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Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
test performed accoring OECD 301D guidelines
Adequacy of study:
key study
Study period:
19-02-2019 - 14-05-2019
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
only minor devation
Principles of method if other than guideline:
Minor deviations from the guidelines of the Closed Bottle test (OECD TG 301D) were introduced; a) ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound) and it improves the precision of the test method.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical name: Sodium cocopropylenediamine propionate
Substance name: β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts
Old CAS no 97659-50-2
New CAS no: 2136366-30-6
Oxygen conditions:
Inoculum or test system:
natural water: freshwater
Details on inoculum:
According to OECD TG 301 river water can be used as other source of inocculum.
The river water was sampled from the Rhine near Heveadorp, The Netherlands (14-02-2019). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992) which improves the precision of the test method. River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
act. ingr.
Initial conc.:
6.8 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test procedures
The Closed Bottle test (OECD TG 301D) was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only river water, 6 bottles containing river water and sodium acetate, 10 bottles containing river water with test substance. The concentrations of the test substance, and sodium acetate in the bottles were 6.8 mg/L (2.0 mg/L active) and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were com¬pletely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.
Reference substance:
acetic acid, sodium salt
Preliminary study:
Results of the screening test are presented in F19018RG where under identical conditions the biodegradability of Sodium cocopropylenediamine propionate was evaluated using two inocula: River water as in this study and Activated sludge which was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to 2.0 mg DW/ in the biological oxygen demand (BOD) bottles (van Ginkel and Stroo, 1992).
Biodegradation of 60% and 63% were found at day 28 in the Closed Bottle test using river water and activated sludge as inoculum, respectively. Sodium cocopropylenediamine propionate should therefore be classified as readily biodegradable. In the prolonged Closed Bottle test the biodegradation percentages for Sodium cocopropylenediamine propionate increased further and reached 79% biodegradation for the test using river water and 86% for the test using activated sludge at day 56.
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
Sodium cocopropylenediamine propionate was biodegraded by 70% at day 28 in the Closed Bottle test (Table II, Figure). Over 60% biodegradation was not achieved within a period of 10 days (14 days for the Closed Bottle test) immediately following the attainment of 10% biodegradation. However, sodium cocopropylenediamine propionate is a chemical consisting of a hydrophilic group linked to a hydrophobic moiety. Biodegradation of both moieties of surfactants requires the concerted action of at least two microorganisms as a single organism usually lacks the full complement of enzymatic capabilities (van Ginkel, 1996). In ready biodegradability tests, the two moieties of this surfactant are therefore 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-day window criterion 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 be ignored as a pass-fail criterion for this surfactant. Sodium cocopropylenediamine propionate is classified as readily biodegradable based on the achievement of 70% biodegradation at day 28.

Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 90


Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of the test substances to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected (Table I). Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.


Test conditions

The pH of the media ranged from 8.7 to 8.8 at the start of the test. The pH of the medium at day 28 was 8.8 (test) and 8.9 (control). The temperature ranged from 22.7 to 22.8°C which is within the prescribed temperature range of 22 to 24°C.


Validity of the test

The validity of the test is demonstrated by an endogenous respiration of 1.2 mg/L at day 28 (Table I). Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 90 (Table II and Figure). Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Validity criteria fulfilled:
The test is valid because of an endogenous respiration of 1.2 mg/L at day 28. Less than 20% difference of replicates day 28. The biodegradation% the reference compound at day 14 was 90. Final oxygen concentrations were >0.5 mg/L.
Interpretation of results:
readily biodegradable
Test performed according guidelines meeting specific criteria. Test performed under GLP and results well documented.
Executive summary:

In order to assess the biotic degradation of sodium cocopropylenediamine propionate, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

The test substancedid not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. The test substance was biodegraded by 70% at day 28 in the OECD 301D Closed Bottle test and should therefore be classified as readily biodegradable.

The test is valid as shown by an endogenous respiration of 1.2 mg/L and by the total mineraliza­tion of the reference compound, sodium acetate. Sodium acetate was degraded by 90% 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.

Description of key information

The substance has been shown to be ready biodegrable in OECD screening studies.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:

Additional information

Sodium cocopropylenediamine propionate was shown to be readily biodegradable. The substance is a UVCB and therefore the 10day window (14 day for the CBT) does not need to be evaluated.

Lebertz (1992) found 69.1% degradation after 28 days in a closed bottle test according to OECD 301D

Danneberg (1993) found 113.8% when testing 5 ppm, 52.4% when testing 10 ppm and 54.5% when testing 20 ppm after 28 days in the modified Sturm test according to OECD 301B.

Roberts (1988) found Sodium cocopropylenediamine propionate to be inherently biodegradable in an OECD 302A test.

The Biodegradability study was repeated in 2019 by Geerts because the results from Danneberg and Robers were not unambiguous. With 70% biodegradation at day 28 the study from Geerts confirms that Sodium cocopropylenediamine propionate is readily biodegradable.