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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:
April - September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of activated sludge: three sewage treatment plants
- Pretreatment: filtered through glass wool (discarding first 200 mL). It was mixed and aerated for 5 days. The filtrate was diluted by using deionized water (ratio 1:1)
- Preperation: The inoculum was prepared by adding the required amount of activated sludge (Maximum COD 6000 mg/L as per ISO 5815-1:2003(E)) and dilution water (based on the COD) in a cleaned glass beaker. The inoculum was stored at about 20 ± 2°C
- Initial cell/biomass concentration: 5.17 mg/L based on ThOD
Duration of test (contact time):
28 d
Initial conc.:
5.17 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Standart OECD medium
- Additional substrate: no
- Solubilising agent: no
- Test temperature: 19.7 - 20.6°C
- pH: 7 - 7.5
- pH adjusted: no
- Aeration of dilution water: yes

TEST SYSTEM
- Culturing apparatus: BOD bottles
- Number of culture flasks/concentration: 3
- Measuring equipment: The dissolved oxygen content in the Control sample (Glucose-glutamic acid), Blank and test solutions were measured initially by Winkler titration. Parallel groups of BOD bottles were prepared for the determination of the reference substances in simultaneous experimental series. All the bottles were incubated at 20 ± 2°C. The BOD bottles were assembled, including inoculum blanks, to allow at least duplicate measurements of oxygen consumption to be made at the desired test intervals, at day 0, 7, 14, 21 and 28 days.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 0.6 mL ATU solution + Inoculum made up to 300 mL.
- Control solution: Glucose-glutamic acid [6 mL] + 0.6 mL ATU solution + Inoculum made up to 300 mL.
- Toxicity control: 50 mL of Reference item solution + 0.6 mL ATU solution + Inoculum made up to 300 mL.
Reference substance:
acetic acid, sodium salt
Preliminary study:
During the preliminary study, determination of oxygen concentrations was performed at the start of the experiment (day 0) and on day 5 and day 7. The temperature in the test room varied between 19.8 and 20.8°C. Measured pH values at the start of the test (using pH strips- found to be 7 - 7.5). The relative biodegradation values were calculated from the O2 measurements performed during the test period of 7 days revealed 16.87%, 16.84% and 19.67% biodegradation of test item at three different concentrations of 1.72 mg/L, 5.17 mg/L and 8.61 mg/L (mean values of duplicates on day 7).
Test performance:
During the definitive study, determination of oxygen concentrations were performed at the start of the experiment (day 0) and on day 7, 14, 21 and 28. The relative biodegradation values were calculated from the O2 measurements performed during the test period of 28 days of three bottles revealed 69.66%, 70.62% and 69.66% biodegradation of test item at 5.17 mg/L concentrations based on ThOD (mean values of duplicates on day 28).
Oxygen depletion in the inoculum blank obtained was 0.19 mg O2/L which is below 1.5 mg O2/L after 28 days.
The residual concentrations of oxygen in the test bottles were found to be > 0.5 mg/L at all durations.
All differences of duplicate biodegradation values expressed as mg O2/L were less than 20% (-1.91 to 4.55%).
Key result
Parameter:
% degradation (O2 consumption)
Value:
69.98
Sampling time:
28 d
Results with reference substance:
The reference substance (sodium acetate) attained a 71.53% biodegradation level (of greater than 60% degradation) within 14 days based on ThOD.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item was readily biodegradable for maximum of 69.98% at 28 days under the conditions of the closed bottle method (OECD 310D).
Executive summary:

The ready biodegradability of the test item was determined using the closed bottle method (OECD guideline No. 301 D, 1992). The ThOD of the test item was calculated to be 2.22 mg of O2/mg. The ThOD of sodium acetate (reference substance) was calculated to be 0.78 mg of O2/mg. The values for the test and reference substances were determined using the molecular formula and the purity of the substances. The test item stocks were prepared in the test media by mixing to accelerate dissolution of the test substance in mineral medium. Required volumes of the stock solution corresponding to the test concentrations and microbial organisms were added to the test medium. During the definitive test, determination of oxygen concentrations were performed at the start of the experiment (day 0) and on day 7, 14, 21 and 28. The temperature in the test room varied between 19.7 and 20.6°C. Measured pH values at the start of the test (using pH strips- found to be 7 - 7.5) and measured oxygen concentrations were between 0.76 and 8.74 mg/L. The relative biodegradation values were calculated from the O2 measurements performed during the test period of 28 days of three bottles revealed 69.66%, 70.62% and 69.66% biodegradation of test item at 5.17 mg/L concentrations based on ThOD (mean values of duplicates on day 28). The test item reached a maximum biodegradability of 69.98% based on ThOD (mean values of three bottles) with 28 days of biodegradation exceeding 60%.Oxygen depletion in the inoculum blank obtained was 0.19 mg O2/L which is below 1.5 mg O2/L after 28 days. The residual concentrations of oxygen in the test bottles were found to be > 0.5 mg/L at all durations. All differences of duplicate biodegradation values expressed as mg O2/L were less than 20% (-1.91 to 4.55%).The reference substance (sodium acetate) attained a 71.53% biodegradation level (more than 60% degradation) within 14 days based on ThOD.Since all criteria for acceptability of the test were met, this study was considered to be valid. The test item was readily biodegradable for maximum of 69.98% at 28 days under the conditions of the closed bottle method.

Description of key information

The test item was readily biodegradable for maximum of 69.98% at 28 days under the conditions of the closed bottle method (OECD 310D).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

The ready biodegradability of the test item was determined using the closed bottle method (OECD guideline No. 301 D, 1992). The ThOD of the test item was calculated to be 2.22 mg of O2/mg. The ThOD of sodium acetate (reference substance) was calculated to be 0.78 mg of O2/mg. The values for the test and reference substances were determined using the molecular formula and the purity of the substances. The test item stocks were prepared in the test media by mixing to accelerate dissolution of the test substance in mineral medium. Required volumes of the stock solution corresponding to the test concentrations and microbial organisms were added to the test medium. During the definitive test, determination of oxygen concentrations were performed at the start of the experiment (day 0) and on day 7, 14, 21 and 28. The temperature in the test room varied between 19.7 and 20.6°C. Measured pH values at the start of the test (using pH strips- found to be 7 - 7.5) and measured oxygen concentrations were between 0.76 and 8.74 mg/L. The relative biodegradation values were calculated from the O2 measurements performed during the test period of 28 days of three bottles revealed 69.66%, 70.62% and 69.66% biodegradation of test item at 5.17 mg/L concentrations based on ThOD (mean values of duplicates on day 28). The test item reached a maximum biodegradability of 69.98% based on ThOD (mean values of three bottles) with 28 days of biodegradation exceeding 60%.Oxygen depletion in the inoculum blank obtained was 0.19 mg O2/L which is below 1.5 mg O2/L after 28 days. The residual concentrations of oxygen in the test bottles were found to be > 0.5 mg/L at all durations. All differences of duplicate biodegradation values expressed as mg O2/L were less than 20% (-1.91 to 4.55%).The reference substance (sodium acetate) attained a 71.53% biodegradation level (more than 60% degradation) within 14 days based on ThOD.Since all criteria for acceptability of the test were met, this study was considered to be valid. The test item was readily biodegradable for maximum of 69.98% at 28 days under the conditions of the closed bottle method.