OECD 306 is an aerobic biodegradation test that determines the biodegradability of an organic material in seawater relying on the microorganisms originally present in seawater without the addition of a specific inoculum. Two methods are specified, i.e., (I)measuring dissolved organic carbon removal in a Shake Flask Method, and (II) determining dissolved oxygen consumption in a Closed Bottle Method. The Shake Flask Method allows a maximum of 60 days of test duration, while the Closed Bottle Method suggests 28 days because no further information can be normally gathered in an extended test.
While most other related OECD guidelines (e.g., 301, 302, 303, and 310) for aqueous aerobic biodegradability tests are only for fresh water environment, OECD 306 was developed due to the growing awareness of the need to protect the marine environment. Although the two methods (i.e., Shake Flask and Closed Bottle) are similar to some of the OECD 301 methods in terms of system setup and parameter analysis, OECD 306 is not a test for ready biodegradability since no inoculum is added in addition to the microorganisms already present in the seawater. Neither do the tests simulate the marine environment since nutrients (mineral medium) are added and the concentration of test substance is much higher than would be present in the sea.
A positive result (> 70% dissolved organic carbon removal for the Shake Flask Method, or > 60% of dissolved oxygen consumption for the Closed Bottle Method) indicates that there is a potential for biodegradation of the test substance in the marine environment.
However, if a sample fails the tests, it does not necessarily mean it cannot be degraded, but indicates further study is necessary, for example, using as low a concentration of the test compound as possible.
Additionally, OECD 306 is equivalent to ISO 16221, a guideline for biodegradability test in seawater but specifies many more methods to use, including DOC die-away, closed bottle, two-phase closed bottle, CO2 evolution, and CO2 headspace tests.
OECD 306 (I) Shake Flask Method
The Shake Flask Method measures the dissolved organic carbon (DOC) removal during aerobic biodegradation in a shaking flask setup. The degradation percentage is calculated as the ratio of DOC removal to the initial DOC dosed into the test medium.
The carbon content of the test substance and its purity may be known to calculate the dosage. 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 (> 50 mg DOC/L), non-volatile, and non-adsorbing.
Compared to the Closed Bottle Method, the Shake Flask Method has a few limitations such as much higher substance dosage (but the inoculum is the same), and incapable of handling volatile or adsorbing materials. Therefore, the Closed Bottle Method is usually our preferred method.
OECD 306 (II) Closed Bottle Method
The Closed Bottle Method 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).
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.
|Sample info required
|OECD 306 (Seawater)
|DOC or dissolved oxygen
|Carbon content or ThOD