OECD 310 Assessment of Ready Biodegradability - CO2 in Sealed Containers (Headspace Test)

The standard “OECD 310 Ready biodegradability - CO2 in sealed containers (headspace test)”, published under the OECD guidelines on responsible business conduct for multinational enterprises (OECD guidelines), describes a screening method for assessing the ready biodegradability of chemicals.

The test substance, normally 20 mg C/L, as the sole carbon and energy source is incubated in closed vials (normally for 28 days) under aerobic conditions containing a buffer-mineral salts medium inoculated with a mixed population of microorganisms. To check the test procedure, a reference substance of known biodegradability (aniline, sodium benzoate or ethylene glycol and 1-octanol) should be tested in parallel.

It is recommended that triplicate vials be analyzed after sufficient time intervals. Additionally, at least five test vials (from test vessels, blank controls, and vessels containing reference material) are analyzed at the end of the test to ensure that 95 percent confidence intervals can be calculated for the mean percent biodegradation value.

The carbon dioxide release resulting from the final aerobic biodegradation of the test substance is determined by measuring the excess of inorganic carbon produced in the test vials over the amount produced in the empty vials containing only inoculated medium. The amount of biodegradation is expressed as a percentage of the theoretical maximum inorganic carbon production based on the initial amount of test substance added. In this test, a biodegradation of greater than 60 percent of the theoretical maximum inorganic carbon within a 10-day window indicates that the test substance is readily biodegradable under aerobic conditions.

The OECD 310 standard provides information similar to the six test methods described in OECD 301 standards A to F. Therefore, a chemical substance that shows positive results in this standard can be considered readily biodegradable and therefore rapidly degradable in the environment.

The well-established OECD carbon dioxide method, based on Sturm's original test and used to assess the biodegradability of organic chemicals by measuring carbon dioxide produced by microbial action, has generally been the first choice for testing poorly soluble chemicals and those with strong adsorption. This method is also preferred for soluble (but non-volatile) chemicals, as carbon dioxide release is considered by many to be the only definitive evidence of microbial activity.

Dissolved organic carbon removal can be accomplished by physical-chemical processes (adsorption, volatilization, precipitation, hydrolysis) and microbial action, and many nonbiological reactions consume oxygen. Carbon dioxide is rarely produced abiotically from organic chemicals.

This test, a standard method developed by the Organisation for Economic Co-operation and Development (OECD), is part of the "OECD Guidelines for the testing of chemicals under the heading of degradation and accumulation." It provides a robust, regulatory-accepted approach to assessing how quickly and completely a chemical degrades under aerobic conditions, particularly for substances with volatile components.

The OECD 310 test is a screening method designed to measure the ready biodegradability of chemicals by monitoring carbon dioxide release in a closed container system. The test substance, typically added at a concentration of 20 mg carbon per liter (mg C/L), serves as the sole carbon and energy source for a mixed population of microorganisms in a buffer-mineral salt medium under aerobic conditions. The test typically lasts 28 days, during which time the carbon dioxide produced from the biodegradation process is measured as inorganic carbon using a total organic carbon (TOC) analyzer. The degree of biodegradation is expressed as a percentage of the theoretical maximum inorganic carbon based on the initial amount of the test substance.

The main features of the OECD 310 standard are:

• Closed-vessel system: Unlike some other OECD tests, the OECD 310 test uses closed vials to prevent the loss of volatile compounds, making it ideal for testing substances that may evaporate during processing. This closed system ensures that all carbon dioxide produced is captured for accurate measurement.
• Carbon dioxide measurement: The test measures aerobic biodegradation by measuring carbon dioxide evolution. This indicates the extent to which the test substance has been mineralized by microorganisms.
• Applicability: This test is suitable for both water-soluble and water-insoluble substances, as long as good dispersion is achieved. It is particularly effective for volatile compounds.
• Repeatability: The method is designed to minimize human error and increase repeatability, providing consistent and reliable data for regulatory purposes.
• Test duration: The standard test lasts 28 days, with extensions possible for final biodegradability assessments.

The OECD 310 test classifies a substance as readily biodegradable if at least 60 percent of its organic carbon is converted to carbon dioxide within a 10-day period during the 28-day test period. This period begins with 10 percent of its organic carbon being converted to carbon dioxide and must end within the 28-day period. If a substance reaches 60 percent biodegradation after the 10-day period, indicating potential degradation under appropriate conditions, such as well-operated wastewater treatment plants, it can ultimately be classified as biodegradable.

The OECD 310 test follows a structured protocol to ensure reliable results:

• Preparation: The test substance is added to closed vials containing a buffered mineral salt medium inoculated with activated sludge obtained from a municipal wastewater treatment plant. A reference substance (e.g., aniline, sodium benzoate, or ethylene glycol) is tested in parallel to validate the procedure.
• Incubation: Bottles are incubated in the dark at a controlled temperature (typically 20-25 degrees) for 28 days, with periodic sampling (at least eight time points) to measure carbon dioxide production.
• Analysis: The carbon dioxide released is converted to carbonate and measured as inorganic carbon using a total organic carbon analyzer. The percentage biodegradation is calculated by comparing the measured IC50 value with the theoretical IC50 value of the test substance.
• Controls: Blank controls (medium and inoculum without test substance) and toxicity controls (test substance combined with reference substance) are included to account for background carbon dioxide production and potential inhibitory effects of the test substance.
• Conclusions: A substance that reaches 60 percent biodegradability within a 10-day window is classified as readily biodegradable. Reaching this threshold after the window indicates ultimate biodegradability.

The OECD 310 test is widely used across many sectors to support environmental claims and regulatory compliance:

• Regulatory compliance: OECD 310 results are accepted by regulatory bodies and certification bodies for lubricants to validate biodegradability claims.
• Product development: Industries such as packaging, personal care, household cleaning products and fertilizers use the OECD 310 standard to demonstrate the environmental compatibility of their products, particularly for volatile or insoluble compounds.
• Environmental risk assessment: This method helps design safer and more sustainable products by evaluating the persistence of chemicals in the environment.
• International certifications: OECD 310 data supports eco-labeling and market acceptance in regions with strict environmental regulations.

The OECD 310 standard is particularly suitable for:

• Substances with volatile components that may be lost in open system tests such as OECD 301B.
• Products requiring international certification or eco-labeling.
• Companies seeking high-quality, reproducible data for regulatory filings.

For poorly soluble or potentially toxic substances, additional considerations may be required, such as the use of silica gel or silicone oil to improve dispersion.

As a result, the OECD 310 standard provides a modern, reliable, and regulatory-compliant method for assessing the readily biodegradable nature of chemicals, particularly those with volatile components. Its closed-container system, direct carbon dioxide measurement, and global acceptance make it a valuable tool for industries seeking to demonstrate environmental compliance. By providing clear criteria for biodegradability, it supports sustainable product development and environmental protection, helping industries meet regulatory requirements and consumer expectations for environmentally friendly products.

Our organization, which has been operating for many years and meticulously follows global developments in science and technology, conducts testing, measurement, and analysis in a wide range of areas for businesses across all sectors with a skilled team and extensive infrastructure. In this context, we also provide testing services in accordance with the OECD 310 "Ready biodegradability - CO2 in sealed containers (headspace testing)" standard.