This test method covers procedures for determining the ready,
ultimate, aerobic biodegradability of organic chemicals by
monitoring CO2 production in sealed vessels containing
the test compound and a dilute sewage inoculum. Because of the
stringency of the test conditions, it can be assumed that a
chemical that is 60 % or better biodegraded in this test method
will biodegrade in most aerobic environmental compartments.
This test method is derived from the sealed vessel procedures of
Birch (1),2 Struijs (2), Boatman (3), and Peterson (4),
which were developed as simpler, more economical alternatives to
the CO2 production techniques reported by Gledhill (5) and Sturm
(6), the Sturm report being the basis of the Modified Sturm Test of
the Organization for Economic Cooperation and Development (OECD)
The procedures are applicable to pure materials, including
sparingly solubles, which can be dissolved or dispersed
homogeneously in aqueous stock solutions of at least 25 ppm of
carbon, or which can be introduced reproducibly to test bottles as
pure test material in 1 to 2-mg portions. The test chemical should
be nontoxic to sewage microorganisms at 10 ppm of carbon. The test
may be applied to volatile materials with Henry's Law Constants of
up to approximately 10−2 atm/m3/mole. The
testing of mixtures, extracts, or fully formulated products can
lead to serious problems in data interpretation.
The procedures involve incubation of the test chemical with a
dilute inoculum of microbes from domestic wastewater secondary
sewage treatment effluent in small, sealed vessels for up to 28
days. Biodegradability is determined by monitoring CO2
production as dissolved inorganic carbon (DIC) in the liquid phase,
and as gaseous CO2 in the head space. Alternatively,
analysis can be performed on just the liquid phase after the
addition of alkali, or on just the headspace following
acidification. The determinations are made using commercial carbon
analyzers based on the IR detection of CO2. The
determination of CO2 production provides unequivocal
proof of biodegradation, barring the unlikely event of abiotic
production of CO2 from the test material.
For water-soluble materials that do not adsorb to glass or
biological solids, biodegradation may be confirmed further by
monitoring the disappearance of dissolved organic carbon (DOC) in
the liquid phase.
The simplicity of the sealed vessel method permits ample
replicate sampling for rate determination or statistical
evaluation, or both.
For a chemical that fails the test as written, the stringency of
the test may be reduced by substituting an acclimated inoculum in
order to provide a measure of inherent biodegradability.
Materials that are toxic to the microbial inoculum at 10 ppm of
carbon may not be amenable to testing by this test method, or they
may require special method modification such as reducing the test
concentration if instrumental sensitivity permits. For some
cationics, complexing the test material with a nondegradable
anionic may reduce toxicity.
The values stated in SI units are to be regarded as the
This standard does not purport to address all of the safety
concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and
health practices and determine the applicability of regulatory
limitations prior to use. For specific precautionary
statements, see Section 6.
2 The boldface numbers in parentheses refer to the
list of references at the end of this standard.