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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:
03.05.2017 to 31.05.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: sewage plant for domestic sewage in Balatonfüred, Hungary
- Storage length: 7 days before the main test
- Preparation of inoculum for exposure: The inoculum 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 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.14, just before use: 7.32.
- Pretreatment: Pre-conditioning consisted of aerating (2 L/minute) activated sludge (in mineral medium) for 7 days at the test temperature (the actual temperature: 20.0 – 21.1 °C). During the pre-conditioning the approximately cell count of aerated inoculum was in the range of ~10^8 - 10^10/L; therefore, before the test the inoculum was further diluted 100 000x with mineral medium to reach the necessary 10^5 - 10^6 cells/L cell concentration.
- Concentration of sludge: 5 g dry material/L
- Initial cell/biomass concentration: ~10^5 - 10^6 cells/L
- Water filtered: no
- Type and size of filter used: After preparation the sludge was filtered through cotton wool.
Duration of test (contact time):
28 d
Initial conc.:
2.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium used. Prepared according to OECD 301.
- Test temperature: 20.0 - 20.1 °C
- pH: 7.63
- pH adjusted: no
- Aeration of dilution water: yes
- Suspended solids concentration: 5 g dry material/L
- Continuous darkness: yes
- Oxygen concentration of the test water: About 8 - 9 mg/L (was measured at the start of the test and found to be 8.90 mg/L).

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL, coded) with special neck and glass stoppers.
- Number of culture flasks/concentration: 10 (+ 2 reserve) bottles containing the test item and inoculum; 16 (+ 2 reserve) bottles containing the reference item and inoculum (procedure control);
- Measuring equipment: The oxygen concentration was measured with an O2 electrode.

SAMPLING
- Sampling frequency: in all duplicate bottles in all groups on days 0, 7, 14, 21 and 28.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 16 (+ 2 reserve) bottles containing only inoculum (inoculum control)
- Toxicity control: 16 (+ 2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The test item solubility, behavior, and toxicity were tested in a 14-day preliminary experiment. The test design was the same as described at the main experiment. In the preliminary experiment the test item was investigated at the concentration of 2.5 mg/L. No toxic effect of the test item was found at this investigated concentration.
Key result
Parameter:
% degradation (O2 consumption)
Value:
15.9
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of Bis(methylthio)methane reached a mean of 15.9 % after 28 days based on its ThODNH4. The threshold for ready biodegradability (60 % biodegradation based on the ThOD) was not reached in the test; therefore, the test item can be considered as not ready biodegradable.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to a mean of 82.9 % after 14 days, and to a mean of 87.9% after 28 days of incubation, based on its ThODNH4.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item is considered to be not ready biodegradable. The pass level for ready biodegradability of 60 % ThOD removal in a 10-day window was not fulfilled. The biodegradation percentage after 28 day was 15.9%.
Executive summary:

The ready biodegradability of the test item was determined with a non adapted activated sludge over a test period of 28 days in the Closed bottle test design according to OECD 301 D, Regulation (EC) No. 440/2008 Method C.4 -E and EPA OPPTS 835.3110. The test item was tested at a concentration of 2.5 mg/L in duplicates.The oxygen concentration was measured with an O2 electrode. The reference item sodium benzoate was sufficiently degraded to a mean of 82.9 % after 14 days, and to a mean of 87.9% after 28 days of incubation, based on its ThODNH4. The biodegradation of the test item reached a mean of 15.9% after 28 days based on its ThODNH4. In conclusion, the test item is not readily biodegradable as the pass level of 60% degradation was not reached in a 10-d-window within the 28 days period of the study.

Description of key information

The test item is considered to be not ready biodegradable, since the pass level for ready biodegradability (removal of 60% ThODNH4 in a 10-day window) was not fulfilled (reference 5.2.1-1).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The ready biodegradation test was conducted according to the test method OECD 301 D (Closed Bottle Test, reference 5.2.1-1). 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. All validity criteria of the study were met. The percentage biodegradation of the test item reached a mean of 15.9% after 28 days based on its ThODNH4. The pass level for ready biodegradability is the removal of 60% ThODNH4 in a 10-day window and therefore the test item is considered to be not ready biodegradable.