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OECD 301B: Ready Biodegradability Test - CO2 Evolution (Modified Sturm Test) (ISO/IEC 17025:2017 Accredited)

OECD 301B is an aqueous aerobic biodegradation test that determines the ready biodegradability of a material by measuring CO2 evolution during biodegradation in continuously aerated bottles. As one of the first-tier tests among OECD 301-303 standard methods, it aims at screening readily biodegradable materials normally within 28 days.


OECD 301B selects chemicals that do not have to be tested further because high biodegradability is expected in sewage treatment plants if they pass the OECD 301B test. Measurement is based on nonspecific parameter -- the evolution of CO2. It was developed to determine whether a chemical is potentially easily biodegradable, rather than to predict the actual rate, of biodegradation in the environment.

A material is considered Readily Biodegradable if 60% of degradation is reached within a 10-day window (normally for materials with single structures) in 28 days. The 10 day window is defined as beginning when 10% of the degradation is reached and ends after 10 days from this point (but before the 28th day).

If a sample passes the tests, it is considered readily biodegradable and is assumed to be able to undergo rapid and ultimate biodegradation in the environment. Therefore no further investigation on the biodegradability, toxicity, or other environmental effects is normally required.

If a sample fails the tests, it does not necessarily mean it cannot be degraded under more environmentally realistic conditions. Instead, higher tiers of tests such as OECD 302 or 303 should be conducted. 

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OECD 301B Key Summary

The OECD 301B CO2 evolution method uses respirometry to determine the biodegradability by measuring the CO2 formation during the biodegradation process over 28 days. As the chemical decomposition process primarily result in the formation of CO2 and H2O, the measurement of generated CO2 can well reflect the biodegradation of the test compounds. With the measured and the theoretical CO2 amount, one can easily calculate the degradation percentages over a specified incubation time.

The aeration of the working solution requires the use of CO2-free air, which can be easily obtained by using a few NaOH washing bottles to remove all the CO2 present in the air. After that, CO2-free air can be sent to aerate the working solution.

OECD 301B CO2 evolution ready biodegradation test scheme

During the aeration, CO2 is generated as a result of test substance biodegradation. Such CO2 can be easily stripped off the working solution during the aeration process. To capture it, three absorption bottles are usually used containing known amount of Ba(OH)2 to convert CO2 to BaCO3 precipitation. An afterwards acid-base titration using HCl as the titrant can determine the amount of the residual Ba(OH)2, which can be used to back calculate the amount of CO2 generated during the biodegradation.

Information on the toxicity of the test substance is helpful in determining the appropriate dosage so that the material does not inhibit bacteria at the concentration tested.

This method is appropriate for highly soluble, poorly soluble (or even insoluble) and/or absorbing materials. However, since aeration is performed throughout the incubation process, the test substance should not be volatile. OECD 301B is similar to ASTM D5864, and the common materials tested by these two methods include lubricants, grease, oil, fuels, surfactants, and personal care products.

OECD 301B Requirement and Applicability

Different testing methods are applicable for materials with different properties. Below is a summary of the applicability of OECD 301B. Please check our Method Selection Guide to select the most appropriate method for your materials. You can also find the applicability for many other methods on Aropha Resource Center

TestAnalytical methodSample info required *Poorly solubleVolatileAdsorbing
OECD 301B (CO2 evolution test)
CO2 evolutionOrganic carbon content *+-+

*"Sample info required" is the information needed to calculate the biodegradation percentages. This must be available for a selected method.
*"Organic carbon content" is the ratio of the organic carbon weight to the weight of the sample. It can be calculated by the sample formula (e.g., acetic acid C2H4O2, organic carbon content is 12*2/(12*2+1*4+16*2)=40%). Try our Online C% Calculator. If the formula is unknown, we can send the sample out to a third party lab for you for analysis (normally $110 with a 10-day turnaround time).

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To learn more about different types of biodegradation tests, their applicability, biodegradation mechanisms, and many other information such as case studies, publications, and blogs, please check our Aropha Resource Center

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