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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Non-adapted activated sludge from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant. 30 mg/L dry matter was present in the final mixture. The activated sludge was used after sampling from the treatment plant without adaptation.

However, the sludge was pre-conditioned (aerated but not fed) for 5 days, to reduce the amount of O2 consumed by the blank controls. Before the pre-conditioning phase, the sludge was washed twice with tap water, by 2 centrifugation steps. Then the sludge was suspended in test medium, at about 2 g/L dry matter. Before the test, this suspension was diluted down to 300 mg/L dry matter, i.e. ten-fold the final concentration, and an appropriate amount of this suspension was added to the flasks.
Duration of test (contact time):
28 d
Initial conc.:
31 mg/L
Based on:
test mat.
Initial conc.:
20.1 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Aerobic mineral salts media prepared with ultrapure water.
- Additional substrate: None
- Solubilising agent (type and concentration if used): None
- Test temperature: 22 +/- 2°C
- pH: 7.3 (mean value of test units)
- pH adjusted: no
- Aeration of dilution water: Yes
- Suspended solids concentration: 30 mg/L dry matter
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: Three replicates
- Method used to create aerobic conditions: water sufficiently aerated before use
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: IC analysis
- Test performed in closed vessels due to significant volatility of test substance: Closed vessels as standard based on test design
- Details of trap for CO2 and volatile organics if used: CO2 trapped in KOH solutions

SAMPLING
- Sampling frequency: Days 0, 1, 7, 10, 14, 17, 21, 24 and 28.
- Sampling method: Samples taken and analysed for IC.
- Sterility check if applicable: NA
- Sample storage before analysis: NA
- Treatment: The test item was applied with the glass fiber method. An aliquot of a stock solution in ethanol was applied on glass fiber filters under a stream of compressed air, and then dried at 40°C for about 3h to remove any traces of solvent. The final concentration in the test flasks was about 20 mg/L with respect to its total organic carbon (TOC) content. Control flasks were also supplemented with glass fiber filters only treated with solvent.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Three replicates (containing inoculum and test medium)
- Procedure control: Three replicates (containing inoculum, test medium and sodium benzoate as ready biodegradable reference item).
- Abiotic sterile control: One replicate (containing test item, ultrapure water and 0.04 mM HgCL2 as a sterilizing agent to prevent microbial decomposition).
- Toxicity control: One replicate (containing inoculum, test medium, test item and sodium benzoate as ready biodegradable reference item).
- Other:

STATISTICAL METHODS: Not performed
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not required
Test performance:
All validity criteria met
Parameter:
% degradation (CO2 evolution)
Value:
30
Sampling time:
28 d
Details on results:
The biodegradability of the test substance, based on CO2 evolution and expressed as % of the theoretical value (ThCO2), was calculated to be 30% after an incubation time of 28 days. At the end of the 10-d window, the biodegradation of the substance reached 21%. Significant biodegradation of the test item was observed after a lag phase of about 1 day.

The positive control, sodium benzoate, reached 100% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.
The substance did not reach the pass level of 60% for ready biodegradability in the CO2 Evolution Test, neither within the 10-d window, nor after 28 days of incubation, and therefore, cannot be termed as readily biodegradable.

According to the OECD guideline, at the applied initial test concentration of about 20 mg/L, the substance is not considered to have any inhibitory effects on the microbial population, since the biodegradation of the mixture (test item + reference item sodium benzoate) exceeded 25% within 14 days.

No significant degradation of the test item in the absence of microorganisms after 28 days of incubation was observed, as indicated by the lack of significant CO2 production.

At the end of the test, the mean pH values of the blank control and of the procedure control were 7.3 and 7.5, respectively. The mean pH values of the test units and the toxicity control were 7.3 and 7.5, respectively. These values lie in the required range.

The temperature was in the required range of 22±2°C during the whole test period.

The degradation of the procedure control exceeded 100%; which cannot be explained. Given that the degradation of the test item was consequently possibly overestimated (according to the procedure control), the conclusion of the present testing remains unchanged (the substance cannot be termed as readily biodegradable).
Results with reference substance:
The results of the procedure control confirmed the acceptance of the test system.

Test substance ready biodegradation:

Time (days)

Blank control

Test unit no. 1 containing test item

Test unit no. 2 containing test item

Mean degradation (%)

Total CO2 release in vessel (mg IC/L)

Total CO2 release in vessel (mg IC/L)

Degradation (%)

Total CO2 release in vessel (mg IC/L)

Degradation (%)

0

0

0

0

0

0

0

1

0.2

0.3

0.3

0.1

-0.7

0

7

1.7

2.4

3.6

3.1

7.3

5

10

2.9

4.7

8.7

4.5

7.6

8

14

3.6

6.7

15.2

6.8

15.7

15

17

4.2

7.9

18.5

7.8

17.7

18

21

4.8

9.1

21.4

8.8

19.9

21

24

5.4

11.2

28.9

9.8

22.3

26

28

5.2

11.5

31.7

10.8

28

30

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
After 28 days the test substance achieved 30% biodegradation based on CO2 evolution in this OECD guideline study. As such the substance cannot be considered readily biodegradable, although some biodegradation was observed.

Description of key information

After 28 days the test substance achieved 30% biodegradation based on CO2 evolution in this OECD guideline study. As such the substance cannot be considered readily biodegradable, although some biodegradation was observed.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information