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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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidelines for the Testing of Chemicals, Effects on Degradation and Accumulation, Version 2, 301F Manometric Respirometry Test, China Environmental Press, China (2013)
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): Fresh activated sludge used in the test was collected from Wuzhong district urban area sewage treatment plant. The address of the plant is No. 1, Baodai East Road, Suzhou city, The People’s Republic of China. The batch number of the inoculum is included in the full study report. The activated sludge of waste water treatment plant is recommended by test guideline.
- Storage conditions: See pretreatment field.
- Storage length: ca. 1 week; 7 days from sampling to test initiation.
- Preparation of inoculum for exposure:
- Pretreatment: The activated sludge after centrifugal separation was washed with test medium after removing the suspended solids and coarse particles on the day of collection, the activated sludge was centrifuged at 1100 g for 5 minutes, and the supernatant was discarded. This process was repeated 3 times. The dry weight content of the activated sludge was determined in triplicates after pre-treatment, and the average value was 6.93%. 115.44 g of the activated sludge (wet weight). This was weighed and the test medium made up to 2 litres (the dry weight concentration was calculated to be 4 g/L based on the dry weight content of 6.93%). After that, the suspension was aerated for 7 days at test temperature. The dry weight content of the activated sludge was determined before inoculation and the average value of triplicate measurements was 6.53%.
- Concentration of sludge: The inoculum was added into the test suspensions, the procedure control, the inoculum blank and toxicity control to give a final concentration of 30.0 mg/L (dry weight).
- Water filtered: Yes.
- Type and size of filter used, if any: The water for preparing medium and solution was made by the Water Purifier (Elix70/Advantage A10). The organic carbon concentration of the water used in the test was 2 ppb. For each series of tests, the same batch of water was used (batch number reported in the full study report).
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Solution A [KH2PO4: 8.50 g; K2HPO4.3H2O: 28.49 g; Na2HPO4.12H2O: 67.15 g; NH4Cl: 0.50 g - in 50 mL water]; Solution B [CaCl2: 36.4 g in 5 mL water]; Solution C [MgSO4.7H2O: 22.5 g in 5 mL water]; Solution D [FeCl3,6H2O: 0.25 g in 5 mL water]. 50 mL of solution A was mixed with 5 ml of solutions B, C and D and made up to 30 L with deionised water.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 22 ±1 °C (actual: 20.8 – 22.3 °C)
- pH: 7.46 (medium)
- pH adjusted: Was not necessary as the pH values of all vessels was: pH 7.4 ± 0.2 at the start of the test
- Aeration of dilution water: Not reported
- Continuous darkness: Yes.

TEST SYSTEM
- Culturing apparatus: Flasks with continuous stirring (magnetic stirrer)
- Number of culture flasks/concentration: In duplicate (Inoculum blank); single flasks (procedure control and toxicity control)
- Method used to create aerobic conditions: Screw sealed flasks wth sensor head/CO2 trap.
- Measuring equipment: The respirometer used during this study is an Oxitop Control System.

SAMPLING
- Sampling frequency: At least every three (3) days
- Sampling method: Respirometry measuring the oxygen uptake of the test medium.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes. See table 1.
- Abiotic sterile control: No.
- Toxicity control: Yes.
- Other: Positive reference control ('Procedure control' ; Sodium Benzoate).
Reference substance:
benzoic acid, sodium salt
Remarks:
45 mg/L
Test performance:
(1) The oxygen uptake of the inoculum blanks were 16.9 mg/L and 14.1 mg/L, the mean was 15.5 mg/L, less than 60 mg/L in 28d.
(2) At the end of the test, the pH values of each test vessel were 7.61-7.62, within the range of 6.0-8.5.
(3) The difference of extremes of replicate values of the removal of the test substance at the plateau, at end of the test or at the end of the 10-d window was less than 20% (actual day-28: 67.81% and 64.60%).
(4) The percentage degradation of the procedure control and the toxicity control were 87.10% and 62.03%, which had reached the pass levels of 60% and 25% of ThOD on 14d.
Parameter:
% degradation (O2 consumption)
Value:
66.21
Sampling time:
28 d
Remarks on result:
other: n=2; 10-d window not met
Results with reference substance:
Degradation of sodium benzoate exceeded 93.4 % after 14 days: the activity of the inoculum was thus verified (validity criterion).
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance mean biodegradation in duplicate was 66.4 % at day 28.
Executive summary:

The ready biodegradability test was carried out according to OECD TG 301F and China Guidelines for the Testing of Chemicals, Effects on Degradation and Accumulation, Version 2, 301F Manometric Respirometry Test (2013) under GLP. The test substance, at a concentration of 30 mg/L was exposed to activated sewage sludge micro-organisms obtained from the Wuzhong district urban area sewage treatment plant of Suzhou city, China, with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in flasks to 30.0 mg/L (dry weight) within the test system and in the negative and toxicity controls. The degradation of the test substance was assessed by the regular (every three days) measurement of oxygen consumption from days 0 and 28 using an Oxitop control system. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The oxygen uptake of the inoculum blanks were 16.9 mg/L and 14.1 mg/L, the mean was 15.5 mg/L, less than 60 mg/L in 28-days. At the end of the test, the pH values of each test vessel were within the range of 6.0-8.5 (actual pH: 7.61 – 7.62). The difference of extremes of replicate values of the removal of the test substance at the plateau, at end of the test or at the end of the 10-d window was less than 20% (actual day-28: 67.81% and 64.60%). The percentage degradation of the procedure control and the toxicity control were 87.10% and 62.03%, which had reached the pass levels of 60% and 25% of ThOD on 14d. The toxicity test attained 60 % degradation after 28 days thereby confirming that the test substance was not inhibitory to the sewage treatment micro-organisms used in the study. Sodium Benzoate attained 87 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The test system therefore met the validation criteria of the guideline. The mean biodegradation for duplicate test flasks at 6 days for the test substance was 10.54 %. The mean biodegradation for duplicate test flasks at 6 days for the test substance was 62.08 %. The mean biodegradation for duplicate test flasks at 28 days for the test substance was 66.21 % (the 10-day window was not met). The test item was biodegraded by 66.21% at day 28 in the Manometric Respiratory Test. The 10-day window that can be applied to Manometric Respiratory Tests, where 60% biodegradation must be achieved following the attainment of 10 % biodegradation, is not applied to multi-constituent substances consisting of structurally similar constituents (OECD, 2006). Under the conditions of the study, test item is considered as readily biodegradable.

Description of key information

Biodegradation: readily biodegradable, mean biodegradation 66.41% (28-days, 10-day window not met). 10-day window not applicable to multi-constituent substances consisting of structurally similar constituents (OECD, 2006); OECD TG 301F, 2016

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Key study: OECD TG 301F, 2016 : The ready biodegradability test was carried out according to OECD TG 301F and China Guidelines for the Testing of Chemicals, Effects on Degradation and Accumulation, Version 2, 301F Manometric Respirometry Test (2013) under GLP. The test substance, at a concentration of 30 mg/L was exposed to activated sewage sludge micro-organisms obtained from the Wuzhong district urban area sewage treatment plant of Suzhou city, China, with culture medium in sealed culture vessels in the dark at 22°C ± 1°C for 28 days. The sludge was diluted in flasks to 30.0 mg/L (dry weight) within the test system and in the negative and toxicity controls. The degradation of the test substance was assessed by the regular (every three days) measurement of oxygen consumption from days 0 and 28 using an Oxitop control system. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The oxygen uptake of the inoculum blanks were 16.9 mg/L and 14.1 mg/L, the mean was 15.5 mg/L, less than 60 mg/L in 28-days. At the end of the test, the pH values of each test vessel were within the range of 6.0-8.5 (actual pH: 7.61 – 7.62). The difference of extremes of replicate values of the removal of the test substance at the plateau, at end of the test or at the end of the 10-d window was less than 20% (actual day-28: 67.81% and 64.60%). The percentage degradation of the procedure control and the toxicity control were 87.10% and 62.03%, which had reached the pass levels of 60% and 25% of ThOD on 14d. The toxicity test attained 60 % degradation after 28 days thereby confirming that the test substance was not inhibitory to the sewage treatment micro-organisms used in the study. Sodium Benzoate attained 87 % degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The test system therefore met the validation criteria of the guideline. The mean biodegradation for duplicate test flasks at 6 days for the test substance was 10.54 %. The mean biodegradation for duplicate test flasks at 6 days for the test substance was 62.08 %. The mean biodegradation for duplicate test flasks at 28 days for the test substance was 66.21 % (the 10-day window was not met). The test item was biodegraded by 66.21% at day 28 in the Manometric Respiratory Test. The 10-day window that can be applied to Manometric Respiratory Tests, where 60% biodegradation must be achieved following the attainment of 10 % biodegradation, is not applied to multi-constituent substances consisting of structurally similar constituents (OECD, 2006). Under the conditions of the study, test item is considered as readily biodegradable.

 

References:

1. ECHA Guidance on Information Requirements and Chemical Safety Assessment (Chapter R.7b: Endpoint Specific Guidance, R.7.9.4, June 2017)

2. Introduction to the section of the OECD Guidelines for Testing of Chemicals on Degradation and Accumulation, Section 3 (OECD, September 2006)

3. GHS revision 4 (2011), Globally harmonized system of classification and labelling of chemicals (GHS). Chapter 4.1. Hazardous to the aquatic Environment. United Nations, 2011

4. ECHA Guidance on Application on the CLP Criteria, section 4.1.3.2.3.2 (v5.0, July 2017)