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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
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Test performed under GLP according guidelines, meeting quality criteria, sufficient info on test substance CAS 28777-98-2. Test substance was first hydrolysed and the product of the hydrolisation was tested in the closed bottle test. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
according to guideline
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
acceptable deviations
Principles of method if other than guideline:
Instead of an effluent/extract mixture, activated sludge was used as an inoculum.
The inoculum was taken from an activated sludge plant treating predominantly domestic waste water. The sludge was preconditioned to reduce endogenous respiration rates. To this end, the sludge (400 mg dry wt/liter) was aerated for a period of 7 days. The sludge was diluted to a concentration in the biochemical oxygen demand (BOD) bottles of 2 mg dry wt/liter.

Ammonium chloride was omitted from the medium to prevent nitrification
GLP compliance:
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Secondary activated sludge was obtained from the WWTP Nieuwgraaf in Duiven, the Netherlands (30-03-2006). The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic wasewater.
-The sludge was preconditioned to reduce endogenous respiration rates. To this end, the sludge (400 mg dry wt/liter) was aerated for a period of 7 days. The sludge was diluted to a concentration in the biochemical oxygen demand (BOD) bottles of 2 mg dry wt/liter.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
other: hydrolysed test material
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: The nutrient medium of the closed bottle test contained per litre of deionized water: 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O.
- Test temperature: 19-21°C
- pH: 7.0
- pH adjusted: no
- Aeration of mineral medium: yes
- Suspended solids concentration: sludge DW in BOD bottles = 2 mg/L
- Continuous darkness: yes

- Culturing apparatus: incubator
- Number of culture flasks/concentration:
10 bottles containing only inoculum = inoculum blank
10 bottles containing test substance and inoculum =test
6 bottles containing sodium acetate and inoculum = reference control
- Method used to create aerobic conditions: aeration with pressured air
- Measuring equipment: oxygen meter

- Sampling frequency: day 0, 7, 14, 21 ,28 (duplicate measurements)
- Sampling method: the bottles were discarded after the oxygen measurement

- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: no
- Reference control: yes

THOD EKA SA 210 = 2.7 mg/mg
THOD sodiumacetate = 0.8 mg/mg

Oxygen consumption (mg/l) (BOD) = mean oxygen concentration (mg/L) inoculum blank - mean oxygen concentration (mg/l) test (or reference)
Biodegradation (%) = BOD/THOD *100

Reference substance:
acetic acid, sodium salt
% degradation (O2 consumption)
Sampling time:
28 d
Results with reference substance:
The reference substance sodium acetate showed 83% degradation after 14 days.

Oxygen consumption (mg/L) and the percentages biodegradation of Eka SA 210, hydrolysed (BOD/Thod) and sodium acetate (BOD/Thod) in the closed bottle test:

 Time (days) Oxygen consumption (mg/l)     Biodegradation (%)    
  Test substance Acetate  Test substance Acetate 
0.0  0.0 
1.3  4.1  24  76 
14  2.1  4.5  39  83 
21  2.8    52   
28  3.2    60   
Validity criteria fulfilled:
endogenous respiration 0.9 mg/L, differences of replicates at day 28 < 20%; biodegradation of reference substance at day 14 = 83% and >0.5 mg/L of oxygen in the closed bottles during the test.
Interpretation of results:
readily biodegradable, but failing 10-day window
Eka SA 210, hydrolysed is biodegraded 60% at day 28 in the closed bottle test and should therefore be classified as readily biodegradable
Test performed under GLP according guidelines with acceptable deviations, meeting quality criteria.
Executive summary:

In order to assess the biotic degradation, 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.

Eka SA 210, hydrolysed did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum.

Eka SA 210, hydrolysed was biodegraded 60% 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 0.9 mg/L and by the total mineralisation of the reference compound, sodium acetate. Sodium acetate was degraded 83% 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

Biodegradation: ASA and the hydrolysis products can be regarded as readily biodegradable (CAS 28777-98-2)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window
Type of water:

Additional information

Because no test with 2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs on biodegradation is available, two tests with the main constituent C18 ASA are assessed.

In a biodegradability test performed according to OECD Guideline 301 D, the test substance C18 ASA was hydrolysed prior to application. Following O2 consumption, the test item did not cause a reduction in the endogenous respiration so that test substance was therefore considered to be non-inhibitory to the inoculum. As an outcome of this test, a biodegradation of 60 % of the test item in 28 days was found, so the test substance and its hydrolysis products can be regarded as ready biodegradable.

In a biodegradability test according to OECD Guideline 301B, 20 % of the C18 ASA was found to biodegraded in 29 days. However, the tested substance could be regarded as inherently biodegradable.

Since the tested substance C18 ASA is a main constituent of 2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs, the same outcome of the tests would be expected for 2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs .

Additionally, the test results can be also regarded valid for the hydrolysis products of C18 ASA and 2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs ., respectively, because the substances hydrolyse immediately upon contact with water. A QSAR assessment on a representative degradation product confirms these findings.