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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:
2014-07-23 to 2014-11-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
(adopted July 17, 1992)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Replicates: Duplicates
Test concentration: 15 mg/L
TOC: 0.70 mg C/mg
ThC02: 2.61 mg C02/mg test item
Carbon content in the vessel: 10.5 mg C/L
Pretreatment: The test item was weighed out. A defined volume of ultrapure water was added to the test item and it was treated with ultrasound (three times for 10 minutes).
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Municipal sewage treatment plant, Hildesheim, Germany
- Pretreatment: 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 and 15 minutes. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free aiur until test start. 10 mL/L of this mixture were used to initiate inoculation.
- Initial cell/biomass concentration: approx. 10^7 - 10^9 CFU/L
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
test mat.
Remarks:
The test item was tested at a concentration of 15 mg/L with 2 replicates, corresponding to a carbon content (TOC) of 10.5 mg C/L in the test vessels.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
- Test temperature: nominal 22 +/- 2°C (measured: 21 - 24 °C)
- pH adjusted: no
- Continuous darkness: low light conditions (brown glass bottles)


TEST SYSTEM
- Culturing apparatus: 5000 mL brown glass flasks
- Method used to create aerobic conditions: Aeration with 30 - 100 mL/min for 24 h
- Details of trap for CO2 and volatile organics if used: CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.

- Course of the study: The following incubation vessels were prepared:
- two for the test item concentration (P1, P2)
- one for the functional control (R1)
- two for the inoculum control (C1, C2)
- one for the toxicity control (T1)
The necessary amounts of ultrapure water, mineral salts medium and inoculum were placed in each of the incubation vessels. The vessels were aerated for 24 hours with CO2 free air. After 24 hours the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles, each containing 100 mL of a 0.0125 mol/L Ba(OH)2 solution.

Test and reference item were weighed out. A defined volume of ultrapure water was added to the test item. This liquid was treated with ultrasound. The test item dispersions and the reference item were transferred to the respective incubation vessels; the vessels were made up to 3 L with ultrapure water and connected to the system for the production of CO2 free air.

On day 28, 1 mL 37 % HCl was added to each of the vessels. Aeration was continued for further 24 h and the quantity of CO2 released was determined.

SAMPLING
- Sampling frequency: Backtitration of the residual Ba(OH)2 with 0.05 N HCL was carried out three times a week during the first ten days and thereafter twice weekly.
- Sampling method: For each titration the first gas wash bottle was removed and a new bottle was connected to the last one.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and reference item
- Toxicity control: Test item and reference item in test concentration


STATISTICAL METHODS:
-The theoretical production of carbon dioxide (ThCO2) of the test item and functional control is calculated by the carbon content and the molecular formula, respectively.
The produced CO2 was calculated by: 1 mL HCl (c = 0.05 mol/L) = 1.1 mg CO2
The net amount of CO2 produced is calculated by correcting the results of the test item and functional control for endogenous CO2 production of the inoculum controls.
The biodegradation is calculated from the ratio theoretical CO2 production to net CO2 production.
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (TOC removal)
Value:
10
Sampling time:
5 d
Parameter:
% degradation (TOC removal)
Value:
60
Sampling time:
11 d
Key result
Parameter:
% degradation (TOC removal)
Value:
81
Sampling time:
28 d
Details on results:
The adaptation phase of the functional control changed after 5 days into degradation phase ( ≥ 10%). The course of the degradation was rapid and the functional control reached the pass level of 60 % after 11 days and a biodegradation of 77% at the end of the study. The validity criterion degradation ≥60% after 14 days is fulfilled.

The beginning of biodegradation (10 % level) was reached on day 5. The pass level of 60% biodegradation has been reached within 11 days. The mean biodegradation was 81% after 28 days. In the inoculum control the total CO2 production was 24.4 mg CO2/L after 28 days.
Results with reference substance:
Sodium benzoate was used as functional control. The pass level of 60% in the functional control was reached after 9 days. The biodegradation of the functional control after 28 days was 77%. In the toxicity control containing both test and reference item a biodegradation of 60 5 was determined within 11 days and it came to 83% after 28 days. The biodegradation of the reference item was not inhibited by the tes item in the toxicity control.
Validity criteria fulfilled:
yes
Remarks:
- total CO2 evolution in the inoculum control <40 mg/L after 28 days. - functional control pass level of ≥ 60 % within 9 days. - differences of replicate values of test item removal <20 % - Toxicity control pass level >25 % degradation within 5 days
Interpretation of results:
readily biodegradable
Conclusions:
The mean 10% level (beginning of biodegradation) was reached on day 5. The 60% pass level was reached within 11 days. The mean biodegradation after 28 days was 81%. Under the test conditions the test item is classified as readily biodegradable within the 10-day-window and within the 28 day period of the study.

Description of key information

The test substance is readily biodegradable within the 10-day window in the biodegradation study according to OECD guideline TG 301B.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The biodegradation of the test substance has been examined according to OECD guideline TG 301B (Modified Sturm Test) with GLP compliance. During the study, the mean 10% level (beginning of biodegradation) was reached on day 5. After 11 days the pass level of 60% has been reached. The mean biodegradation after 28 days has been 81%. Therefore, the test substance is classified as readily biodegradable within the 10-day-window and within the 28 day period of the study under the test conditions.