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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 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
α-d-Glucopyranosiduronic acid, (3β,20β)-20-carboxy-11-oxo-30-norolean-12-en-3-yl 2-O-β-d-glucopyranuronosyl-, dipotassium salt
EC Number:
EC Name:
α-d-Glucopyranosiduronic acid, (3β,20β)-20-carboxy-11-oxo-30-norolean-12-en-3-yl 2-O-β-d-glucopyranuronosyl-, dipotassium salt
Cas Number:
Molecular formula:
α-d-Glucopyranosiduronic acid, (3β,20β)-20-carboxy-11-oxo-30-norolean-12-en-3-yl 2-O-β-d-glucopyranuronosyl-, dipotassium salt
Specific details on test material used for the study:
CAS number: 68797-35-3
EC number: 272-296-1
Batch number: 3194/001/A17
Appearance: White to yellowish Powder
Purity: 95% (102.7% according to the Certificate of Analysis)
Expiry date: 11 July 2019
Storage conditions: Controlled room temperature (15-25oC, ≤70% relative humidity), protected from humidity (store in a tightly closed container)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety.

Study design

Oxygen conditions:
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
The inoculum: Secondary effluent, microorganisms from a domestic waste water treatment plant.
Origin: The secondary effluent was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The secondary effluent used for this study was allowed to settle approximately for an hour, then the decanted effluent was aerated until use (not later than an hour).
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 3.7 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:

The test was carried out in an incubator and controlled environment room (during the formulation and oxygen measuring) at a temperature of 22  2C according to guideline. The test flasks were placed into an incubator and kept at 21.1 - 22.1C, in the dark. The temperature was measured on weekdays during the experiment.
The oxygen concentration of test water was 8.7 mg/L at the start of the test.
The pH value of the test water was checked prior start of the experiment. The pH of the test water was 7.45.

The test conditions were measured with suitable instruments and documented in the raw data.


Before the preparation of the respective test solutions with Dipotassium Glycyrrhizate an aqueous stock solution of the test item with a concentration of 37.0 mg/L was prepared and diluted accordingly (using ultrasonic bath for 2 minutes). During the preparation of the test solutions they were mixed by mechanical stirring. The test item concentration in the test solutions was 3.7 mg/L.
The chosen test item concentration was based on the measured chemical oxygen demand (COD): 1.64 ±0.044 mg O2/mg test item and on the performed 14-day preliminary test.

The components were applied in the amounts/volumes in the test flasks:

1.) Test Item (flasks 1a and 1b)

Based on the measured chemical oxygen demand (COD) of 1.64 mg O2/mg test item,
test item stock solution* (corresponding to 12.58 mg of Dipotassium Glycyrrhizate) was thoroughly mixed into 3.40 litres of aqueous test medium (corresponding to 3.7 mg/L test item, with a COD of about 6.068 mg O2/L).

* The concentration of the stock solution was: 37.0 mg/L.

2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)

Based on the theoretical oxygen demand (ThOD) of Sodium benzoate (1.67 mg
O2 per mg; details on calculation are given in the guidelines), stock solution* corresponding to 12.24 mg of Sodium benzoate was mixed into 3.40 litres of aqueous test medium (corresponding to 3.6 mg/L reference item, respectively a ThOD of about 6.012 mg O2/L).

* The concentration of the stock solution was: 360.0 mg/L.

3.) Inoculum Control (flasks 3a and 3b)

Only filtered inoculum was added to 3.40 litres of aqueous test medium.

4.) Toxicity Control (flasks 4a and 4b)

The Test item* (340.0 mL) and the reference item stock solutions** (34.0 mL) were mixed into 3.40 litres of aqueous test medium corresponding to 3.7 mg/L test item (COD of 6.068 mg O2/L) and 3.6 mg/L reference item (ThOD of 6.012 mg O2/L).

* The concentration of the stock solution was: 37.0 mg/L.
** The concentration of the reference item stock solution was: 360 mg/L.

Microbial inoculum (0.5 mL per litre) was added to each preparation bottle.


1/ Preparation of Test Flasks

Sufficient number of BOD flasks was cleaned with 5 - 10 mL of a wash liquid (2.5 g iodine and 12.5 g potassium iodide per litre of 1 % w/v sulphuric acid) by shaking well to coat the bottle walls. After allowing to stand for 15 minutes, the wash liquid was poured off, and the bottles were thoroughly rinsed with tap water and deionised water. Then, the previously described test solutions were filled into the bottles bubble-free until the bottles were completely filled. Then they were tightly closed with glass stoppers.

3.9.2. The Test Bottles

The number of test bottles was the follow:
 10 bottles containing the test item and inoculum
 10 bottles containing the reference item and inoculum (procedure control)
 10 bottles containing only inoculum (inoculum control)
 10 bottles containing the test item, reference item and inoculum (toxicity control)
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Key result
% degradation (O2 consumption)
ca. -2.5
Sampling time:
28 d

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
Executive summary:

The test item Dipotassium Glycyrrhizate was investigated for its ready biodegradability in a Closed Bottle Test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

The test system was a microbial inoculum of good quality, collected from a sewage plant for domestic sewage. The sludge was allowed to settle and then aerated until use.

 Under the test conditions the percentage biodegradation ofDipotassium Glycyrrhizatereached a mean of -2.5 % after 28 days based on the COD of the test item.

According to the test guidelines the pass level for ready biodegradability, 60 % ofThOD/COD.

Therefore, the test item is considered not readily biodegradable.