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Environmental fate & pathways

Biodegradation in water: screening tests

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Description of key information

The test substance, cocobis(2-hydroxyethyl)methylammonium chloride was biodegraded by 70% at day 28 in the Closed Bottle test, and can therefore be classified as readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

In order to assess the biotic degradation, five ready biodegradability tests are performed which allow the biodegradability to be measured in aerobic aqueous media. Four out of five studies were performed with (modified) Closed Bottle Tests (CBT, according to OECD TG 301D) with the substance as registered. For the other study no information on test method was reported. Various biodegradation results were reported.

In the oldest study (1974) a biodegradation percentage of 82.3% was reported for the substance. Details on test method are lacking. In the absence of a full study report, the study was assigned reliability 4.

In another study (1989), a biodegradation percentage of 20-23% was reported (OECD TG 301D, GLP, reliability 2). The low biodegradation results were explained by the very poor solubility and therefore very poor bioavailability. Inhibition of respiration was not observed at the test concentration. It was concluded that the biodegradation of the substance should be assessed using a test design with a slow release method. This study can be used as evidence of biodegradability and this report can be considered reliable with restrictions.

In a third study (2009) a combination of a semi-continuous activated sludge test with the closed bottle test was employed to search for a suitable screening test method taking into account the poor solubility and poor bioavailability of the substance. The tests were performed according to OECD guidelines with acceptable minor deviations (non GLP). The biodegradation percentage was reported to be 63% after 28d. The results can be used as supporting evidence. The optimal conditions found in this screening work were used in two subsequent GLP studies.

In the key study (2009), the ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD TG301D and in compliance with GLP. Ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission did not result in nitrogen limitation as shown by the biodegradation of the reference compound) and river water was used as inoculum. The test was performed in the presence of silica gel to allow for a slow release of the test substance.


Cocobis(2-hydroxyethyl)methylammonium chloride did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be noninhibitory to the inoculum. Cocobis(2-hydroxyethyl)methylammonium chloride was biodegraded 70% at day 28 in the Closed Bottle test. Hence this compound should be classified as readily biodegradable. The test is valid as shown by an endogenous respiration of 1.4 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded 70% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

In the fifth test (2009), a biodegradation percentage of 64% at day 28 was reported in a CBT (OECD 301D, GLP) under similar conditions as the key study. The test substance did not meet the 10-day window validation criteria as mentioned in the OECD guideline 301D, but this concept is not considered valid to quantify biodegradation rates of mixtures of homolgue surfactants as the cocobis(2-hydroxyethyl)methylammonium chlorides.The 10-day window concept assumes that biodegradation of a single organic compound in a ready biodegradability test is a growth-linked process which follows an S-shaped growth curve. Cocobis(2-hydroxyethyl)methylammonium chlorides are mixtures of homologues with C12-C18 alkyl chains. The biodegradation kinetics (lag period, growth rate, yield etc.) of the individual homologues in the mixture are not necessarily the same. The biodegradation of a surfactant consisting of a mixture of homologues is therefore an addition of different biodegradation curves. It is thus possible that individual compounds meet the 10-day time window criterion whereas the biodegradability curve of the mixture of homologues suggests that the surfactant is not readily biodegradable. 

Based on the latter two studies (OECD 301D, GLP, reliability 1) it can be concluded that the substance fullfills the criterion for readily biodegradability. Therefore, the biodegradation half lives proposed in the REACH guidance for readily biodegradable substances are used in the Chemical Safety Assessment.