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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 June - 4 October 2006
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
according to guideline
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
Adopted January 1998.
GLP compliance:
yes (incl. QA statement)
The study report included a current certificate of GLP compliance for the test facility.
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Aeration tank at Worlingworth sewage treatment works (Suffolk, UK)
- Preparation of inoculum for exposure: aliquots (25 mL) of a homogenised sample were filtered through dried (approximately 105°C) and pre-weighed Whatman GF/C filter papers. Filters were dried and re-weighed. The solids in the sludge determined and an appropriate volume used to inoculate control and test vessels.
- Initial cell/biomass concentration: 30 mg/L
- Water filtered: yes
- Type and size of filter used, if any: Whatman GF/C
Duration of test (contact time):
29 d
Initial conc.:
10 mg/L
Based on:
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: Composition of Mineral Salts Medium was consistant with the medium described in OECD test guideline 301 (301B).
- Test temperature: 20.9 to 23.8°C
- pH: 7.4 ± 0.2
- pH adjusted: yes
- Aeration of dilution water: Inoculum was aerated prior to test initiation.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes (test performed in amber glass bottles)

- Number of culture flasks/concentration: Duplicate test flasks used.
- Method used to create aerobic conditions: Vessels were continuously flushed with air (air has been treated before to remove carbon dioxide)
- Details of trap for CO2 and volatile organics if used: 3 Dreschel bottles in series, each containing 0.025N, nominal barium hydroxide (110 mL) were connected to the air outlet from each test vessel.

- Sampling frequency: daily during the first week of the test and hereafter on day 10, 14, 21, 28 and 29
- Sampling method: 20 mL samples in duplicate were taken from the Dreschler bottle nearest to the test vessel, which was .

- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
Reference substance:
benzoic acid, sodium salt
% degradation (CO2 evolution)
ca. 10
Sampling time:
2 d
Key result
% degradation (CO2 evolution)
Sampling time:
7 d
Key result
% degradation (CO2 evolution)
Sampling time:
29 d
Results with reference substance:
The degradation of the reference substance had achieved 64% of its TCO2 after 6 days of incubation and 79% of its TCO2 after 29 days of incubation.
Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The Subtilisin was considered to be readily biodegradable under the conditions of this test. The test substance reached 102% degradation by the end of the 29-day test period.
Executive summary:

The readily biodegradability of Subtilisin, batch PXI 48489 was assessed in the CO2 Evolution (Modified Sturm) test.
The total organic carbon was measured to calculate the weight of the test substance required to achieve a concentration of 10 mg C/L. Subtilisin, batch PXI 48489 was added to two, five-liter vessels containing three liters of mineral salts medium inoculated with activated sludge (39 mg solids/L).

Two control vessels contained inoculated mineral medium alone and one contained inoculated mineral salts medium plus the reference substance sodium benzoate (10 mg C/L). An inhibition assay comprised sodium benzoate (10 mg C/L) and Subtilisin, batch PXI 49489 (10 mg C/L).

Test, control and reference mixtures were aerated for 29 days with air that had been treated to remove carbon dioxide (CO2). The CO2 produced by each culture was trapped in a series of Dreschel bottles containing barium hydroxide, which were connected to the outlet from each test vessel. The residual barium hydroxide was determined at intervals by titration.

The pH of control, reference and test mixtures was measured at the start of the test and after 28 days. The pH of the test plus reference mixture was measured at the start of the test and on the day of its termination (Day 5).

Sodium benzoate had been biodegraded by 64% after 6 days and 79% after 29 days in the absence of Subtilisin, batch PXI 48489, and by 61% after 5 days in its presence, which confirmed that of Subtilisin, batch PXI 48489 was not inhibitory to the activity of the microbial inoculum. Cumulative levels of CO2 production in the controls adter 29 days were within the acceptable range for this assay system. These results conformed the viability of the inoculum and the test validity.

Mean cumulative CO2 production by mixtures containing Subtilisin, batch PXI 48489 was equivalent to 10% of the theoretical value after ca. 2 days of incubation, 60% by Day 7 and reached 102% by the end of the test on Day 29.

Substances are considered to be readily biodegradable in this test if CO2 production is equal to or greater than 69% of the theoretical value within ten days of the level achieving 10%. Subtilisin, batch PXI 48489 can therefore be considered to be readily biodegradable.

Description of key information

The test substance was considered to be readily biodegradable under the conditions of the OECD 301B test. The test substance reached 102% degradation by the end of the 29-day test period.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A ready biodegradability test (OECD test 301B, Modified Sturm Test) was performed to determine the biodegradability characteristics of Subtilisin.

The study was conducted according to OECD and EC test guidelines, and was performed in accordance with GLP.

The results obtained for the degradation of sodium benzoate (reference substance) and for cumulative CO2production by the control mixtures (81.4 mg CO2) fulfil the validity criteria for this test.

The mean cumulative CO2 production by mixtures containing the test substance at 10 mg C/L was equivalent to appr. 10% of the TCO2 after 2 days, 60% on Day 7 and 102% by the end of the test on Day 29.

On this basis, the test substance was considered readily biodegradable under the conditions of the test.