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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
Study period:
2020-03-03 to 2020-03-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
January 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
30 may 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
adopted on 17 July 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge, microorganisms from a domestic waste water treatment plant.

- Origin: Sewage plant for domestic sewage in Balatonfüred, Hungary

- Conditioning: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with test water (mineral medium, see above) and then aerated under test conditions (for 6 days) until use. The pH of the activated sludge inoculum after preparation was 7.39, just before use: 7.30. A pH adjustment of the activated sludge inoculum was not performed.
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: According to guideline
- Test temperature: 20.1 – 20.7 °C (during the preparation, aeration and incubation of the mineral medium); 20.3 - 20.7 °C (during incubation of test units)
- pH: 7.37
- pH adjusted: no
- Aeration of dilution water: yes
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Large glass tank (volume: ~30 L) and Large glass bottles (volume: at least 5 L)
- Number of culture flasks/concentration: 16
- Method used to create aerobic conditions: Aeration system
- Measuring equipment: Oxygen and pH meter with appropriate O2 and pH electrode

SAMPLING
- Sampling frequency: Oxygen concentration measurements were performed on days 0, 2, 5, 7, 12, 14, 21 and 28.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The biodegradation in % was calculated based on ThODNH4 of the test item
Test performance:
The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. (The percentage degradation of the reference item was 75.3 % on the 14th day.)
Key result
Parameter:
% degradation (O2 consumption)
Value:
17.1
Sampling time:
28 d
Results with reference substance:
The biodegradation of the test item did not reach 60 % after 28 days of incubation. Thus, the test item is not readily biodegradable.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
In a study performed according to OECD TG 301 D, the test item is considered to be not readily biodegradable. The pass level for ready biodegradability is removal of 60 % theoretical oxygen demand (ThODNH4) in a 10-day window.
Executive summary:

The ready biodegradability of the test item in a Closed Bottle Test was determined in a test according OECD 301 D. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item was investigated at the concentration of 3.0 mg/L. The test item concentration was chosen based on the theoretical oxygen demand of the test item (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 2.04 mg O2/mg. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met. Under the applied test conditions, no ready biodegradation of this test item was noticed. The percentage biodegradation of the test item reached a mean of 17.1 % after 28 days based on its theoretical oxygen demand (ThODNH4). The highest biodegradation value of 17.4 % was obtained on the 21st day of the test. The reference item, sodium benzoate, was sufficiently degraded to a mean of 75.3 % after 14 days, and to a mean of 77.5 % after 28 days of incubation, based on ThODNH4. (The biodegradation reached its plateau on about the 7th day and from this day onwards the observed slight changes were considered as being within the biological variability range of the applied test system). In the toxicity control containing both, the test item and the reference item, a mean of 38.7 % biodegradation was noted within 14 days and 39.5 % biodegradation after 28 days of incubation (from about the 7th day of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system). The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.


In a study performed according to OECD TG 301 D, the test item is considered to be not readily biodegradable. The pass level for ready biodegradability is removal of 60 % theoretical oxygen demand (ThODNH4) in a 10-day window.


According to the test guidelines the test item can be assumed as not inhibitory (at the applied concentration level) on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.

Description of key information

In a study performed according to OECD TG 301 D, the test item is considered to be not readily biodegradable. The pass level for ready biodegradability is removal of 60 % theoretical oxygen demand (ThODNH4) in a 10-day window.

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information

The ready biodegradability of the test item in a Closed Bottle Test was determined in a test according OECD 301 D. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 °C) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item was investigated at the concentration of 3.0 mg/L. The test item concentration was chosen based on the theoretical oxygen demand of the test item (ThODNH4 calculated according to equation given in the guidelines, assuming that no nitrification occurs) of 2.04 mg O2/mg. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. All validity criteria of the study were met. Under the applied test conditions, no ready biodegradation of this test item was noticed. The percentage biodegradation of the test item reached a mean of 17.1 % after 28 days based on its theoretical oxygen demand (ThODNH4). The highest biodegradation value of 17.4 % was obtained on the 21st day of the test. The reference item, sodium benzoate, was sufficiently degraded to a mean of 75.3 % after 14 days, and to a mean of 77.5 % after 28 days of incubation, based on ThODNH4. (The biodegradation reached its plateau on about the 7th day and from this day onwards the observed slight changes were considered as being within the biological variability range of the applied test system). In the toxicity control containing both, the test item and the reference item, a mean of 38.7 % biodegradation was noted within 14 days and 39.5 % biodegradation after 28 days of incubation (from about the 7th day of the test onwards the obtained slight changes in the biodegradability values were considered as being within the biological variability range of the applied test system). The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum.


The test item is considered to be not readily biodegradable, since the pass level for ready biodegradability is removal of 60 % theoretical oxygen demand (ThODNH4) in a 10-day window.


According to the test guidelines the test item can be assumed as not inhibitory (at the applied concentration level) on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days.