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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:
May 2017 - June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
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 activated sludge: sewage plant for domestic sewage in Balatonfüred, Hungary, on 26 April 2017
- Storage length: 7 days
- Preparation of inoculum for exposure: 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. A pH adjustment of activated sludge inoculum was not performed.
- Pretreatment: Pre-conditioning (26 April-03 May 2017) consisted of aerating (2 L/minute) activated sludge (in mineral medium) at the test temperature (the actual temperature: 20.0 – 21.1 °C). During the aeration the cell count of inoculum was checked as follows: the viability of the cultured sludge was determined by plating 0.1 mL of the different, usually 10E-1, 10E-2, 10E-3 and 10E-4 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by these plating experiments by manual colony counting. During the pre-conditioning the approximately cell count of aerated inoculum was in the range of ~10E8 - 10E10/L; therefore, before the test the inoculum was further diluted 100 000 x with mineral medium to reach the necessary 10E5 - 10E6 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improved the precision of the test methods by reducing blank values.
Duration of test (contact time):
28 d
Initial conc.:
5.8 mg/L
Based on:
test mat.
Initial conc.:
5.15 other: mg O2/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 301 D
- Additional substrate: no
- Solubilising agent: no
- Test temperature: 20.0 - 20.9 °C
- pH: 7.63
- pH adjusted: no
- Aeration of dilution water: for 20 min
- DOC: 8.90 mg/L
- Illumination: no

TEST SYSTEM
- Culturing apparatus: Winkler bottles (300 mL, coded) with special neck and glass stoppers
- Number of culture flasks/concentration: 16 (+ 2 reserve) bottles
- Method used to create anaerobic conditions: bottles completely filled and tightly closed
- Measuring equipment: Oxygen and pH meter with appropriate O2 and pH electrode
- Test performed in open system: no

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

CONTROL AND BLANK SYSTEM
- Inoculum blank: Only filtered inoculum was added to the aqueous test medium
- Procedure control: sodium benzoate with aqueous test medium
- Toxicity control: Test and reference item with aqueous test medium
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 5.15 mg 5-sulfosalicylic acid /L (corresponds to 6 mg/L 5-sulfosalicylic acid dihydrate). No toxic effect of the test item was found at this investigated concentration.
Key result
Parameter:
% degradation (DOC removal)
Value:
12
Sampling time:
28 d
Details on results:
The pass level for ready biodegradability is the removal of 60 % ThODNH4 in a 10-day window. Minimal biodegradation of the test item occurred in this study; however the value remained far below the pass level; therefore the test item was considered to be not ready biodegradable.
Results with reference substance:
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 82.9 % on the 14th day.)
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item is considered to be not ready biodegradable, since the pass level for ready biodegradability that is the removal of 60 % ThODNH4 in a 10-day window was not fulfilled.
Executive summary:

The ready biodegradation test was conducted according to the test method OECD 301 D (Closed Bottle Test). 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. Under the test conditions minimal biodegradation of this test item was noticed. The percentage biodegradation of the test item reached a mean of 12.0 % after 28 days based on its ThODNH4. The pass level for ready biodegradability is the removal of 60 % ThODNH4 in a 10-day window. Minimal biodegradation of the test item occurred in this study; however the value remained far below the pass level; therefore the test item is considered to be not ready biodegradable.

Description of key information

The test item is considered to be not ready biodegradable, since the pass level for ready biodegradability that is the 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. Under the test conditions minimal biodegradation of this test item was noticed. The percentage biodegradation of the test item reached a mean of 12.0 % after 28 days based on its ThODNH4. The pass level for ready biodegradability is the removal of 60 % ThODNH4 in a 10-day window. Minimal biodegradation of the test item occurred in this study; however the value remained far below the pass level; therefore the test item is considered to be not ready biodegradable.