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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP according guidelines, all quality criteria were met, no CoA but sufficient info on test substance in report.
The test uses an emulsifier (1.5% of total organic carbon), that allows dispersion of the insoluble substance. The emulsified dispersion is the only form in which the substance is commerscially available and used. In this test 96% biodegradation was found at day 28.
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge from laboratory wastewater plant treating municipal sewage. Concentration of activated sludge in the test assay: 30 mg/l (dry substance) was pre-aerated for about two days before the start of the test
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The biologically produced carbon dioxide is trapped and measured at day 0,1,3,5,7,10,12,14,17,21,24,27 and 28
At the end of the test (day 28) the pH in the test vessels is lowered to remove all the dissolved carbon dioxide.
To determine the removal of dissolved organic carbon, the dissolved organic carbon content is determined at the end (day 28) of the test.

Test set-up:
two test bottles (with test substance and inoculum) (110 mg ThCO2/test vessel)
two blanc controls (with inoculum but without test substance) -
one inhibiton control (with test substance and reference substance and inoculum) (220 mg ThCO2/test vessel)
one abiotioc control (without test substance and without inoculum) -
one reference control (with reference substance and with inoculum) (109.8 mg ThCO2 / test vessel)

% degradation (CO2 evolution)
Sampling time:
28 d
% degradation (DOC removal)
Sampling time:
28 d
Details on results:
biodegradation reference substance on day 28 : CO2/ThCO2 = 105 % ; DOC = 92%
biodegradation test substance on day 28: CO2/ThCO2 = 96%; DOC = 94% ; 10 day window met = yes
biodegradation inhibition control : CO2/ThCO2 = 91% ; DOC = 99%
The substance also met the ten day window criterion for ready biodegradable substances.

validity criteria:
differences between replicates at day 28 < 20%,
degradation of reference substance at day 14 >60%,
degradation of inhibition control after day 14 > 25%,
DIC in the blank control by test begin at 20 mg/l TOC test concentration <1 mg/l
CO2 production in the blank control <70 mg/l
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
Tamol NN2901 was used as an emulsifier to increase the watersolubility and herewith the bioavailability of AKD. The contribution of the emulsifier to the carbon dioxide evolved will be negligilbe relative to that from AKD. AKD dispersion is biodegraded 96% (CO2 to ThCO2) at day 28 in the CO2 evolution test (OECD 301B) and should therefore be classified as readily biodegradable. AKD dispersion is also meeting the 10 day window criteria. Test is performed under GLP according guidelines and all the quality criteria are met.
Executive summary:

The test substance (AKD Dispersion) is readily biodegradable (according the OECD criteria).

Description of key information

Key study is OECD 301 B (ready biodegradability) study (BASF 2003). 
Two peer reviewed articles are used as supporting evidence for ready biodegradability of AKD.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

2-Oxetanone, 3-C12-16-alkyl-4-C13-17-alkylidene (CAS No. 84989-41-3) is commonly referred to in the industry as alkyl ketene dimer (AKD). Alkyl ketene dimers are produced with linear alkyl chains, the alkyl chain lengths vary from C14-C18.

The ready biodegradability of AKD was tested in the CO2 evolution test (OECD 301B). Due to the poor water solubility of the substance, a dispersion containing linear alkyl chains (C16-C18) and a small amount of emulsifier (1,5% of total organic carbon) was tested. The emulsified dispersion is the only form in which AKD is commercially available and used. In this test 96% biodegradation was found at day 28. The substance also met the ten day window criterion for ready biodegradable substances.

The biodegradation of dialkyl ketones formed upon hydrolysis of AKD is probably initiated by monoxygenases (enzyme) catalyzing a Baeyer-Villiger reaction (Kamerbeek, 2003). The ester formed by this reaction is readily hydrolyzed into an alcohol and a fatty acid. The further degradation of these compounds proceeds by b-oxidation with subsequent formation of two-carbon unit shorter fatty acids and acetyl-CoA, with eventual liberation of carbon dioxide. The b-oxidation cycle also handles unsaturated fatty acids. Isomerization of the double bonds occurs to ensure the correct positioning of the double bonds in the substrate for the b-oxidation sequence (Ratledge, 1994). Based on this biodegradation mechanism, the potential of microorganisms to degrade AKD with respect to varying alkyl chain lengths is expected to be comparable.

Alkyl ketene dimers are classified as readily biodegradable based on the ready biodegradability test result of 2-Oxetanone, 3-C12-16-alkyl-4-C13-17-alkylidene and the biodegradation mechanism of ketones.