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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Remarks:
Determination of biodegradability in the CO2 headspace tes
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-02-13 - 2003-03-18 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
OECD Test Guideline 310 (Proposal) "Ready biodegradability - C02 in sealed vessels (Headspace Test)" (2001)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
ISO 14593:1999 (Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test))
Version / remarks:
ISO/DIS 14593 "Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium - Method by analysis of released inorganic carbon in sealed flasks" (1997)
Deviations:
yes
Remarks:
In contrary to the ISO/DIS 14593, the reference item stock solution was added directly to the test vessels.
GLP compliance:
yes (incl. QA statement)
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): The inoculum used was activated non-adapted sludge from the Marl-Ost (Germany) municipal sewage treatment plant taken off on 14.02.03.
- Storage conditions: The freshly obtained sludge was used immediately
- Preparation of inoculum for exposure: Five days prior to the beginning of the test (addition of test item), 15 I of the mineral medium were prepared and aerated overnight. On the following day the inoculum was diluted in the mineral medium (9980 ml mineral medium were inoculated with 20 ml of inoculum). These inoculum batches were aerated with moist air for four days in the dark. At the 17.02.2003 the inoculum was determined by a TOC-anaiysis (TC: 1.800 mg C/l, IC: 0.651 mg C/l, TOC: 1.149 mg C/l). At the test start (18.02.03) 130 ml of the Inoculum batches were added In each of the test batches by means of a dosing pump. Then, the test item was added.
- Pretreatment: The sewage sludge was treated in accordance with specifications in the laboratory.
- Concentration of sludge: The content of suspended solids in the test batches was 6.6 mg/l. The content of dry matter in the inoculum used was 3.318 g/l.
- Initial cell/biomass concentration: The number of bacteria in the inoculum was determined by the Koch pour-plate method. The inoculum had a bacterial count of 100 * 104 CFU/ml (colony forming units/ml).
- Water filtered: no
Duration of test (contact time):
28 d
Initial conc.:
34.3 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Defined liquid mineral medium (composition not specified). The bidest. water, which we used for the preparation of the mineral medium was determined by a TOC-anaiysis (TC: 0.687 mg C/l, IC: 0.262 mg C/l, TOC: 0.425 mg C/l).
- Additional substrate: One hour before analysis 1.3 ml sodium hydroxide, c(NaOH) = 7 mol/l, was injected through the septum of each test vessel sampled and shake for one hour.
- Test temperature: 21.6 - 22.8°C (mean: 22.3°C)
- pH: At the end of the test (28th day), the pH was measured.
Test vessel 20 (blank): pH 7.5
Test vessel 40 (sodium benzoate): pH 7.1
Test vessel 60 (Trimethyl orthoacetate (TMOA)): pH 6.8
- pH adjusted: not specified
- Suspended solids concentration: The content of suspended solids in the test batches was 6.6 mg/l.
- Continuous darkness: yes
- Other: Test vessels were placed on an orbital shaker and shake with 180 rpm.

TEST SYSTEM
- Culturing apparatus: Approximately 210 ml gas-tight glass vessels sealed with butyl rubber septa and aluminium crimp seals.
- Number of culture flasks/concentration:
Test suspension: 20 vessels without test item with inoculum, 20 vessels containing reference item and inoculum, 20 vessels containing test item and inoculum.
Application: The test item was added directly with a Gilson pipetman and pipette tip. In contrary to the ISO/DIS 14593, the reference item stock solution was added directly to the test vessels also.
- Method used to create aerobic conditions: Test vessels were sealed with butyl rubber septa and aluminium crimp seals, with a headspace of air.
- Measuring equipment: The CO2 content was measured via TIC analysis in a carbon analyser (Shimadzu). The concentration of the compound used normally yields an initial organic concentration in the medium of 2 - 40 mg DOC or TOC/I. Biodegradation (mineralisation to CO2) is determined by measuring the net increase in organic carbon (IC) levels over time compared with unamended blanks. The extent of biodegradation is expressed as a percentage of the theoretical IC production based on the amount of test item (as organic carbon) added initially.

SAMPLING
- Sampling frequency: TIC analyses were carried out after 0, 3, 7, 10, 14, 17, 21 and 28 days in duplicate determination on prediluted samples if necessary..
- Sampling method: One hour before analysis 1.3 ml sodium hydroxide, c(NaOH) = 7 mol/l, was injected through the septum of each test vessel sampled and shake for one hour.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 20 vessels without test item with inoculum.
- Toxicity control: 20 vessels containing reference item and inoculum. Sodium benzoate was used as a suitable reference item to monitor the activity of the inoculum.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
No information about a preliminary study are available.
Test performance:
No unusual observations during test period observed.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
116
Sampling time:
28 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
78
Sampling time:
10 d
Details on results:
The test item Trimethyl orthoacetate (TMOA) studied reached a degree of degradation of 116 % in the course of 28 days (mean of the test item batches). Since Trimethyl orthoacetate (TMOA) is degraded by > 60 % in the course of 10 days after the time when biodegradation had achieved 10 %, Trimethyl orthoacetate (TMOA) is considered as „readily biodegradable".
Results with reference substance:
Sodium benzoate was used as a suitable reference item to monitor the activity of the inoculum. The degree of biodégradation (in %) of this reference item must achieve the plateau phase for ready biodegradability in the course of 14 days. In this test the reference item sodium benzoate achieved a degree of degradation of 96% within 14 days and 100 % in the course of 28 days. Therefore it can be concluded that the inoculum used had a sufficient biological activity (validity criterion fullfilled).
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The study was conducted under GLP according to OECD 310 without deviations on the registered substance itself. The method is to be considered scientifically reasonable and suitable for the test item, the available information allows the conclusion that the test was properly conducted, the positive control showed the appropriate results. Hence, the results can be considered as reliable to assess ready biodegradability of Trimethyl orthoacetate. The test item attained 116% biodegradation after 28 days and therefore can be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 310 ("Ready biodegradability - C02 in sealed vessels (Headspace Test)").
Executive summary:

The objective of the study was to evaluate Trimethyl orthoacetate (TMOA) for its ready biodegradability in an aerobic aqueous medium with microbial activity introduced by inoculation with the supernatant of activated sludge; Carbon dioxide (CO2) headspace test. The study procedures described in this report were in compliance with the OECD guideline No. 310, 2001, under GLP. In addition, the procedures were designed to meet the test methods of the ISO standard 14593, 1997.

The test item Trimethyl orthoacetate (TMOA) studied reached a degree of degradation of 116 % in the course of 28 days (mean of the test item batches). Since Trimethyl orthoacetate (TMOA) is degraded by > 60 % in the course of 10 days after the time when biodegradation had achieved 10 %, Trimethyl orthoacetate (TMOA) is considered as „readily biodegradable".

The reference item sodium benzoate achieved a degree of degradation of 100 % in the course of 28 days. Therefore it can be concluded that the inoculum used had a sufficient biological activity.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Description of key information

Biodegradation in water, screening test: readily biodegradable: 116 % (CO2 evolution) in 28 days (OECD TG 310)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Ready biodegradation of the test item was determined according to OECD TG 310, sodium benzoate was the positive control and gave the appropriate results. Biodegradation of the test item was determined to be 116 % (CO2 evolution) after 28 days, accordingly it is readily biodegradable.

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