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Environmental fate & pathways

Biodegradation in water: screening tests

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Description of key information

Di (2-hydroxypropyl) tallow amine is at normal test substance concentrations toxic to the microorganisms in the inoculum. Silica gel may be added to reduce the test substance concentration in the aqueous phase and facilitating slow release of the test substance. Under these conditions, the substance has been demonstrated to be readily biodegradable in Closed Bottle tests.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Di (2-hydroxypropyl) tallow amine is a surfactant consisting of a hydrophilic part i.e. di (2-hydroxypropyl) amine linked to a hydrophobic tallow-alkyl moiety. Biodegradation of both moieties of surfactants usually requires the concerted action of at least two species of microorganisms as one single micro-organism usually lacks the full complement of enzymatic capabilities (van Ginkel, 1996). The biodegradation mechanism of di (2-hydroxypropyl) tallow amine was deduced from semi-continuous activated sludge (SCAS) test results (Ginkel et al. 2009).

In the SCAS test activated sludge was exposed to a specified concentration of the test substance and the non-purgeable organic carbon (NPOC) was analysed frequently to determine the carbon removal. This test has been performed according to OECD Test Guidelines (OECD, 1981). The calculated organic carbon concentration of di (2-hydroxypropyl) tallow in the influent of the SCAS test was 37.2 mg/L. The measured concentrations of the non purgeable organic carbon (NPOC) in the filtrated and unfiltrated diluted stocks (1:20) was 33.5 mg/L and 34.0 mg/L, respectively. 34.0 mg/L (CT) was used to calculate the removal percentages. The test substance caused no reduction of the removal of organic carbon present in the primary settled sewage. Di (2-hydroxypropyl) tallow amine present at a concentration of 50 mg/L is therefore considered to be non-inhibitory to the activated sludge present in full-scale biological treatment plants. These test conditions allow biodegradation of di (2-hydroxypropyl) tallow amine by microorganisms present in activated sludge.

Effluent NPOC values obtained from the test substance and control units were comparable and constant before additions of di (2-hydroxypropyl) tallow amine. During the first 10 days, the carbon removal ranged from 60 to 72% suggesting the formation of hydrophilic compound(s). Thereafter the NPOC values of effluent of the unit fed with domestic wastewater spiked with di (2-hydroxypropyl) tallow amine did decrease further. During the last weeks of the test the removal of di (2-hydroxypropyl) tallow amine increased to a level of >90% indicating degradation of the hydrophilic moiety.The most likely mechanism derived from the SCAS test results is the oxidation of the hydrophobic part (alkyl chain) from the far-end. This oxidation leads to formation of water-soluble amines with two 2-hydroxypropyl and an acetate or butyrate group. These water-soluble amines with two 2-hydroxypropyl and an acetate or butyrate group represent approximately 40% of the carbon of the surfactant. The partial degradation of the alkyl chain and the hydrophilic moiety are degraded by a consortium of microorganisms.

The high carbon removal percentages after an initial partial removal strongly indicate that di (2-hydroxypropyl) tallow amine is ultimately biodegradable.Closed Bottle tests inoculated with adapted activated sludge from the SCAS test are suitable to predict ultimate biodegradation of di (2-hydroxypropyl) tallow amine, which is not in the aqueous phase because of their capacity to adsorb. In a Closed Bottle test with adapted sludge, di (2-hydroxypropyl) tallow amine is biodegraded 63% at day 28. The biodegradation percentage in excess of 60% also indicates ultimate (complete) degradation.

In ready biodegradability tests, the two moieties of this fatty amine derivative are degraded sequentially. The degradation curve will therefore be the sum of two growth curves. The biodegradation of the two moieties may be fully in line with the time window criterion (10-day) when judged as separate chemicals. The time window criterion was developed on the assumption that a compound is degraded according to the “standard” growth curve in ready biodegradability tests. The time-window should therefore be ignored as a pass fail criterion for this surfactant in ready biodegradability tests.

The Theoretical Oxygen Demand (ThOD) of di (2-hydroxypropyl) tallow amine is 2.4 mg/mg. This ThOD has been used to calculate the biodegradation percentages. The pH of the medium was 7.0 at the start of the test. The pH of the medium with sludge at day 28 was 6.8 to 7.2. The pH in the bottles with river water was 7.8 to 8.1. Temperatures ranged from 22 to 24°C. The validity of the tests is demonstrated by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Di (2-hydroxypropyl) tallow amine was not observed to be readily biodegradable in a “standard” Closed Bottle test. The inhibition of the endogenous respiration clearly demonstrates that biodegradation is jeopardized by the toxicity of the test substance (Table). Inhibition of biodegradation by the test substance can in some cases be prevented through the addition of humic acid, lignosulphonic acid or silica gel, all known for their capacity to reduce the toxicity of cationic surfactants (van Ginkel et al, 2008). Inhibition of the biodegradation in OECD tests is an artefact because environmental concentrations are in the range of µg/L. The best result was obtained with silica gel (Table). The toxicity of the test substance was not or only slightly counteracted by lignosulphonates and humic acid.

A final Closed Bottle test incompliance with the OECD principles of Good Laboratory Practice was carried out with river water as inoculum and silica gel to prevent toxicity. Di (2-hydroxypropyl) tallow amine was biodegraded 62% at day 28 in the Closed Bottle test. The time-window was ignored as a pass fail criterion because of the biodegradation kinetics. Hence this substance should be classified as readily biodegradable.


Percentages biodegradation of di (2-hydroxypropyl) tallow amine (BOD/ThOD) in Closed Bottle tests with unacclimated and acclimated sludge. The best result was obtained with river water (without particles), with only 1 mg/L test substance and through addition of silica gel (2 g/bottle).

Time (days)

Biodegradation (%)


Unadapted (sludge)

Unadapted (river water and silica gel)







-4 (toxicity)




-13 (toxicity)




-14 (toxicity)