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Environmental fate & pathways

Biodegradation in water: screening tests

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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:
other: GLP compliant guideline study without restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Municipal activated sludge from a wastewater treatment plant. The inoculum was collected from the aeration tank of the plant. In the laboratory the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm and aerated in the laboratory until use. To reduce the blank value effect in the test assays the activated sludge was aerated with pressured air for about 48 h at 23.1 to 23.2 °C.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The manometric respiration test was performed in 500 mL test vessels filled up to a volume of 250 mL. For the preparation of the blank controls, the reference substance, the inhibition control and the test substance 240 mL mineral medium were dosed into the incubation bottles. The test vessel for the verification of physical elimination was filled up with 250 mL deionized water. The required amount of test substance was weighed to small glass plates and completely added with the glass plates to the test substance assay, the inhibition control assay and the physical elimination assay. For the preparation of the reference substance assay and inhibition control assays about 25 mg of aniline were added to the test vessels.
The physical elimination was tested without inoculum. To obtain an activated sludge concentration of 30 mg/L dry substance 10 mL of the prepared suspension of municipal activated sludge were added at all other test assays.
The incubation bottles were closed with a specifically stopper filled with soda lime pellets for adsorption of the biogenic produced carbon dioxide. All test vessels were implemented to the respirometer. The assays were stirred by magnetic stirrers and the consumed oxygen was automatically delivered and registered
additionally.
The data on oxygen consumption were read daily and summarized over the test period of 28 days. With consideration of the oxygen consumption of the blank control assays the BOD values were calculated for the reference substance assay, the inhibition control and for the test substance assays and related to the inserted amounts of test- and reference substance, respectively. The BOD value of the physical elimination assay was calculated without consideration of the mean value BOD of the blank controls assays.
The BOD values were compared with the corresponding ThOD values and the relations of BOD to ThOD were specified as per cent biodegradation.
Reference substance:
aniline
Test performance:
Validity criteria fulfilled:
The difference of extremes of replicate values of the removal of the test substance at the end of the experimental phase, should be less than 20 %: yes
Degradation degree of the reference substance should be > 60 % BOD/ThOD after 14 days: yes
The highest oxygen uptake of the single blank controls should be < 60 mg/L at the end of the exposure period: yes
The degree of biodegradation in the inhibition control should be > 25 % BOD/ThOD after 14 days: yes
pH values in the test assays with test substance at the end of exposure should be in a range from pH 6 to pH 8.5: yes
Parameter:
other: BOD/ThOD
Value:
3
Sampling time:
28 d
Details on results:
The degree of biodegradation was calculated at the end of exposure as mean value. Based on the degree of biodegradation of < 10 % BOD/ThOD at the end of exposure, the test substance can be evaluated as poorly biodegradable in this study.
Results with reference substance:
Degree of biodegradation of the reference substance aniline after 14 days (mean value of two single values): 64 % BOD/ThOD
Degree of biodegradation in the inhibition control after 14 days: 42 % BOD/ThOD
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2013-01-11 to 2013-02-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study without restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
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 from a municipal sewage plant, waste water sewage plant Mannheim, Germany
- Storage conditions: aerated with carbon dioxide free air for about 48 hours at 22 ± 2° C
- Pretreatment: No
- Concentration of sludge: 800 mg/L dry weight
- Initial biomass concentration: 4 mg/L
- Water filtered: yes
- Type and size of filter used, if any: woven mesh with a mesh size of about 1 mm
Duration of test (contact time):
28 d
Initial conc.:
20 other: mg/L TOC
Based on:
act. ingr.
Initial conc.:
ca. 26 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: Test medium was prepared in accordance to the OECD Guideline.
- Solubilising agent: Acetone, the solvent was removed by evaporation before the mineral medium was added to the test vessels.
- Test temperature: 22 +/- 2 °C
- pH: 7.4
- pH adjusted: yes
- Aeration of dilution water: No
- Suspended solids concentration: 4 mg/L

TEST SYSTEM
- Culturing apparatus: 160 mL incubation bottles filled up to a volume of 100 mL
- Number of culture flasks/concentration: 29 blank controls, 31 bottels treated with test substance, 29 inhibition controls, 17 toxicity controls, 5 abiotic controls
- Method used to create aerobic conditions: oxygen, available in the headspace of the bottles, was used for the aerobic mineralization processes
- Measuring equipment: Total Inorganic Carbon (TIC) was determined with a TOC-analyzer. The measured TIC-values were compared with the added amount of TOC and expressed as biodegradation degree and plotted by a biodegradation curve.

SAMPLING
- Sampling frequency: Samples were taken after 3, 5, 7, 11, 14, 18, 21, 25 and 28 days of exposure
- Sampling method: at each sampling date three bottles were taken. 1 mL 7M sodium hydroxide solution was injected with a syringe trough the septum in each test bottle. The bottles were shaken for about one hour. The carbon dioxide in the headspace, evolved by biological processes, was solved in the alkaline solution. For the measurement at the end of exposure after 28 days five test item bottles were prepared.
- Sample storage before analysis: The samples were analyzed on the day of sampling without further treatment

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control: Yes
Reference substance:
aniline
Remarks:
initial weight: 401 mg/L, corresponding ThOC-value: 310 mg/L
Preliminary study:
A pH- shift in the test vessels, induced by addition of the test substance to the test medium was checked in a preliminary test. No shift of the pH-value was detected. The pH-value from the mineral test medium was therefore assumed for the test assays at start of exposure
Parameter:
% degradation (inorg. C analysis)
Value:
3
St. dev.:
1
Sampling time:
28 d
Details on results:
Validity criteria
TIC-value in the blank control at the end of exposure (mean value): 1.0 mg/L
Deviation of degree of biodegradation of the test substance at end of exposure should be <20%: yes
Degree of biodegradation of the reference substance should be > 60% TIC/TOC after 14 days: yes
Degree of biodegradation in the inhibition control should be > 25 % TIC/TOC after 60 days: yes
TIC-value in the blank control at the end of exposure should be <15% of the amount of test substance added initially (mg TOC/L): yes
Test temperature in the range of 22 ± 2 °C: yes
Results with reference substance:
Degree of biodegradation in the reference item bottles was 79 % after 14 days of exposure.
Degree of biodegradation in the inhibition control was 44 % after 28 days of exposure.
Degree of biodegradation in the abiotic control was 2 % after 28 days of exposure.

Description of key information

Two tests according to OECD Guideline 301 F and OECD Guideline 310 indicate that the test substance is poorly biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Two tests according to OECD Guideline 301 F and OECD Guideline 310 indicate that the test substance is poorly biodegradable.

In the key study (BASF, 2013) the ready biodegradability of the test item was assessed over a 28 day period by the Manometric Respiration Test (OECD 301F). The nominal test concentration was 100 mg a.s./L. The test material attained up to 3 % biodegradation after 28 days whereas the positive control substance (Aniline) was 68 % degraded after 28 days.

These results are confirmed by a supportive study (BASF, 2013) using the CO2 Headspace Test (OECD 310). The nominal test concentrations was 20 mg a.s./L. The test material attained up to 3 % biodegradation after 28 days whereas the positive control substance (Aniline) was 79 % degraded after 14 days.

These results indicate that the test item cannot be classified readily biodegradable under conditions of the test.