Registration Dossier

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
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
liquid: viscous
Specific details on test material used for the study:
Name: Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine
Batch/Lot No.: 0002293298
Appearance: High-viscous, colourless to yellowish liquid
Purity: Considered as 100%
Expiry date: 22 November 2019
Storage conditions: Controlled room temperature (15-25ºC, below 70 relative humidity (RH) %)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

Study design

Inoculum or test system:
sewage, domestic, 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. 3.4 mg/L
Based on:
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
An aqueous stock solution of the test item with a concentration of 34.0 mg/L was prepared and diluted accordingly. The test item concentration in the test solutions was 3.4 mg/L.
The chosen test item concentration was based on the measured chemical oxygen demand (COD): 1.79 ±0.031 mg test item and on the performed 14-d 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 the test item (1.79 mg O2/mg), stock solution (34 mg/L) was prepared and diluted with aqueous test medium to get a concentration level of 3.4 mg/L test item, with a COD of about 6.086 mg O2/L).
2.) Procedure Control: Sodium benzoate (flasks 2a and 2b)
Based on the theoretical oxygen demand (ThOD) of Sodium benzoate (1.67 mg O2/mg; details of calculation are given in the guidelines), stock solution (360 mg/L) was prepared and diluted with aqueous test medium to get a concentration level of 3.6 mg/L reference item, respectively a ThOD of about 6.012 mg O2/L).
3.) Inoculum Control (flasks 3a and 3b)
Only filtered inoculum was added to 3.38 litres of aqueous test medium.
4.) Toxicity Control (flasks 4a and 4b)
340 mL test item stock solution and 34.0 mL reference item stock solution were mixed into 3.40 litres of aqueous test medium corresponding to 3.4 mg/L test item (COD of 6.086 mg O2/L) and 3.6 mg/L reference item (ThOD of 6.012 mg O2/L).
Microbial inoculum (0.5 mL per litre) was added to each preparation bottle.

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.

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)
The reference item and inoculum (procedure control) were also part of a concurrent studies (Citoxlab study codes: 18/163-322AN, 18/104-322AN and 18/144-322AN) performed in the same experimental period using the same batch of chemicals and solutions. Raw data sheets are clearly associate data with the appropriate study number. Copies of all raw data of positive and negative controls run in parallel were placed in the study file.

Results and discussion

% Degradation
Key result
% degradation (O2 consumption)
ca. 8.2
Sampling time:
28 d

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The test item was not considered to have significant inhibitory effects on the secondary effluent microorganisms.
Based on the results of this study, the test item is considered not readily biodegradable.
Executive summary:

The test item 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 of Ethanol, 2-(2-butoxyethoxy)-, reaction products with phosphorus oxide (P2O5), compds. with N,N-dimethylcyclohexanamine reached a mean of 8.2% after 28 days based on the COD of the test item.

According to the test guidelines the pass level for ready biodegradability, 60 % of ThOD/COD. Therefore, the test item is considered not readily biodegradable.