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ISO 9439: Ultimate Biodegradability Test - CO2 Evolution (ISO/IEC 17025:2017 Accredited)

ISO 9439 is an aqueous aerobic biodegradability test that determines the ultimate aerobic biodegradability of organic compounds in aqueous medium by measuring CO2 evolution during biodegradation in continuously aerated bottles. The test normally lasts for 28 days.

ISO 9439 Key Summary

The ISO 9439 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.

ISO 9439

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. ISO 9439 is similar to OECD 301B and ASTM D5864, and the common materials tested by these two methods include lubricants, grease, oil, fuels, surfactants, and personal care products.

ISO 9439 Requirement and Applicability

Different testing methods are applicable for materials with different properties. Below is a summary of the applicability of ISO 9439. 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
ISO 9439 (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, 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).

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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