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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:
24 November 2010 - 23 December 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
(concentration of suspended solids of the sludge was outside the range 3-5 g/l and therefore slightly diluted to obtain an acceptable value; acceptability criteria for positive control substance and background level (total CO2 release in blank) were met)
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
(see OECD 301B remarks)
according to guideline
ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)
Version / remarks:
(see OECD 301B remarks)
according to guideline
other: ISO Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium" (1995)
(see OECD 301B remarks)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2-ethylbutyl)cyclohexane-1-carboxylic acid
EC Number:
Cas Number:
Molecular formula:
C13 H24 O2
1-(2-ethylbutyl)cyclohexane-1-carboxylic acid
Details on test material:
- Name of test material (as cited in study report): CAT-Acid
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark

Study design

Oxygen conditions:
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage
- Storage conditions: The freshly obtained sludge was slightly diluted with ISO medium and kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: Before use, the sludge was allowed to settle (30 minutes) and the liquid was decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
- Pretreatment:
- Concentration of suspended solid in the concentrated sludge: 5.1 g/L (1)

(1) The concentration of suspended solids of the sludge as obtained from the municipal sewage treatment plant was not within the range as described by the protocol (3-5 g/l). The original concentration of suspended solids was 5.1 g/l. Therefore, the freshly obtained sludge was slightly diluted with ISO medium and kept under continuous aeration until further treatment. The amount of suspended solids of the sludge as used for the test was 3.9 g/l. A logical correction on the amount of inoculum was made. Furthermore, the acceptability criteria for the positive control substance and the background level (the total CO2 release in the blank) were met. Therefore, the deviating amount of suspended solids was considered to have no effect on the outcome of this study.
Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
ca. 16.5 mg/L
Based on:
test mat.
Initial conc.:
12 other: mg TOC/L
Based on:
other: organic carbon content was based on the molecular formula
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: according to OECD 301
- Additional substrate: no
- Test temperature: between 21.2 and 22.5°C
- pH: 7.5-7.6 at start, 7.3 -7.9 at the end
- pH adjusted: no
- Aeration of dilution water: yes
- Suspended solids concentration: not reported but expected to meet the guideline criteria because inoculum was prepared in agreement with the guideline
- Continuous darkness: yes (brown coloured bottles)

- Culturing apparatus: 2 litre all-glass brown coloured bottles.
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: stirring, continuous aeration with CO2 free air
- Measuring equipment: none
- Details of trap for CO2 and volatile organics if used: 3 traps, filled with 100 ml 0.0125 M Ba(OH)2

- Sampling frequency CO2 traps: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day, for the inoculum blank and test suspension.
- Sample storage before analysis: none

- Inoculum blank: Yes, duplicate
- Reference: Yes, single
- Toxicity control: Yes, single
- Abiotic sterile control: no

Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

Test performance:
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of CAT-acid.

The degradation curve of the reference substance sodium acetate was as expected.

The temperature recorded in a vessel with water in the same room varied between 21.2 and 22.5°C in both tests.

At the end of the test, the pH values of the different test media were in the range 7.3-7.9 (start: 7.5-7.6).
% Degradation
other: % biodegradation calculated as the ratio between CO2 produced (cumulative) and the ThCO2 of the test substance: 88.3 mg CO2/2l (bottle A), 88.6 mg CO2/2l (bottle B)
3 - 7
Sampling time:
29 d
Details on results:
The ThCO2 of CAT-Acid was calculated to be 2.70 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

The criterion for ready biodegradability (at least 60% biodegradation within a 10-day window) was not met.

The difference of duplicate values for %-degradation of the test substance was always less than 20 (4 at the most).

In the toxicity control more than 25% biodegradation occurred within 14 days (33% (mean of 2 measurements), based on ThCO2). Therefore, the test substance was assumed not to inhibit microbial activity.

The total CO2 release in the blank at the end of the test did not exceed 40 mg/l (54.3 mg CO2 per 2 litres of medium, corresponding to 27.2 mg CO2/l).

The Inorganic Carbon content (IC) of the test substance (suspension) in the mineral medium at the beginning of the test was less than 5% of the Total Carbon content (TC).

BOD5 / COD results

Results with reference substance:
In the positive control (sodium acetate) more than 60% biodegradation occurred within 14 days (61% (mean of 2 measurements), based on ThCO2).

Applicant's summary and conclusion

Validity criteria fulfilled:
Interpretation of results:
under test conditions no biodegradation observed
CAT-Acid was not readily biodegradable under the conditions of the modified Sturm test performed.