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Test Method Selection Guide

Aerobic biodegradation is a natural process where complex organic substances are broken down into smaller and simpler compounds by the enzymes produced by the microorganisms when oxygen is present. It usually involves the metabolic and enzymatic processes with the final products of carbon dioxide and water. Although complicated, the biodegradation tests usually usually measure relatively simple and non-specific parameters such as oxygen uptake, CO2 production, or dissolved organic carbon (DOC).

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Requirement and Applicability of Different Methods

Despite all other facts about the material compositions, local or regional regulatory requirements, and customers' own needs, the most important factors that determine which method to use for the aqueous biodegradation test are the related physicochemical properties of the materials including solubility, volatility, and adsorptivity

As the adsorbing substances are usually hard to deal with and can easily lead to falsely high results, we do not use any methods that cannot handle adsorbing substances (e.g., OECD 301A).

The table below summarizes the applicability of different methods for test substances with different physicochemical properties. The methods OECD 302 and 303 are not included as their scopes are different compared to these below.

Test Analytical method Sample info required* Poorly soluble Volatile Adsorbing
301B (CO2 evolution test)
CO2 evolution Carbon content + - +
301D (Closed bottle test)
Dissolved oxygen
- + +
301F (Closed respirometer test)
Oxygen consumption
+ +/- +
ASTM D5864 (CO2 evolution test)
CO2 evolution Carbon content + - +
ASTM D6731 (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.
*"COD" analysis is available and free at Aropha.

Recommended Methods for Different Types of Substances

As all our methods are appropriate for adsorbing substances, here we only discuss two properties: solubility and adsorptivity

1. If the test substances are soluble in water to at least 100 mg/L and are non-volatile, they may be assessed by all the methods. The OECD 301F, ASTM D6731, and OECD 301D are recommended due to their high reliability and simple experimental setup.

2. For soluble and volatile (e.g., Henry's law constant < 50 L·atm/mol) substances, only the closed bottle (OECD 301D) or closed respirometer (OECD 301F, ASTM D6731) methods can be used.

3. For insoluble and non-volatile substances such as lubricant, oil, and grease, the CO2 evolution (OECD 301B, ASTM D5864) or closed respirometer (OECD 301F, ASTM D6731) methods can be used. OECD 301F and ASTM D6731 are recommended.

4. For insoluble and volatile substances, only the closed respirometer methods (OECD 301F and ASTM D6731) are applicable.

5. For highly volatile substances (e.g., Henry's law constant > 50 L·atm/mol), the closed respirometer methods (OECD 301F and ASTM D6731) may not be applicable. Instead, the closed bottle test (OECD 301D) is more appropriate.

Substance properties Example substances Applicable methods Recommended methods (ranked)
Soluble, non-volatile
Personal care product... All OECD 301F, ASTM D6731, OECD 301D
Soluble, volatile
Pharmaceuticals, pesticides, herbicides... OECD 301D, OECD 301F, ASTM D6731
OECD 301D, OECD 301F, ASTM D6731
Insoluble, non-volatile
Lubricant, oil, grease...
OECD 301B, OECD 301F, ASTM D5864, ASTM D6731 OECD 301F, ASTM D6731
Insoluble, volatile
Petroleum fuels, paints, dry cleaning agents...
OECD 301F, ASTM D6731 OECD 301F, ASTM D6731
Paints, pharmaceuticals, and refrigerants...

Note: ASTM D5864 and ASTM D6731 are specified for the biodegradation tests of lubricants or their components. They may not be selected if the test substances are not such materials.

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

Aropha resource center


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