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Environmental fate & pathways

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:
2009-11-23 to 2009-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Batch Number: TL00910581
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater. 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. During the holding period of one day prior to use, the sludge was aerated at room temperature. Prior to use, the dry weight of this diluted activated sludge was determined again and defined amounts were added to test water to obtain a final concentration of 30 mg dry material per liter.

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 combined and made up to 1000 mL with purified water. The pH was adjusted from 7.6 to 7.4 with a diluted hydrochloric acid solution.
Duration of test (contact time):
> 0 - <= 28 d
Initial conc.:
ca. 30 other: mg C/L
Based on:
DOC
Initial conc.:
ca. 80 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Test temperature: 21-23°C. The inoculated flasks were incubated in a temperature-controlled room. The temperature was checked on each sampling date in one of the inoculum controls. Additionally, the room temperature was continuously recorded.
- pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum and was pH 7.4 in all test flasks. At the end of incubation, the pH was measured again in each test flask and was between pH 7.2 and pH 7.5.
- pH adjusted: No (not necessary)
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 2000-mL Erlenmeyer flasks, cleaned with alcoholic hydrochloric acid, rinsed with deionized water and dried. The final volume was 1000 mL per flask. Each flask was loosely covered with aluminium foil to allow the exchange of air between the flask and the surrounding atmosphere. The test media were continuously stirred by magnetic stirrers. The test vessels were labeled with all necessary information to ensure unmistakable identification.
- Number of culture flasks/concentration: 2
- Test performed in open system: Yes
- Other: The test flasks were incubated in a dark room

SAMPLING
- Sampling frequency: One sample of about 10 mL was taken from each test flask per sampling date. Prior to sampling, water evaporation losses were determined by weighing the flasks and were compensated by adding purified water. Deposits on the test vessels were resuspended.
- Sample storage before analysis: Samples were filtered through a 0.45-µm filter. The first 34 mL of the filtrate was discarded. Thereafter, the samples were analyzed for DOC on the day of sampling.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, two flasks
- Toxicity control: Yes, one flask
- Other: Positive control with reference compound, two flasks
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The amount of test item was based on the measured DOC content of 0.39 mg C per mg test item (calculated carbon content: 0.36 mg C/mg). The DOC determination was performed in a solubility pre-test of the test item (without GLP). During the study, the carbon content of the test item was verified under GLP.
Key result
Parameter:
% degradation (DOC removal)
Value:
> 99
Sampling time:
28 d
Remarks on result:
other: Sampling Dates: Exposure Day 0, 3, 7, 10, 14, 21 and 28
Details on results:
In the test flasks, containing the test item and activated sludge (inoculum), the mean concentration of DOC (dissolved organic carbon) continuously decreased from initially 27 mg/L on Day 0 to 0.4 mg/L on Day 28 (Table 1). The mean biodegradation of the test item based on DOC decrease amounted to 99%. Therefore, the test item was found to be biodegradable under the test conditions over the 28 day exposure to activated sludge. Moreover, the pass level for ready biodegradability, i.e. biodegradation of at least 70% removal of DOC in a 10 day window within the 28 day period of the test, was reached.

Therefore, the test item Sodium ascorbate crystalline is readily biodegradable under the test conditions.
Results with reference substance:
In the procedure controls, containing the reference item sodium benzoate and activated sludge (inoculum), sodium benzoate was completely biodegraded (>90%) within seven days of exposure, thus confirming suitability of the activated sludge.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item is readily biodegradable in a 10-day window and after 28 days of exposure. All validity criteria of the test guidelines were fulfilled.
Executive summary:

The test item was investigated for its ready biodegradability in a GLP-compliant 28-day DOC Die-Away Test according to EU-Method C.4-A and the OECD Guideline for Testing of Chemicals, No. 301 A (1992). In the test flasks, containing the test item and activated sludge (inoculum), the mean concentrations of dissolved organic carbon (DOC) continuously decreased by 99% of the initial values within the test period of 28 days. Therefore, the test item was found to be biodegradable under the test conditions. Moreover, the pass level for ready biodegradability (70% removal of DOC in a 10-day window within a 28-day period) was reached. Therefore, the test item was found to be readily biodegradable under the test conditions. The reference item sodium benzoate was completely (>90%) biodegraded within seven days of exposure, thus confirming suitability of the activated sludge. In the toxicity control, containing the test item, the reference item sodium benzoate and activated sludge (inoculum), the initial DOC decreased by 100% within 14 days of exposure. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration of 80 mg/L because degradation was >35% within 14 days. All validity criteria of the test guideline were fulfilled.

Description of key information

The test item is readily biodegradable (> 99% DOC removal) in a 10-day window and after 28 days of exposure.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The test item was investigated for its ready biodegradability in a GLP-compliant 28-day DOC Die-Away Test according to EU-Method C.4-A and the OECD Guideline for Testing of Chemicals, No. 301 A (1992). In the test flasks, containing the test item and activated sludge (inoculum), the mean concentrations of dissolved organic carbon (DOC) continuously decreased by 99% of the initial values within the test period of 28 days. Therefore, the test item was found to be biodegradable under the test conditions. Moreover, the pass level for ready biodegradability (70% removal of DOC in a 10-day window within a 28-day period) was reached. Therefore, the test item was found to be readily biodegradable under the test conditions. The reference item sodium benzoate was completely (>90%) biodegraded within seven days of exposure, thus confirming suitability of the activated sludge. In the toxicity control, containing the test item, the reference item sodium benzoate and activated sludge (inoculum), the initial DOC decreased by 100% within 14 days of exposure. Thus, according to the test guidelines the test item was not inhibitory to activated sludge at the tested concentration of 80 mg/L because degradation was >35% within 14 days. All validity criteria of the test guideline were fulfilled.