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OECD 301D: Ready Biodegradability Test - Closed Bottle (ISO/IEC 17025:2017 Accredited)

OECD 301D is an aqueous aerobic biodegradation test that determines the ready biodegradability of a material by measuring dissolved oxygen consumption during biodegradation in closed 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 301D selects chemicals that do not have to be tested further because high biodegradability is expected in sewage treatment plants if they pass the OECD 301D test. Measurement is based on nonspecific parameter -- the consumption of dissolved oxygen. 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 301D Key Summary

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

Compared to some other methods, OECD 301D has the top levels of simplicity and applicability.

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.

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 capable of testing samples that are highly soluble, volatile, and/or adsorbing

OECD 301D Requirement and Applicability

Different testing methods are applicable for materials with different properties. Below is a summary of the applicability of 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

TestAnalytical methodSample info required *Poorly solubleVolatileAdsorbing
OECD 301D (Closed bottle test)
Dissolved oxygen
ThOD or COD *

*"Sample info required" is the information needed to calculate the biodegradation percentages. This must be available for a selected method.
*"ThOD" can be easily calculated based on the formula of the sample. Try our Online ThOD Calculator.
*"COD" can be used as an alternative to ThOD if the formula of the material is unknown. We provide COD analysis at Aropha.

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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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