to avoid photodegradation.
- The solution is passed through a continuous stream of air that is free from CO2 for the maintenance of aerobic conditions.
OECD 301B test requirements
In order to carry out the test, certain key requirements must be met:
- Chemical information
Before testing, it is necessary to understand the chemical structure, formula, and additional properties like solubility, vapor pressure, and adsorption characteristics. This information helps in deciding the type of testing to be performed as well as the correct interpretation of results.
- Duration
The test is performed for a period of 28 days, during which the CO2 levels are measured intermittently to monitor the progress of biodegradation.
- Parallel controls
Blanks, reference substances, and, when necessary, toxicity and abiotic controls are set up in parallel to ensure the validity of the test.
OECD 301B test methodology
The test follows a defined protocol:
- Preparation
A test concentration is prepared by dissolving the test substance in a mineral medium and inoculating it with a microbial population. Only the inoculum is added to the control flasks to measure the endogenous respiration.
- Aeration
CO2-free air is bubbled through the test as well as control flasks to ensure the supply of oxygen required for microbial activity.
- CO2 trapping
The CO2 produced from microbial degradation is trapped in a series of jars containing barium or sodium hydroxide. The CO2 reacts with the hydroxide and forms a precipitate or measurable inorganic carbon.
- Measurement
The amount of CO2 in the traps is determined either through titration (for barium hydroxide) or by using an inorganic carbon analyzer (for sodium hydroxide). The CO2 produced by the test substance is calculated by subtracting the CO2 generated by the inoculum control.
- Interpretation of results
The biodegradation percent is obtained by comparing the measured CO2 with the theoretical CO2 (ThCO2) that would be produced, assuming the test material was completely degraded.
- Acceptance criteria
At least 60% of the theoretical CO2 production (ThCO2) must be achieved within a 10-day period during the total 28-day test duration.
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.
Test organisms
Inoculum used in the test is usually obtained 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 product information
This test applies to a wide variety of organic chemicals. This may include but is not limited to:
- Surfactants
- Detergents
- Pesticides
- Pharmaceuticals
- Industrial chemicals
The test substance must be water-soluble and non-volatile. Occasionally, special handling techniques, such as the use of emulsifying agents, are required when the chemicals tested are poorly soluble.
Compliance and industry standards for OECD 301B
OECD 301 is a well-recognised standard used for testing biodegradation of chemical substances. It is considered a key standard in environmental risk assessment and regulatory compliance. The test has been adopted by many international regulatory authorities because of its reliability and scientific integrity. Below are some significant regulatory frameworks and standards that acknowledge and employ the OECD 301 standards:
- REACH (EU Regulation)
OECD 301 is one of the majorly adopted methods for testing under REACH (Registration, Evaluation, Authorisation, and Restriction of Chemicals) regulation in the EU. REACH mandates biodegradability testing as an essential step for chemical registration and risk assessment. Testing by this standard allows manufacturers and suppliers to prove that their chemicals are safe enough to meet environmental standards, ensuring that hazardous materials are handled appropriately at any point during production, usage, or disposal.
- EPA (United States)
In the United States, the Environmental Protection Agency accepts OECD 301 B data as part of the criteria for assessing the environmental impact of chemicals. According to the Environmental Protection Agency (EPA), chemicals especially those used in industrial and consumer products, must meet specific environmental safety guidelines before being allowed to be used in industrial and consumer products.
- ISO Standards
The OECD 301 B test aligns with various ISO standards for environmental testing with a focus on the biodegradability testing of chemicals in aquatic systems. For example, ISO 7827:1994 is similar to the OECD 301 B test in many aspects. This allows manufacturers to follow this standard to satisfy the international regulatory requirements for biodegradability. Global companies often depend on ISO standards and OECD tests to ensure their products meet environmental safety regulations worldwide.
- CLP Regulation (Classification, Labelling and Packaging)
The European CLP regulation which aligns with the GHS (Globally Harmonised System) of the United Nations for the classification of chemicals, also acknowledges the OECD 301 tests for biodegradability. This recognition enables manufacturers to classify their products correctly and ensures proper product identification using an environmental hazard label regarding the biodegradable capability of the chemical under consideration. This promotes safer use and handling of chemicals at all stages in the supply chain.
- Global Regulatory Compliance
Besides the EU and the U.S., the OECD 301 B test is recognized by many regulatory agencies of various countries, including Japan’s Ministry of the Environment and Environmental Protection Act (CEPA) in Canada. Thus this type of international recognition makes the test essential to companies that wish to sell their products internationally.
Advantages of OECD 301B Test Standard
- Widely accepted: This standard is widely adopted and recognized by regulatory bodies around the world for the assessment of biodegradability of chemicals.
- Cost-effective: It offers a relatively inexpensive way to assess environmental persistence and thus enables business organizations to comply with regulatory and legal stipulations effectively.
- Reliable: The test delivers a reliable means of assessing biodegradation under uniform conditions, guaranteeing consistent results across various laboratories.
Conclusion
The OECD 301B test is a highly valid, standardized method to check ready biodegradation of organic compounds. Because of its universal acceptance and ease of use, OECD 301 has been widely used by organizations for environmental risk assessment and to meet regulatory compliance requirements. Knowledge about the biodegradability of chemicals also enables companies to make informed decisions that further reduce their environmental footprint.
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 skilled team delivers precise and prompt results, facilitating your product development and compliance needs.
Reach out to us today to discover more about our testing services or to schedule your consultation.
FAQs
The test assesses the amount of CO2 emitted as a substance degrades biologically in an aerobic aquatic ecosystem.
The test duration is 28 days.
The primary components consist of the test substance, inoculum, mineral medium, and the CO2 measurement system.
This experiment is performed under aerobic conditions with a constant temperature at 22°C ± 2°C.
This standard applies to water-soluble, non-volatile organic materials, such as detergents, surfactants, and other industrial chemicals.
The critical elements of the test include the preparation of the test solution, inoculation, aeration, CO2 trapping, and determination of the outcome.
Biodegradation is quantified by measuring the amount of CO2 produced throughout the degradation process using the titration method or by using an inorganic carbon analyzer.
For water-insoluble test substances, specific preparation techniques, such as using emulsifying agents, may be needed for testing.
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