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OECD 301B, OECD 301D, OECD 301F Ready Biodegradability Test

As the first tier of tests in OECD standard methods, OECD 301B, OECD 301D and OECD 301F are aerobic biodegradation tests aiming at screening readily biodegradable materials normally within 28 days. Measurement is based on nonspecific parameters including CO2 and oxygen consumption.


This series of tests select chemicals that do not have to be tested further because high biodegradability is expected in sewage treatment plants if they pass the OECD 301 tests. Measurement is based on nonspecific parameters like DOC, BOD, or CO2. They were 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% (for OECD 301 B-D and F) or 70% (for OECD301 A and E) of degradation is reached within a 10 day window 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 301 CO2 evolution and closed bottle test overview

OECD 301B CO2 Evolution Test

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 may be 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 into 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.

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 CO2 evolution ready biodegradability test laboratory setup

OECD 301D Closed Bottle Test

The OECD 301D closed bottle test measures the DO consumption during aerobic biodegradation in a closed bottle setup without head space and the degradation percentage is calculated as the ratio of DO consumption to the theoretical oxygen demand (ThOD).

The formula of the test substance and its purity may be known to calculate the ThOD. If the ThOD cannot be obtained, the chemical oxygen demand (COD) may be experimentally determined and used as a less satisfactory alternative.

This method is capable of testing samples that are highly soluble, volatile, and/or adsorbing. While testing poorly soluble compounds, however, the shaking or stirring of the solutions during incubation might be helpful to improve the dissolution/dispersion.

OECD 301D closed bottle ready biodegradation test setup

OECD 301F Closed Respirometer Test (most recommended)

The OECD 301F closed respirometer method determines the biodegradability of a material by measuring the oxygen consumption during the biodegradation process in a period of 28 days in a closed respirometer setup. The degradation percentage is calculated as the ratio of oxygen consumption to the theoretical oxygen demand (ThOD). It is similar to the 301C, mainly differing in the inoculum employed.

Different from the OECD 301D closed bottle test, this method employs much higher dosages of the test substances and inoculum and therefore requires headspace in the reactors to provide adequate oxygen for the microorganisms.

The oxygen consumption is usually determined by measuring the quantity of oxygen (produced electrolytically) required to compensate the consumed one to maintain a constant gas volume/pressure in the respirometer flask, or from the change in the volume or pressure in the flask without compensating the consumed oxygen. The evolved CO2 is absorbed by sodium hydroxide solution present in a tube in the flask.

Similar to OECD 301D method, the formula of the test substance and its purity may be known to calculate the ThOD. If the ThOD cannot be obtained, the chemical oxygen demand (COD) may be experimentally determined and used as a less satisfactory alternative.

This method is capable of testing samples that are highly soluble, poorly soluble, insoluble, volatile, involatile, and/or adsorbing. It has therefore been often used for materials that the OECD 301B and OECD 301D methods cannot handle (e.g., insoluble and volatile).

OECD 301F is our first recommended method (followed by ASTM D6731) due to its simple experimental setup and high applicability.

Requirement and Applicability

Different testing methods are applicable for materials with different properties. Below is a summary of the applicability of OECD 301B and OECD 301D. 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

Test Analytical method Sample info required * Poorly soluble Volatile Adsorbing
301B (CO2 evolution test)
CO2 evolution Carbon content * + - +
301D (Closed bottle test)
Dissolved oxygen
ThOD or COD *
- + +
301F (Closed respirometer test)
Oxygen consumption
ThOD or COD *
+ +/- +

*"Sample info required" is the information needed to calculate the biodegradation percentages. This must be available for a selected method.
*"Carbon content" is the ratio of the carbon weight to the weight of the sample. It can be calculated by the sample formula (e.g., acetic acid C2H4O2, carbon content is 12*2/(12*2+1*4+16*2)=40%) or measured experimentally. At Aropha, we currently do not provide such an analysis service.
*"ThOD" can be easily calculated based on the formula of the sample. Calculate it now.
*"COD" analysis is available and free at Aropha.

Aropha Resource Center

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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