Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 02-Aug-2010 to 02-Sep-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with international guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

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): aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater
- Laboratory culture:/
- Method of cultivation:/
- Storage conditions:/
- Storage length:/
- Preparation of inoculum for exposure:The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per liter.
- Pretreatment:During the holding period of one day prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was diluted with test water to a concentration of about 1 g dry material per liter. Defined volumes of this diluted activated sludge were added to test water to obtain a final concentration of 30 mg dry material per liter.
- Concentration of sludge:30 mg dry material per liter
- Initial cell/biomass concentration:/
- Water filtered: yes/no: no data
- Type and size of filter used, if any:/
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
32 - 32.2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:
1) KH2PO4 8.50 g/L K2HPO4 21.75 g/L Na2HPO4 × 2H2O 33.40 g/L NH4Cl 0.50 g/L
The pH of this solution was 7.4.
2) MgSO4 × 7H2O 22.50 g/L
3) CaCl2 × 2H2O 36.40 g/L
4) FeCl3 × 6H2O 0.25 g/L, stabilized with one drop of concentrated HCl per liter
To obtain the final test water, 10 mL of stock solution 1) and 1 mL each of stock solutions 2) - 4) were added to about 800 mL purified water and made up to 1000 mL with purified water. The pH of the final test water was adjusted from 8.0 to 7.4 with a diluted hydrochloric acid solution.
- Additional substrate:/
- Solubilising agent (type and concentration if used):no
- Test temperature: 23°C
- pH:7.4
- pH adjusted: yes
- CEC (meq/100 g):/
- Aeration of dilution water:no
- Suspended solids concentration:/
- Continuous darkness: yes
- Other:

TEST SYSTEM
- Culturing apparatus: 5-liter all-glass amber bottles.
- Number of culture flasks/concentration:2
- Method used to create aerobic conditions:During the holding period of one day prior to use, the sludge was aerated at room temperature.
- Method used to create anaerobic conditions: /
- Measuring equipment: The samples were analyzed for inorganic carbon (IC) using a TOC analyzer (Shimadzu TOC-5000A) equipped with an automatic sampler.
- Test performed in closed vessels due to significant volatility of test substance:/
- Test performed in open system:no
- Details of trap for CO2 and volatile organics if used:Air was led through a bottle containing about 750 mL of a 2 M NaOH solution to trap CO2. The CO2-free air was passed through the test solutions at a rate corresponding to about 30–100 mL/min. Two absorber flasks, the first one containing 300 mL 0.05 M NaOH and the second one containing 200 mL 0.05 M NaOH, were connected in series to the exit air line of each test flask.
- Other:

SAMPLING
- Sampling frequency:Test item and inoculum control: Exposure Day 2, 5, 7, 9, 12, 14, 19, 23, 27, 28 and 29. Procedure control: Exposure Day 2, 7, 14, 28 and 29. Toxicity control: Exposure Day 7, 14, 28 and 29.
- Sampling method:On each sampling day, an aliquot of 5.0 mL was withdrawn from the absorber flask nearest to the test flask for analysis of inorganic carbon (IC). Additional samples for analysis of IC were withdrawn from the second absorber flask of all test vessels on Exposure Day 14 and at the end of the exposure period on Exposure Day 28 in order to correct for any carry over of CO2. After sampling on Exposure Day 28, the pH was measured in each test flask. Next, 1 mL of concentrated HCl was added to each test flask and the flasks were aerated overnight with CO2-free air to drive off any residual CO2 present. On Day 29, a sample from each absorber flask was withdrawn and analyzed for IC to determine residual CO2 that was present in the test suspensions on Exposure Day 28. In this way, any residual CO2 remaining in the test suspensions was determined as the difference between the amounts of IC found before and after acidification.
- Sterility check if applicable:/
- Sample storage before analysis:
- Other:

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Abiotic sterile control:/
- Toxicity control:1
- Other: procedure control: 2

STATISTICAL METHODS:
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
3.6
Sampling time:
2 d
Parameter:
% degradation (CO2 evolution)
Value:
11
Sampling time:
5 d
Parameter:
% degradation (CO2 evolution)
Value:
16
Sampling time:
14 d
Parameter:
% degradation (CO2 evolution)
Value:
19.2
Sampling time:
28 d
Details on results:
The percent biodegradation of the test item was calculated based on a total organic carbon content (TOC) of 0.47 mg C/mg test item.
The CO2 formation of test item in the test media slowly increased from the start of the test until test termination after 28 days. At the end of the 28-day exposure period, average biodegradation was 19%.
Consequently, test item was found to be biodegradable by 19% under the test conditions within 28 days. However, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was not reached.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
At the start of the test, the IC of the test item was less than 5% of the TC. However no indication about the total CO2 evolution in the inoculum blank at the end of the test which should not normally exceed 40 mg/l medium.
Interpretation of results:
inherently biodegradable
Conclusions:
The test item was found to be biodegradable by 19% under the test conditions within 28 days. However, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was not reached.
Executive summary:

The test item was investigated for its ready biodegradability in a 28-Day CO2 Evolution (Modified Sturm) Test according to EU Commission Directive 92/69/EEC C.4-C, Commission Regulation (EC) No 440/2008, C.4-C and OECD Guideline for Testing of Chemicals, No. 301 B (1992).

The test item was found to be biodegradable by 19% under the test conditions within the 28-day exposure period. However, the pass level for ready biodegradability, i.e. a CO2 formation of at least 60% of the TOC in a 10-day window within the 28-day period of the test was not reached.

In the toxicity control, containing both the test item and the reference item sodium benzoate, no inhibitory effect on the biodegradation of the reference item was determined. Thus, the test item had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 32 mg/L.

In the procedure controls, average biodegradation of the reference item was 84% by Exposure Day 14, thus confirming suitability of the activated sludge (≥60% degradation by Exposure Day 14).