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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:
January 2021 to May 2021
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 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
Molecular formula:
Test material form:
Details on test material:
Test substance was expected to be stable for the duration of testing.
Specific details on test material used for the study:
Batch CH-DA-01-18

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
The study was performed with aerobic activated sewage sludge from the aeration stage of the local wastewater treatment plant which treats predominantly domestic sewage.
Details on inoculum:
Aerobic activated sewage sludge was washed twice by centrifugation, decantation of the supernatant liquid phase and resuspension of the solid material in tap water and finally the mineral medium.

A calculated amount of wet sludge was suspended in mineral medium to obtain a concentration equivalent to 4 g dry material per litre. During the holding period of one day prior to use, the sludge was aerated at room temperature. Prior to use, the dry weight of the sludge was again determined and the sludge was diluted with mineral medium to 802 mg dry material per litre. Defined volumes of the diluted activated sludge were added to the mineral medium in the test vesesl to obtain a final concentration of 30 mg dry material per litre.

Mineral medium was prepared according to testing guidelines.
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:

The mineral medium was prepared according to test guidelines. Purified water was used from an ELGA PURELAB Option-Q water purification system. The following stock solutions were obtained by dissolving analytical grade salts in ultrapure water:

Stock Solution 1 (pH was 7.4):

KH2PO4 - 8.50 g/L
K2HPO4 - 21.75 g/L
Na2HPO4 - 33.40 g/L
NH4Cl - 0.5 g/L

Stock solution 2:

CaCl2.2H2O - 36.40 g/L

Stock Solution 3:

MgSO4.7H2O - 225.50 g/L

Stock Solution 4:

FeCl3.6H2O - 0.25 g/L, stabilised with one drop concentrated HCl per litre

The final 1L mineral medium was prepared by combining stock solution 1 (10 mL), stock solution 2, 3 and 4 (1 mL of each) and ultrapure water (800 mL). The resulting solution was mixed and made up to 1L with ultra-pure water.

The pH was adjusted from 7.6 to 7.4 with dilute hydrochloride acid.

The test temperature was 22.0-22.2 oC

The pH at the start of the test was 7.4

The test vessels were incubated under diffused lighting


For the measurement of BOD, the OxiTop Control system from WTW GmbH, Weilheim, Germany consisting of pressure measuring heads, test bottles, inductive stirring system and OxiTop OC 110 controller was used.

The CO2 was absorbed by an alkali trap which caused a net reduction in the gas pressure within the test vessel. The pressure reduction was continuously detected by the OxiTop pressure measuring heads and recorded every four hours. The OxiTop controller collected the pressure values from the measuring heads and processed them to BOD values.


The prepared test vessels were air-tight closed with the OxiTop measuring heads and the measurement of oxygen uptake started. The vessels were incubated under experimental conditions for 28 days the the BOD values automatically recorded every four hours.


The toxicity control was sodium benzoate (99.5% purity, Sigma-Aldrich) and was tested simultaneously under the same conditions as the test item and functioned as a procedure control.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Key result
% degradation (O2 consumption)
Sampling time:
28 d
Remarks on result:
other: Average of results of two test replicates
% degradation (O2 consumption)
Sampling time:
21 d
Remarks on result:
other: Average of results of two test replicates
Key result
% degradation (O2 consumption)
Sampling time:
14 d
Remarks on result:
other: Average of results of two test replicates
% degradation (O2 consumption)
Sampling time:
7 d
Remarks on result:
other: Average of results of two test replicates

BOD5 / COD results

Results with reference substance:
The percentage degradation of the reference item reached 69% by Exposure Day 4. The pass level for ready biodegradability is at least 60% in a 10 day window (by Day 14).

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
inherently biodegradable
The pass level for ready biodegradability for dihydronepetalactone (biodegradation of at least 60% of the ThOD in a 10-day window within the 28-day period) was not reached. Therefore, dihydronepetalactone was not readily biodegradable under the test conditions within 28 days. The obtained biodegradation value of 71% can be used to indicate inherent biodegradability based on Paragraph 36 of the "Revised Introduction to the OECD Guidelines for Testing Chemicals, Section 3".
Executive summary:

The ready biodegradability of test substance dihydronepetalactone was determined during a GLP-compliant study performed in accordance with OECD Test Guideline 301F (Manometric Respirometry).


Sodium benzoate was used as a reference substance and the activated sludge was taken from a domestic sewage treatment plant.  Oxygen consumption was evaluated on Day 0, 7, 14, 21 and 28.  All validation criteria were met.

On Day 14 (10-day window) the test substance did not achieve the 60% of the ThOD and therefore dihydronepetalactone was not readily biodegradable.  The obtained biodegradation value of 71% indicates that dihydronepetalactone is inherently biodegradable.