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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
according to
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
GLP compliance:
yes (incl. certificate)

Test material


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): mixed population of aquatic microorganisms (activated sludge) was obtained on 2016-10-04 from the aeration tank of a wastewater plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)

- Preparation of inoculum for exposure:
- Before use, the inoculum was stored for one day at room temperature under continuous stirring with aeration.
- The sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm and 20 °C and decanting off the supernatant.
- An aliquot of the wet sludge was dried in order to determine the wet weight / dry weight ratio of the sludge and to prepare a stock suspension (activated sludge) of 3 g dw/L.
- The calculated amount of sludge, needed to achieve 300 mL of this stock suspension, was dissolved in mineral medium and then filled up to a defined end volume.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
- Composition of medium: mineral medium:
The mineral medium was prepared from stock solutions of mineral components described as follows.
1. Mineral salt solution:
KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H2O 33.40 g/L
NH4CI 0.50 g/L
The components were dissolved in deionised water and filled up to 1 litre.
The pH of the solution was in the recommended range of pH 7.4 ± 0.2.

2. Magnesium sulphate solution:
MgSO4.7H2O 22.50 g/L
The substance was dissolved in deionised water and filled up to 1 litre.

3. Calcium chloride solution:
CaCl2.2H2O 36.40 g/L
The substance was dissolved in deionised water and filled up to 1 litre.

4. Iron (III) chloride solution:
FeCl3.6H2O 0.25 g/L
The substance was dissolved in deionised water and filled up to 1 litre. To stabilise the solution 1 drop of concentrated hydrochloride solution (37%) was added.

To prepare the mineral medium 10 mL of the mineral salt solution were mixed with 800 mL deionised water. Then 1 mL each of the magnesium sulphate-, calcium chloride- and the iron (III) chloride solution were added and filled up to 1 litre with deionised water.

- Test temperature: 22 ± 1 °C
- pH adjusted: no
- Suspended solids concentration: 30 mg/L suspended solids
- Continuous darkness: yes

- Culturing apparatus: 250 mL flasks
- Number of culture flasks/concentration: 3 per test item & control (but only 2 considered for results for test item and control), 2 per reference item and one for the toxicity control
- Measuring equipment: OxiTopControl System (WTW); mixing: 1 magnetic stirrer per test vessel
- Measuring frequency: Daily

- Inoculum blank: The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item.
- Toxicity control: 25 mg of the test item and 25 mg of the reference compound were weighed out and added to the test flasks, filled with 200 mL of mineral medium. Afterwards the flask volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg test item and reference compound/L.
- Other: NO3-N/NO2-N chemical analysis: Determination of nitrite nitrogen and nitrate nitrogen and the sum of both by flow analysis (CFA and FIA) and spectrometric detection; test apparatus: Continuous Flow Analyser
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradation
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Details on results:
All validity criteria of the test method were met:
The reference compound reached the level of ≥ 60 percent for ready biodegradability within 14 days.
The toxicity control exhibited degradation rates > 25 % within 14 days.
At the end of the test, at the plateau, or the end of the 10-d window, biodegradation in parallels with test item did not differ by more than 20 percentage points.
The oxygen uptake of the inoculum blank was ≤ 60 mg/L.
In cases where the degradation was ≤ 60 %, the pH values were in between 6.0 and 8.5 at the end of the test.

BOD5 / COD results

Results with reference substance:
The reference compound sodium benzoate showed 85 % degradation after 14 days.

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The study was conducted under GLP according to OECD 301 F without deviations on the registered substance itself. The method is to be considered scientifically reasonable and suitable for the test item, the available information allows the conclusion that the test was properly conducted, the positive control showed the appropriate results. Hence, the results can be considered as reliable to assess ready biodegradability of the test substance. The test item attained 81% biodegradation after 28 days and can therefore be considered as readily biodegradable under the terms and conditions of OECD Guideline No. 301F.
Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, "Ready Biodegradability; Manometric Respirometry Test" and EU method C.4-D.

A suspension of 100 mg/L test item in a mineral medium, equalling to 50-100 mg ThOD or COD/Litre as the nominal sole source of organic carbon, was stirred in a closed flask and inoculated at a constant temperature (22 ± 1 °C) for up to 28 days under aerobic conditions in the dark.

The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item. A reference compound (sodium benzoate) was run in parallel to check the operation of the procedures.

A toxicity control (test item and reference compound mixed, one replicate) was run in parallel, to ensure that the chosen concentration of the test item was not inhibitory to microorganisms.

Degradation was followed by the determination of oxygen uptake and measurements were taken at frequent intervals to allow the identification of the beginning and end of biodegradation and the slope of the biodegradation curve.

Hydroxytoluic acid showed:

0 % degradation after 7 days

61 % degradation after 14 days

75 % degradation after 21 days

81 % degradation after 28 days

Therefore, Hydroxytoluic acid is considered to be “Readily Biodegradable“.

The reference compound sodium benzoate showed 85 % degradation after 14 days. All validity criteria of the test method were met.