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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:
01 - 29 Apr 2009
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
insoluble test material was visible during the test.
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Due to low water solubility, an approach endorsed by ISO1995 is to absorb the test material onto an inert support prior to dispersion in the test vessels, ensuring an even distribution and surface area exposed to test organisms is increased.
GLP compliance:
yes (incl. QA statement)
The Department of Health of the Government of the United Kingdom, date of inspection 19 Aug 2008
Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge was obtained on 30 Mar 2009 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, UK, which treats predominantly domestic sewage.
- Storage conditions: The washed sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present.
- Concentration of suspended solids: 2.3 g/L prior to use

Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
other: TOC
Initial conc.:
22.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: as recommended by the guideline OECD 209
- Solubilising agent: silica gel
- Test temperature: 21 °C
- pH: 7.4 - 7.7
- Aeration of dilution water: 24 h prior to addition of test and standard materials, the inoculated culture media were aerated overnight.
- Suspended solids concentration: 30 mg/L medium
- Continuous darkness: yes

- Culturing apparatus: 5 L glass culture vessels, containing 3 L of solution
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The culture vessels were aerated with CO2-free air (40 mL/min) and continuously stirred during the test.
- Measuring equipment: Tekmar-Dohrmann Apollo 9000 TOC analyser, Shimadzu TOC-V CSH TOC analyser
- Details of trap for CO2 and volatile organics if used: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH.

- Sampling frequency: Samples (2 mL) were taken from the control, standard and test material first CO2 absorber vessels on days 0, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on days 0, 2, 6, 8, 10 and 14. The second absorber vessel was sampled on days 0 and 29 for the control, standard and test material and on day 0 for the toxicity control. On day 28, 1 mL of concentrated HCl was added to each vessel to drive off any inorganic carbon. The vessels were resealed, aerated overnight and both absorber vessels were sampled on day 29.
- Sample storage before analysis: All samples were analysed for CO2 immediately.

- Inoculum blank: yes, 2 vessels with inoculated medium and 100 mg silica gel
- Toxicity control: yes, 1 vessel containing 100 mg silica gel, 10 mg TOC/L test material and 10 mg TOC/L reference substance
- Reference control: yes, 2 vessels with inoculated medium, sodium benzoate and 100 mg silica gel

Reference substance:
benzoic acid, sodium salt
test concentration 10 mg TOC/L
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
- Toxicity control: 55% degradation after 14 days (required: ≥ 25%)
- Inorganic carbon contents of the test suspensions in the mineral medium: 2 - 3% of total carbon (required: < 5%)
- From the biodegradation curve of the test substance it is evident that the test substance meets the 10-day window requirement.
- Observations made throughout the test period showed the contents of the control vessels to be light brown, slightly cloudy dispersions and the contents of the standard material vessels were light brown, slightly cloudy dispersions with no undissolved standard material visible. The test material vessels were observed to contain cloudy dispersions with very small globules of test material on the surface. The toxicity control vessel contained a cloudy dispersion with very small globules of test material on the surface and no undissolved standard material visible.
Results with reference substance:
Sodium benzoate attained 84% degradation after 14 days and 86% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Table 1: Percentage Biodegradation Values


% Degradation

Sodium Benzoate

Test Material

Toxicity Control





































Validity criteria fulfilled:
Interpretation of results:
readily biodegradable
The test material attained 70% degradation after 28 days and therefore can be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301B.
Executive summary:


A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)).


The test material, at a concentration of 10 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al, 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms.

The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.


The test material attained 70% degradation after 28 days and satisfied the
10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test material can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Description of key information

Readily biodegradable: 70% (ThCO2) in 28 days (OECD 301B)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

One key study investigating the ready biodegradability of the substance according to OECD 301B and GLP is available (Showa Denko, 2009). In the test domestic activated sludge was used as inoculum. Due to the low solubility of the test material in water, it was adsorbed on silica gel prior to dispersal in medium applying high shear mixing. Cloudy dispersions with very small globules of test material on the surface were observed in the test vessels.

Based on the measurement of generated CO2, the test item attained 70% degradation after 28 days and can therefore be classified as readily biodegradable under the strict OECD conditions. The test meets all validity criteria required by the guideline.