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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
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
TEST ORGANISMS:
- Source of inoculum/activated sludge: Activated sludge from the municipal sewage treatment plant, 31137 Hildesheim (Germany)
- Preparation of inoculum for exposure: The activated sludge was washed twice with chlorine free tap water; after the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration for 2 hours and 15 minutes; thereafter the sludge was homogenized with a blender; after sedimentation the supernatant was decant-ed and maintained in an aerobic condition by aeration with CO2-free air for 7 days; 10 mL/L were used to initiate inoculation.
- Initial cell/biomass concentration: 1.35 x 10E+09 CFU/L
Duration of test (contact time):
28 d
Initial conc.:
23 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301F
- Application: Once at test start
- Test vessels: Brown glass bottles (volume 500 ml)
- Test volume: 250 ml
- Test temperature: Nominal: 20 - 24, ± 1 °C; Actual: 21.5 – 21.7 °C
- Dispersion treatment: Continuous stirring
- Photoperiod: Dark, in an incubator
- Initial cell/biomass concentration in the final test solution: 1.35 x 10E+07 CFU/L

TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Closed bottles with a rubber sleeve with soda lime
- Measuring equipment: The bottles were closed with OxiTOP measuring heads.
- Oxygen consumption: determined in the incubation vessels by the OxiTop® measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period

SAMPLING
- Sampling frequency: 360 measuring points (every 112 minutes) during the 28 d incubation period.
- Sampling method: Pressure measurements directly from the test vessels
- pH value: at test start and end

CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and reference item
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentrations

STATISTICAL METHODS:
- Calculation of the theoretical oxygen demand (ThOD) of the test and reference item
- According to OECD-guideline 301, the index of the atoms of the item was used for the calculation of the ThOD
- The oxygen depletion of the test item, functional control and toxicity control was calculated out of the difference of the test and inoculum control replicates
- The biodegradation of the test item, functional control and toxicity control was calculated out of the BOD in relation to the ThOD

Reference substance:
benzoic acid, sodium salt
Remarks:
(FLUKA, batch no.: BCBL4472V, purity: 99.7 %, 45 mg/L)
Preliminary study:
No preliminary study
Test performance:
TEST DESCRIPTION:
- Based on the calculated oxygen demand, the test concentration of 25 mg/L, corresponding to an oxygen demand of 50.8 mg O2/L (58.3 mgO2/L with nitrification) in the vessel, was selected
- The test solutions were prepared in measuring flasks and were placed in brown glass bottles as incubation vessels (inoculum control) or were prepared directly in brown glass bottles (test item, functional control, toxicity control): 2 for the test item, 1 for the functional control, 1 for the toxicity control, 2 for the inoculum control
- Separate replicates of test item and toxicity control were prepared for measurement of the pH-value at test start: 1 for the test item, 1 for the toxicity control
- The inoculated test medium, consisting of the required volumes of mineral medium stock solutions, ultrapure water and 10 mL/L inoculum, were prepared in a measuring flask. 250 mL of this solution were filled in the brown glass bottles of the inoculum control, using a 250 mL measuring flask
- For the test item replicates the test item was weighed out for each replicate and directly transferred into the test vessel with 250 mL inoculated test medium
- For the toxicity control, the test and reference item were weighed out and directly transferred into the test vessel with 250 mL inoculated test medium
- For the functional control the reference item was weighed out and di-rectly transferred into the test vessel with 250 mL inoculated test medium
- A rubber sleeve with soda lime was hung into the opening of the bot-tles to absorb evolved CO2; the bottles were closed with OxiTop measuring heads and the measuring system was activated
Key result
Parameter:
% degradation (O2 consumption)
Value:
3
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
1 - 4
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
1
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
6
Sampling time:
21 d
Details on results:
RESULTS:
- Toxicity control: 55 % after 14 days
- Functional group: > 60 % degradation was reached within 2 days
- Theoretical Oxygen Demand: ThOD of 2.03 mg 02/mg test item
Results with reference substance:
Kinetic of reference substance (% Degradation)
82 after 7 days
89 after 14 days
97 after 21 days
96 after 28 days
Validity criteria fulfilled:
yes
Remarks:
(- Ready biodegradation of reference compound ≥ 60 % within 14 days. - Oxygen uptake of blank inoculum was ≤ 60 mg/L.)
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Within 28 days, a degradation rate of 5 % was determined. The substance is classified as not readily biodegradable in the 10-day-window and within 28 days.. The reference compound sodium benzoate showed 79 % degradation after 14 days.
Executive summary:

The ready biodegradability was determined with non-adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted according to OECD guideline 301 F. The test item concentration selected as appropriate was 25 mg/L, corresponding to a ThOD of 50.8 mg 02/L (58.3 mg 02/L with nitrification) per test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage SOD (biological oxygen demand) and was calculated for each study day. The mean oxygen depletion in the inoculum control was 2.8 mg 02/L on day 28. In order to check the activity of the test system sodium benzoate was used as functional control. The pass level > 60 % was reached within 2 days. The biodegradation reached a maximum of 98 % degradation on day 23. In the toxicity control containing both test and reference item 55 % degradation occurred within 14 days. After 28 days the biodegradation came to 59 %. The degradation of the reference item was not inhibited by the test item. The biodegradation remained in the range 0-8 % until test end and the 60 % pass level was not reached within 28 days. The validity criteria of the guideline are fulfilled.

Description of key information

Within 28 days, a degradation rate of 5 % was determined. Solvent Violet 59 is classified as not readily biodegradable in the 10-day-window and within 28 days. The reference compound sodium benzoate showed 79 % degradation after 14 days.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information