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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-03-26 - 2001-04-24 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 301 A, Ready Biodegradability: DOC Die-Away Test, 1992.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
Version / remarks:
EU Commission Directive 92/69 EEC C.4-A, DOC Die-Away Test, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature at about 20 °C, away from direct sunlight
- Stability under test conditions: Unlimited in water
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
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
- Preparation of inoculum for exposure: The sludge was washed once 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 holding, the sludge was aerated at room temperature until use.
- Concentration of sludge: Test water was inoculated with defined volumes of this diluted activated sludge to give a final concentration of 30 mg dry material per liter.
Duration of test (contact time):
28 d
Initial conc.:
63 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: The test water was prepared according to the testing guidelines.
- Solubilising agent (type and concentration if used): The amounts of test item were directly weighed into the test flasks. No emulsifiers or solvents were used but ultrasound dispersion was employed for fifteen minutes to obtain a clear solution of the test item.
- Test temperature: 22 - 23 °C. The inoculated flasks were incubated in a temperature-controlled room. The temperature was checked on each sampling date. Additionally, the room temperature was continuously recorded.
- pH: The pH of the test water was adjusted to 7.4 with a diluted hydrochloric acid solution. Prior to test start the pH was 7.4 (measured in all test flasks before the addition of activated sludge (inoculum)). At the end of incubation the pH was 7.3 in all test flasks.
- Continuous darkness: yes, the test flasks were incubated in a dark room.

TEST SYSTEM
- Culturing apparatus: Test vessels: 2000-ml Erienmeyer flasks, cleaned with alcoholic hydrochloric acid, rinsed with deionized water and dried. The test vessels were filled with 1000 ml of test medium, or test medium containing test item and/or reference item. 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.
- Number of culture flasks/concentration: 2 for each test item and reference item, each 1 for Inoculum blank, Abiotic control and Toxicity control
- Measuring equipment: DOC-analyses were performed on a Shimadzu TOC-500 Analyser (in triplicate per sample).
- Test performed in open system: Yes, but water evaporation losses were determined by weighing the flasks and were compensated by adding purified water.

SAMPLING
- Sampling frequency: Samples were taken on Day 0 (treatment day), 3, 7, 10, 14, 21, 27 and 28 of the incubation period for DOC analysis.
- Sampling method: 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 scraped off and resuspended in the test vessel. Samples were filtered through a 0.45 µm filter.
- Sample storage before analysis: Samples were analyzed for DOC on the day of sampling.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
Reference substance:
benzoic acid, sodium salt
Remarks:
50 mg/l
Test performance:
In the abiotic control, containing the test item and poisoned medium, the DOC concentrations varied from 29.0 to 31.2 mg/l over the exposure period of 28 days and were not significantly different from the initial DOC concentration of 28.4 mg/l measured on Day 0. Thus, no abiotic degradation occurred under the test conditions.
In the procedure controls, containing the reference item sodium benzoate and activated sludge (inoculum), sodium benzoate was readily biodegraded by an average of 99% within 7 days of exposure, confirming suitability of the activated sludge.
In the toxicity control, containing the test item (corresponding to 48.9% of total DOC), the reference item (corresponding to 51.1% of total DOC) and activated sludge (inoculum), the initial DOC concentration of 57.1 mg/l measured on Day 0 decreased by 50% within 14 days of exposure. Thus, according to the test guidelines the test item can be assumed to not be inhibitory to activated sludge because degradation was clearly >35% within 14 days.
Key result
Parameter:
% degradation (DOC removal)
Value:
>= -6 - <= 2
Sampling time:
28 d
Remarks on result:
other: not biodegradable under the test conditions
Details on results:
In the test flasks containing the test item and activated sludge (inoculum) the mean concentration of DOC (dissolved organic carbon) varied between 27.9 and 30.2 mg/l over the exposure period of 28 days and was not significantly different from the initial mean DOC concentration of 28.5 mg/l measured on Day 0. Expressed as percentage DOC-removal, mean values in the range from -6 to 2% were noted. Therefore, the test item was found to be not 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 readily biodegraded by an average of 99% within 7 days of exposure, confirming suitability of the activated sludge.
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The study was conducted acc. OECD TG 301A under GLP, controls gave the appropriate results. Hence, the results canbe considered sufficiently reliable to assess the ready biodegradability of 6-amino-1,3-dimethyluracil. In the test flasks containing the test item and activated sludge (inoculum) the mean concentration of DOC (dissolved organic carbon) varied between 27.9 and 30.2 mg/l over the exposure period of 28 days and was not significantly different from the initial mean DOC concentration of 28.5 mg/l measured on Day 0. Expressed as percentage DOC-removal, mean values in the range from -6 to 2% were noted. Therefore, the test item was found to be not biodegradable under the test conditions.
Executive summary:

1,3-Dimethyl-4-aminouracil was investigated for its ready biodegradability in a "28-Day DOC Die-Away Test" according to the EU Commission Directive 92/69 EEC, C.4-A, 1992, and the OECD Guideline for Testing of Chemicals, No. 301 A, 1992, under GLP.

In the test flasks containing the test item 1,3-Dimethyl-4-aminouracil and activated sludge (inoculum) the mean concentrations of dissolved organic carbon (DOC) were not significantly different from the initial mean DOC concentration measured on Day 0. Therefore, 1,3-Dimethyl-4-aminouracil was found to be not biodegradable under the test conditions.

In the abiotic control, containing the test item and poisoned mineral medium, no degradation was noted after 28 days of exposure (based on DOC-measurements).

The reference item sodium benzoate was biodegraded by 99% within 7 days of exposure, 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 50% within 14 days of exposure. Thus, according to the test guidelines the test item can be assumed to not be inhibitory to activated sludge because degradation was >35% within 14 days.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline Study, scientifically acceptable
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
lnoculum: activated sludge from laboratory wastewater plants treating municipal sewage Inoculum concentration in the test: 30 mg/l dry weight
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
DOC
Initial conc.:
43 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Test temperature: Incubation: at room temperature (22 +/- 2 °C)

TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: 2

SAMPLING
- Sampling frequency: daily


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
Reference substance:
aniline
Key result
Parameter:
% degradation (DOC removal)
Value:
< 10
Sampling time:
28 d
Details on results:
Kinetic of test substance (in %):
= 0 after 1 day(s)
= 0 after 10 day(s)
= 0 after 21 day(s)
= 0 after 28 day(s)
Kinetic of control substance (in %):
< 10 after 1 day(s)
= 97 after 10 day(s)
Interpretation of results:
under test conditions no biodegradation observed

Description of key information

Biodegradation in water: Screening tests: Not readily biodegradable: -6 – 2 % (DOC removal) after 28 days (OECD 301A, GLP)

Biodegradation in water: Screening tests: Not readily biodegradable: <10% (DOC removal) after 28 days (OECD 301A, GLP)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information