Biodegradability in Seawater (Shake Flask method and Closed Bottle Methods)

The shake flask method consists on dissolution of a pre-determined amount of the test substance in the test medium to yield a concentration of 5-40 mg/l dissolved organic carbon (DOC). Five flasks, at least, are used: two for the test suspension, two for the blank and one for procedure control. The solution of the test substance in the test medium is incubated, under agitation in the dark or in diffuse light under aerobic conditions, at a fixed temperature which will normally be within the range 15-20ºC.

The recommended maximum test duration is about 60 days. Degradation is followed by DOC measurements (ultimate degradation) and, in some cases, by specific analysis (primary degradation). The closed bottle method consists of dissolution of a pre-determined amount of the test substance in the test medium in a concentration of usually 2-10 mg/l (one or more concentrations are used).The solution is kept in a filled closed bottle in the dark in a constant temperature bath or enclosure controlled within a range of 15-20ºC. The degradation is followed by oxygen analyses over a 28-day period but if the blank biological oxygen demand value remains within the 30 per cent limit, the test could be prolonged.

Twenty-four bottles at least are used (8 for test substance, 8 for reference compound and 8 for sweater plus nutriment). All analyses are performed on duplicate bottles. Four determinations of dissolved oxygen, at least, are performed (day 0, 5, 15 and 28) using a chemical or electrochemical method.

OECD (1992), Test No. 306: Biodegradability in Seawater, OECD Guidelines for the Testing of Chemicals, Section 3, OECD Publishing, Paris, https://doi.org/10.1787/9789264070486-en.