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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012-11-22 to 2013-01-21 (experimental start and end)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed under GLP and according to appropriate methods to address this endpoint.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from municipal sewage STP D-31137 Hildesheim. The sludge of that STP comprises mostly municipal sewage and hardly industrial chemical waste.
- Laboratory culture: Not applicable.
- Method of cultivation: Not applicable.
- Storage conditions: In the dark at test temperature 20 to 24°C, under aeration with CO2 free air.
- Storage length: 4 hours under aerobic conditions, prior to use.
- 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. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air until test start. 20 mL/L of this mixture were used to initiate inoculation.
- Pretreatment: No data.
- Concentration of sludge: See below, biomass concentration.
- Initial cell/biomass concentration: 10^7 to 10^8 CFU/L under test conditions.
- Water filtered: See above.
- Type and size of filter used, if any: No data.
Duration of test (contact time):
28 d
Initial conc.:
8.81 mg/L
Based on:
other: Organic Carbon (mg C/L)
Initial conc.:
17 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
TOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 301.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 20 to 22.5 °C.
- pH adjusted: No.
- pH during the test: 7.7 to 7.8 in all test groups.
- CEC (meq/100 g): No data are reported.
- Aeration of dilution water: Aeration with CO2 free air.
- Suspended solids concentration: Not determined.
- Continuous darkness: Low light conditions.

TEST SYSTEM
- Culturing apparatus: Several bottles in series (reactor system): 5 L reactor connected to 3 x 250 mL gas-washing bottles (in series), filled with Ba (OH2) adsorption solution.
- Number of culture flasks/concentration:
Reactors 1 and 2: Test concentration 8.81 mg C/L.
Reactors 3 and 4: Blank control.
Reactor 5: Reference substance sodium benzoate at 11.6 mg C/L.
Reactor 6: Toxicity control, i.e. 8.81 mg C/L + 11.6 mg C/L reference substance.
- Method used to create aerobic conditions: Continuous aeration with CO2 free air, mixing of the test solutions by magnetic stirring.
- Method used to create anaerobic conditions: NA.
- Measuring equipment: Titration equipment (type not specified).
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: Sealed vessels, but aerated with CO2 free air.
- Details of trap for CO2 and volatile organics if used: Yes. Aliquots from these traps were removed to measure TIC (indirect method via non precipitated Ba(OH2).

SAMPLING
- Sampling frequency: days 1, 4, 6, 8, 11, 14, 18, 21, 25 and 28 after incubation initiation.
- Sampling method: Non precipitated Ba (OH)2 was titrated by hydrochloric acid to the phenolphthalein endpoint.On day 28 TOC was determined after removal of CO2 from test solutions by HCl.
- Sterility check if applicable: NA.

- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, two replicates.
- Abiotic sterile control: No.
- Toxicity control: Yes (one replicate).
- Reference substance: Yes (one replicate).
- Other:

STATISTICAL METHODS: NA
Reference substance:
benzoic acid, sodium salt
Test performance:
No events are reported, which might have affected the quality of the study.
Parameter:
% degradation (TOC removal)
Value:
12
Sampling time:
28 d
Remarks on result:
other: Mean of 5% and 18%
Details on results:
The substance was degraded within 28 days by average 12% (mean of 18% and 5%). Trigger for ready biodegradation is 60%. Biodegradation of the reference substance reached 44 % within 14 days of incubation. Trigger for validity: 60%. The substance was not toxic to the activated sludge, since 44% of of the reference substance was degraded within 14 days in the presence of the test substance (trigger is 25%).
Results with reference substance:
Biodegradation of the reference substance reached 77 % within 14 days of incubation. Hence the test is rendered valid.
Validity criteria fulfilled:
yes
Interpretation of results:
other: Not readily biodegradable
Conclusions:
The substance is not readily biodegradable: Average biodegradation (12%) is below the guideline trigger value of 60%. However biodegradation was observed: The 10% generic trigger value for biodegradation initiation of one replicate was reached within 11 days after test initiation. For both replicates a biodegradation plateau had been reached on day 18.
Executive summary:

In a valid GLP study according to OECD 301B the test item was aerobically exposed in duplicate at 17 mg/L test substance (8.81 mg C/L, 1.90 ThCO2 per mg test item) to standard OECD mineral medium and non-adapted, activated sludge obtained from a domestic STP. Final concentration of bacteria in test: 10^7 - 10^8 CFU/L. Exposure was for 28 days at low light conditions and permanent stirring at 20 to 22.5°C. Six brown glass reactors with a volume of 5 L were stocked with 3 L volume each which was constantly aerated with CO2 free air. Each reactor had received inocculum and mineral medium and was equipped with 3 gas washing bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution. Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in an alkaline solution (0.0125 mol/L Ba(OH)2). Five test groups were incubated: Test item (8.81 mg C/L, two replicates), blank control (two replicates), reference substance (11.6 mg C/L of sodium benzoate, one replicate) and a toxicity control (8.81 mg C/L test substance plus 11.6 mg C/L reference substance, one replicate). Biodegradation was determined on days 1, 4, 6, 8, 11, 14, 18, 21, 25 and 28. In addition residual CO2 in the inocculum was determined on day 28 after release of CO2 from the inoculum by HCL and measurement of trapped CO2 after purging the inoculum for 24 hours. The results show that all validity criteria of the test guideline were met. Biodegradation of the reference substance reached 44 % within 14 days of incubation. Trigger for validity: 60%. The substance was not toxic to the activated sludge, since 44% of of the reference substance was degraded within 14 days in the presence of the test substance (trigger is 25%). Hence the results obtained for the test item are valid. The results show that the substance was degraded within 28 days by average 12% (mean of 18% and 5%). Trigger for ready biodegradation is 60%. In conclusion, the substance is not readily biodegradable: Average biodegradation (12%) is below the trigger value of 60%. However biodegradation was observed: The 10% generic trigger value for biodegradation initiation of one replicate was reached within 11 days after test initiation. For both replicates a biodegradation plateau had been reached on day 18.

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-01-29 to 2013-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed under GLP and according to appropriate methods to address this endpoint.
Qualifier:
according to
Guideline:
OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
other: non-adapted and adapted activated domestic sludge (test groups 1 and 2, respectively)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge from municipal sewage STP D-31137 Hildesheim. The sludge of that STP comprises mostly municipal sewage and hardly any industrial chemical waste.
- Laboratory culture: Not applicable.
- Method of cultivation: Not applicable.
- Storage conditions: Not applicable.
- Storage length: Not applicable.
- Preparation of inoculum for exposure: The activated sludge was washed twice with tap water. The dry sludge concentration was determined and an appropriate volume of inoculum was chosen to reach a dry sludge concentration of 0.2 - 1.0 g/L in the test vessels and a ratio of DOC of test compound / inoculum in the range of 1:2.5 - 1:4.
- Pretreatment: Side experiment with adapted sludge: On day 0, 1 L of activated sludge was spiked with 50 mg/L yeast extract. 4 mg C/L of the test item was added. The inoculum was covered and stirred continuously. On day 7 and 10 after preparation of the inoculum 8 mg C/L of the test item was added. Prior to application the dry sludge concentration was determined and an appropriate volume of inoculum was chosen to reach a dry sludge concentration of 0.2 - 1.0 g/L in the test vessels and a ratio of DOC of test compound / inoculum in the range of 1:2.5 - 1:4.
- Concentration of sludge: See below, biomass concentration.
- Initial cell/biomass concentration: Non-adapted inoculum: 6.86 g dry sludge/L. Adapted inoculum: 5.87 g dry sludge/L. Ratio of DOC of test compound to inoculum dry weight: 1:4.0 (non-adapted and adapted inoculum).
- Water filtered: See above.
- Type and size of filter used, if any: No data.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Initial conc.:
51.8 mg/L
Based on:
other: Carbon
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium according to OECD 302 B.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 20.3 to 23 °C.
- pH adjusted: The pH was adjusted in every test vessel to 6.5 - 8.0, if necessary.
- pH value during the test: 6.7 to 7.8 in all test vessels.
- CEC (meq/100 g): No data are reported.
- Aeration of dilution water: Aeration with CO2 free air under continuous stirring.
- Suspended solids concentration: Not determined, assumed: ambient conditions.
- Continuous darkness: No information, ambient conditions assumed.

TEST SYSTEM
- Culturing apparatus: Several bottles in series (reactor system): 2 L test vessels.
- Number of culture flasks/concentration:
Test group 1 (non-adapted sludge)
Reactors 1 and 2: Test concentration 100 mg/L test material (51.8 mg C/L).
Reactors 3 and 4: Blank control.
Reactor 5: Reference substance - Diethylene glycol 120 mg/L (54.4 mg C/L).
Reactor 6: Abiotic control - Test item in test concentration without inoculum, poisoned with
10 mL/L HgCl2 solution (10 g/L).
- Number of culture flasks/concentration:
Test group 2 (adapted activated sludge):
Reactors 1 and 2: Test concentration 100 mg/L test material (51.8 mg C/L).
Reactor 3: Blank control.
Reactors 4 and 5: Reference substance - Diethylene glycol 120 mg/L (54.4 mg C/L).
- Method used to create aerobic conditions: Continuous aeration with CO2 free air, mixing of the test solutions by magnetic stirring (continuously).
- Method used to create anaerobic conditions: NA.
- Measuring equipment: DOC Analyzer.
- Test performed in closed vessels due to significant volatility of test substance: No.
- Test performed in open system: Yes, but aerated with CO2 free air.
- Details of trap for CO2 and volatile organics if used: Not applicable.

SAMPLING
- Sampling frequency:
Non adapted activated sludge: days 0 (3 hours), 2, 7, 14, 20, 28 after incubation initiation.
Adapted activated sludge: days 0 (3 hours), 3, 7, 14, 21, 28 after incubation initiation.
- Sampling method: Removal and filtration of aliquots.
- Sterility check if applicable: Not reported.

- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, two replicates.
- Abiotic sterile control: Yes (one replicate).
- Toxicity control: No.
- Reference substance: Yes (one replicate).
- Other:

STATISTICAL METHODS: NA
Reference substance:
benzoic acid, sodium salt
Test performance:
No events are reported, which might have affected the quality of the study.
Parameter:
% degradation (DOC removal)
Value:
100
Sampling time:
14 d
Remarks on result:
other: Non-adapted sludge
Parameter:
% degradation (DOC removal)
Value:
100
Sampling time:
14 d
Remarks on result:
other: Adapted sludge
Details on results:
The biodegradation for the replicates with non-adapted activated sludge reached the 10 % level (beginning of biodegradation) after 3 days. After 7 days the biodegradation came to 52 %. The pass level of 70 % was reached on day 10 and the biodegradation came to a maximum of 100 % after 14 days. For the replicates with adapted activated sludge, the start of the biodegradation (10% level) was reached after one day. After 7 days, a biodegradation of 78 % was determined and the pass level of 70 % was reached after 5 days. The maximum of degradation was 100 % after 14 days. No significant physico-chemical elimination occurred in the sterile control after 28 days (maximum 3%).
Results with reference substance:
Biodegradation of the reference substance reached 100 % within 14 days of incubation. Hence the test is rendered valid.
Interpretation of results:
other: Inherently ultimately biodegradable
Conclusions:
The substance is inherently ultimately biodegradable by non-adapted sludge. Biodegradation was reached within 3 days (10% level passed) and the pass level of 70% was reached on day 10. There was no elimination by sorption and abiotic degradation and hence elimination is due to mineralization of the test substance.
Executive summary:

In a valid GLP study according to OECD 302 B the test item was aerobically exposed at 100 mg/L test material (51.8 mg C/L) to standard OECD mineral medium and activated sludge obtained from a domestic STP. Two test groups were analyzed: Test group 1 used non-adapted sludge and test groups 2 used adapted sludge. The dry sludge concentration was 6.86 g/L (adapted sludge) and 5.87 g/L (non-adapted sludge), respectively. The ratio DOC to activated sludge dry weight was 1:4 at the beginning. For sludge adaptation, 50 mg/L yeast and 4 mg C/L (test material) were added to the inocculum which was then maintained under test conditions until start of the main test. On days 7 and 10 further 8 mg C/L test substance were added. The thus adapted sludge was used after 14 days to run test group 2. Exposure of both groups was for 28 days at ambient light conditions, under permanent stirring and between 20.3 and 23°C. Per test group, six test vessels with a volume of 2 L were stocked with 2 L volume, which was constantly aerated with CO2 free air and stirred permanently. Each reactor had received inocculum and mineral medium. Per test, the following replicates were incubated: Test item (100 mg C/L, two replicates), blank control (two replicates), reference substance (120 mg C/L of diethylene glycol, one replicate) and an abiotic control (sterilized with a 10 mL/L HgCl2 solution). DOC of filtered samples was analyzed by a DOC analyzer at 3 hours (non-adapted sludge only) and on days 2(3), 7, 14, 20(21) and 28 (number in brackets: adapted sludge). The 14-day results of the functional control showed that a biodegradation rate of ≥ 99 % was reached with non-adapted and adapted activated sludge. These result validates the testing procedure. The biodegradation for the replicates with non-adapted activated sludge reached the 10 % level (beginning of biodegradation) after 3 days. After 7 days the biodegradation came to 52 %. The pass level of 70 % was reached on day 10 and the biodegradation came to a maximum of 100 % after 14 days. For the replicates with adapted activated sludge, the start of the biodegradation (10% level) was reached after one day. After 7 days, a biodegradation of 78 % was determined and the pass level of 70 % was reached after 5 days. The maximum of degradation was 100 % after 14 days. No significant physico-chemical elimination occurred in the sterile control after 28 days (maximum 3%). Therefore it can be concluded, that the decrease of the test item is due to biodegradation. It can be concluded, that the substance is inherently ultimately biodegradable.

Endpoint:
biodegradation in water: screening tests
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Biodegradation was estimated using the BIOWIN™ v4.10 module of EPI Suite™ v.4.11. BIOWIN™ estimates aerobic and anaerobic biodegradability of organic chemicals using 7 different models. Two of these are the original Biodegradation Probability Program (BPP™). The seventh and newest model estimates anaerobic biodegradation potential. The Estimation Programs Interface EPI Suite™ was developed by the US Environmental Protection Agency's Office of Pollution Prevention and Toxics and Syracuse Research Corporation (SRC).
Justification for type of information:
QSAR prediction: migrated from IUCLID 5.6
Principles of method if other than guideline:
QSAR.
GLP compliance:
no
Remarks on result:
other: Based on the QSAR, the substane is readily biodegradable
Interpretation of results:
readily biodegradable
Conclusions:
The substance is biodegradable based on BIOWIN™ v4.10 module of EPI Suite™ v.4.11.
Executive summary:

Biodegradability was estimated using the BIOWIN™ v4.10 module of EPI Suite™ v.4.11. KOCWIN™. The conclusion of the BIOWIN™ v4.10 assessment is that the substance is readily biodegradable.

Description of key information

The results of the key study, which is appropriate for the assessment of inherent biodegradability, shows that the substance is inherently ultimately biodegradable. The substance is mineralized by non-adapted sludges from 10% on day 3 to 70% on day 10 after test initiation. Totally 100% were degraded within 14 days. Mineralization is fast in adapted sludges: The 10% pass level for biodegradation was reached within 1 day and 70% mineralization was reached within 5 days of incubation. On day 14, 100% of the test material had been mineralized. Hence mineralization is considered fast under the test conditions of an inherent biodegradation study.  

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, fulfilling specific criteria

Additional information

Inherent biodegradation is supported by the result of a valid ready biodegradation study according to OECD 301 B, which shows that up to 18% are biologically degraded within 28 days of incubation under ready biodegradation conditions. Furthermore, the result of a QSAR estimation states that the substance is readily biodegradable. The BIOWIN™ v4.10 module of EPI Suite™ v.4.11 was applied to do the calculations. The results of two other ready biodegradation studies (OECD 301 B study by Anonymous 2005 and OECD 301 F by Drozdowski 1988) are not used for this hazard assessment, since the studies are not reliable (Klimisch 3).