Registration Dossier

Diss Factsheets

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Dipotassium propanedioate
EC Number:
Cas Number:
Molecular formula:
Dipotassium propanedioate

Study design

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): activated sludge from municipal wastewater treatment plant AZV Staufener Bucht (predominantly domestic wastewater).
- Dry solid content of the activated sludge: 4.7 g/L after drying for 6.75h at 105°C.
- Preparation of the inoculum: activated sludge was washed 2x with tap water by settling the sludge, decanting the supernatant and re-suspending the sludge.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
150 - 150.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: 10 mL A + 900 mL demineralised water + 1 mL B + 1 mL C + 1 mL D, and subseqently the volume is made up to 1L.
A: 8.50g KH2PO4, 21.75g K2HPO4, 33.40g Na2HPO4*2H2O, 0.50g NH4Cl are dissolved in demineralised water and made up to 1L.
B: 36.4g CaCl2*2H2O is dissolved in demineralised water and made up to 1L.
C: 22.5g MgSO4*7H2O is dissolved in deminseralised water and made up to 1L.
D: 0.25g FeCl3*6H2O is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 1L.
- Test temperature: 20.1-22.2°C
- pH: not determined
- pH adjusted: yes/no

- Culturing apparatus: 1000 mL gas wash bottles
- Number of culture flasks/concentration: 3
- Method used to create aerobic conditions: overnight aeration with CO2-free air on the day before addition of test substance.
- Measuring equipment: inorganic carbon measurement by means of total carbon analyser TOC 5050A, Shimadzu.
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: absorber wash bottles containing 0.2M NaOH

- Determination of inorganic carbon concentration of the 0.2M NaOH solution: IC = 6.32 mg/L.
- Sampling frequency: sampling on day 4, 7, 11, 14, 21 and 28
- Sampling method: 4 mL NaOH solution from the first of two CO2-absorber flasks was samples and the IC content was determined. On day 28, 2 mL of 4M HCl was added to the reactor in order to release the CO2 dissolved in water. On day 29, IC was determined in both CO2 absorber flasks in line.

- Inoculum blank: yes
- Reference substance: yes: sodium benzoate, conc. 20 mg organic carbon /L
- Abiotic sterile control: no
- Toxicity control: yes: 150.2 mg of test item + 5.15 mL of 10.0g/L sodium benzoate stock solution, to yield a total organic carbon concentration of 40 mg organic carbon /L.

Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
not applicable
% Degradation
Key result
% degradation (CO2 evolution)
St. dev.:
Sampling time:
28 d
Details on results:
The degradation extent of the test item at the end of the test was 81.0% (28 d after acidification, mean of three replicates.
Within 7 days the degradation of the test item was already 62.5% and was therefore higher than 60%. The 10-day window is met.

- Toxicity control: The degradation extent in the toxicity control was 76.2% within 14 days, the test substance had no inhibitory effect on the inoculum
- Reference item: The reference compound sodium benzoate reached the pass level for ready biodegradability within 4 days
- Blank: The mean CO2-evolution of the blank flasks was 20.0 mg/L on day 28 after acidification

- Criteria of validity:
- The IC content in the test vessel was less than 5% of the TOC introduced with the test item
- The CO2 evolution in the inoculum blank at the end of the test was above below 40 mg/L
-The difference of extremes of replicate values of the test item at the end of the test was less than 20% in two of three test vessels
- The biodegradation of the reference compound reached the pass level of 60% ThCO2 by day 7
- The degradation extent in the toxicity control was above 25% in 14 days based on ThCO2
The test is valid according to OECD Test Guideline 301 B (July 1992).

Any other information on results incl. tables

Ultimate biodegradation after x days [% of ThCO2]

reactor day 0 4 7 11 14 21 28 29
17 test flasks 0 45.3 61.1 71.4 73.8 77.3 78.8 80.1
18 0 46.1 61.9 71.4 75.2 79.4 79.3 81.9
19 0 51.6 64.6 72.5 74.4 77.9 79.6 80.9
4 Reference flasks 0 66.2 82 88.8 92.3 96.2 94.2 93.7
5 0 71.7 86.4 92.3 96 100.9 100.4 99.9
6 0 67.1 81.9 88.8 92.1 96 93.9 93
20 Toxicity control  0 53.7 66.2 73.3 76.2 79.2 79.4 84.9
Test item
Reference item

Mean CO2-evolution of blank flasks after x days

Day 0 4 7 11 14 21 28 29
CO2-evolution (mg/L) 0 6.7 9.9 12.4 14.7 16.6 20.3 20

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The degradation extent of the test item at the end of the test was 81.0% (28 d after acidification, mean of three replicates).
Therefore the test item reached the criteria for ready biodegradability (60% of ThCO2 within a 10-d window).
Executive summary:

A CO2 Evolution Test according to OECD 301 B (July 1992) was conducted in order to investigate the ready biodegradability of Dipotassium malonate. The test was performed in compliance to GLP.

A solution or suspension of the test item in a mineral medium, corresponding to 10 - 20 mg TOC/L, is inoculated with activated sludge (30 mg d. s./L). The test vessels are aerated by the passage of carbon dioxide-free air and are incubated under aerobic conditions in diffuse light for 28 days. Degradation is followed by determining the carbon dioxide produced and absorbed to sodium hydroxide via IC-measurement (IC = inorganic carbon). The amount of carbon dioxide produced from the test item less the amount derived from the blank inoculum is expressed as a percentage of ThCO2 (theoretical amount of CO2). The pass level for ready biodegradability is 60% of ThCO2 and must be reached within a 10-d window. The 10-d window begins when the degree of biodegradation reaches 10%. The test method is suitable for water soluble and non-soluble test items which are non-volatile and not toxic under the test conditions.

In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound and one reactor containing test item and reference compound (toxicity control) were set up.

The degradation extent of the test item at the end of the test was 81.0% (28 d after acidification, mean of three replicates. Within 7 days the degradation of the test item was already 62.5% and was therefore higher than 60%.

The test item Dipotassium malonate reached the pass level for ready biodegradability and passed all validity criteria according to Guideline OECD 301B (July 1992).

Dipotassium malonate was found to be readily biodegradable.