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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
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidelines for the Testing of Chemicals, Effects on Degradation and Accumulation, Version 2, 310 Ready Biodegradabilty-C02 in Sealed Vessels (Headspace Test), China Environmental Press (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): Activated sludge from Wuzhong district urban area sewage treatment plant, Suzhou, China. The inoculum was selected as it was recommended by "The Guidelines for the Testing of Chemicals". The batch number of the inoculum is included in the full study report.
- Storage conditions: See pretreatment field.
- Storage length: < 1 week from sampling to test initiation.
- Preparation of inoculum for exposure:
- Pretreatment: The sludge was removed any coarse particles and impurities on the surface, then was washed with the test medium (1100 g, 5min, repeated three times), the supernatant liquid phase was decanted and the suspended solids re-suspended in the test medium. The dry weight of the suspended solid was determined in triplicate and the mean value was 8.24%. 97.087 g of the activated slude (wet weight) was weighed and suspended made up to 2 litres (dry weight concentration was 4 g/L based on the dry weight contend of 8.24%). The suspension was aerated for 7 days at test temperature. The dry weight of the activated sludge was remeasured in triplicate and the mean value was 5.44%.
- Concentration of sludge: The sludge was diluted in flasks to 4 mg/L within the test systems based on dry weight; this included approximately 4 mg/L in the negative and toxicity controls.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
inorg. C analysis
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: See table 1. Solution A [KH2PO4: 8.50 g; K2HPO4.3H2O: 28.49 g; Na2HPO4.12H2O: 67.15 g; NH4Cl: 0.50 g - in 1 L water]; Solution B [CaCl2: 36.4 g in 1 L water]; Solution C [MgSO4.7H2O: 22.5 g in 1 L water]; Solution D [FeCl3,6H2O: 0.25 g in 1 L water]. 200 mL of solution A was mixed with 20 mL solutions A, B and C; then this solution was made up to 20 L with deionised water (the batch number of the water used is listed in the full study report).
- Solubilising agent (type and concentration if used): None.
- Test temperature: 20 ±1 °C
- pH: The pH of the test water used to prepare the test medium was pH = 7.46 ; See table 1 for details of the test item solution, blank, toxicity and reference controls.
- pH adjusted: Not required, the pH of the test solutions was pH = 7.4 ± 0.2 at the start of the test ; See table 1 for details.
- Aeration of dilution water: Not reported
- Suspended solids concentration: 4 mg SS/L inoculum concentration within the test vessels.
- Continuous darkness: Yes.

TEST SYSTEM
- Culturing apparatus: Glass serum bottles, 250 mL; with continuous stirring. Fill volume 200 mL. The volume of headspace was approximately 100 mL.
- Number of culture flasks/concentration: In triplicate (test item; Inoculum blank; procedure control and toxicity control)
- Method used to create aerobic conditions: Sealed flasks with butyl rubber.
- Measuring equipment: The TOC analyser used during this study is a Shimadzu TOC-LCSH/SSM-5000A analyser.

SAMPLING
- Sampling frequency: For inoculum blank and test suspension: days 1, 4, 7, 11, 14, 18, 21, 25 and day 28 after culture. For reference substance, day 14 and 25. For toxicity control, day 14 and 28.
- Sampling method: The TOC analyser was calibrated using an appropriate standard before each batch of analyses. For each sample, 2 mL of 7 mol/L NaOH solution was injected and the bottles were shaken for 1 hour at the test temperature. The bottles were removed from the shaker and allowed to settle. Suitable volumes of the liquid phase in each vessel and analysed for DOC determination after filtering.

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:
20 mg/L
Test performance:
(1) The concentration of TIC in inoculum blank should be < 3 mg/L (by carbon) after 28-days. Actual: 1.6136 mg/L (by carbon)
(2) The percent degradation of the reference substance should be larger than 60% at 14-days. Actual: 82.79%
Parameter:
% degradation (inorg. C analysis)
Value:
72.65
Sampling time:
28 d
Remarks on result:
other:
Remarks:
10-day window met
Details on results:
1. 14-day of the test, the percentage degradation of the procedure control and the toxicity control were 82.79% and 76.21%, respectively. On day 25 the procedure control was 81.37% and on day 28, the toxicity control was 80.14%. This showed that the inoculum activity could meet the requirement of the test, and the test substance was considered not to have a inhibitory effect on the sewage sludge micro-organisms used in the study.
2. 14-day, the mean percentage biodegradation of the test substance was: 74.91%.
28d, the mean percentage biodegradation of the test substance was: 72.65%.
3. The biodegradation curve for test item indicated that the 10-day window was achieved: by > 60% degradation within ten days of achieving 10% degradation on day 2.
Results with reference substance:
Degradation of sodium benzoate exceeded 82.79 % 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 item mean biodegradation was 72.26 % at day 28 (10-day window met).
Executive summary:

The ready biodegradability test was carried out according to OECD TG 310 guideline under GLP. The test item, at a concentration of 20 mg C/L was exposed to activated sewage sludge micro-organisms obtained from the Wuzhong district urban area sewage treatment plant, Suzhou, China, following seven day pre-conditioning with culture medium in sealed culture vessels in the dark at 22°C ± 1°C (actual temperature: 19.5 to 20.8 °C) for 28 days. The inoculum was diluted in flasks to 4 mg SS/L. within the test systems and controls. Biodegradation was determined by analysis of IC concentration over the 28-day test period. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The concentration of the reference substance, sodium benzoate, was 20 mg C/L in a procedure control group 20 mg C/L of sodium benzoate and 20 mg C/L of test substance within a toxicity control group. The test condition in procedure control and toxicity control was the same as in the test substance groups. Parallel blank inoculum controls were additionally completed. The mean percentage biodegradation of test substance in duplicates was 74.91% on day 14, and 72.65% at the end of the test (day 28), and the percentage biodegradation of the test substance was up to the pass level at the end of 10-day window. The percentage biodegradation of reference substance were 82.79% on day 14, and 76.21% in the toxicity control on day 14, indicating that the inoculum was not inhibited by the test substance and met the test requirements. Under the conditions of the study, test substance is considered to be readily biodegradable.

Description of key information

Biodegradation: mean biodegradation 72.65% (28-days; 10-day window met), OECD TG 310, 2017

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Key study: OECD TG 310, 2017 : The ready biodegradability test was carried out according to OECD TG 310 guideline under GLP. The test item, at a concentration of 20 mg C/L was exposed to activated sewage sludge micro-organisms obtained from the Wuzhong district urban area sewage treatment plant, Suzhou, China, following seven day pre-conditioning with culture medium in sealed culture vessels in the dark at 22°C ± 1°C (actual temperature: 19.5 to 20.8 °C) for 28 days. The inoculum was diluted in flasks to 4 mg SS/L. within the test systems and controls. Biodegradation was determined by analysis of IC concentration over the 28-day test period. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The concentration of the reference substance, sodium benzoate, was 20 mg C/L in a procedure control group 20 mg C/L of sodium benzoate and 20 mg C/L of test substance within a toxicity control group. The test condition in procedure control and toxicity control was the same as in the test substance groups. Parallel blank inoculum controls were additionally completed. The mean percentage biodegradation of test substance in duplicates was 74.91% on day 14, and 72.65% at the end of the test (day 28), and the percentage biodegradation of the test substance was up to the pass level at the end of 10-day window. The percentage biodegradation of reference substance were 82.79% on day 14, and 76.21% in the toxicity control on day 14, indicating that the inoculum was not inhibited by the test substance and met the test requirements. Under the conditions of the study, test substance is considered to be 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)

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