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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without restrictions.
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
TEST ORGANISMS:
- Origin: aeration tank of a wastewater plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal)
- Date of collection: 2014-02-03
- Concentration of inoculum: 30 mg/L suspended solids

PRE-TREATMENT OF THE INOCULUM:
− Before use, the inoculum was stored for three days at room temperature under continuous shaking with aeration
− The sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm and 20 °C and decanting off the supernatant
− An aliquot of the wet sludge was dried in order to determine the wet weight / dry weight ratio of the sludge and to prepare a stock suspension (activated sludge) of 3 g dw/L
− The calculated amount of sludge, needed to achieve 300 mL of this stock suspension, was dissolved in mineral medium and then filled up to a defined end volume
Duration of test (contact time):
40 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral medium was prepared from stock solutions of mineral components (i.e. mineral salt solution, magnesium sulphate solution, calcium chloride solution and Iron (III) chloride solution) in accordance with the guideline. To prepare the mineral medium 10 mL of mineral salt solution were mixed with 800 mL deionised water, 1 mL each of the magnesium sulphate-, calcium chloride- and the iron (III) chloride solution were added and filled up to 1 litre with deionised water
- Solubilising agent (type and concentration if used): None
- Pretreatment of the test item: 25 mg of the test item were weighed out and directly added to the test flasks, filled with 200 mL of mineral medium; afterwards the volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg/L
- Test temperature: 22+/- 1°C
- pH: 7.6 - 8.4 (measured in the test flasks after 28 days)
- pH adjusted: no
- Aeration of dilution water: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: closed flasks with a test volume of 250 mL
- Number of culture flasks/concentration: 2 (test item & reference compound), 3 (inoculum blank)
- Measuring equipment: OxiTopControl System (WTW)
- Test performed in closed vessels: Yes
- Test performed in open system: not applicable
- Mixing: 1 magnetic stirrer per test vessel
- Incubation time: 40 days
- Details of trap for CO2 and volatile organics if used: not applicable

SAMPLING
- Sampling frequency: The BOD (in mg O2/L) was measured daily

CONTROL AND BLANK SYSTEM
- Inoculum blank: Blanks with inoculum and without test item were run in parallel (3 replicates)
- Reference substance: a reference compound (sodium benzoate) was run in parallel, to check the operations of the procedures. The concentration of the reference substance used in the test was 100 mg/L
- Toxicity control: 25 mg of the test item and 25 mg of the reference compound were weighed out and added to the test flasks, filled with 200 mL of mineral medium. Afterwards the flask volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg test item and reference compound.
Reference substance:
benzoic acid, sodium salt
Remarks:
(Sigma-Aldrich), purity: 99.7 %, Batch no.: 1438955
Test performance:
STUDY PERIOD:
- Start of the incubation period: 2014-02-06
- End of the incubation period: 2014-03-18

PRE-TREATMENT OF THE TEST SUBSTANCE:
- 25 mg of the test item were weighed out and filled with 200 mL of mineral medium
- afterwards the volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg test item/L

PRE-TREATMENT OF THE REFERENCE COMPOUND:
- 25 mg of the reference compound were weighed out and filled with 200 mL of mineral medium
- afterwards the volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg reference compound/L

PRE-TREATMENT OF THE TOXICITY CONTROL:
- 25 mg of the test item and 25 mg of the reference compound were weighed out and filled with 200 mL of mineral medium
- afterwards the flask volume was made up to 250 mL with mineral medium containing the inoculum to give a test concentration of 100 mg test item and reference compound/L
Parameter:
% degradation (O2 consumption)
Value:
19
Sampling time:
7 d
Parameter:
% degradation (O2 consumption)
Value:
36
Sampling time:
14 d
Parameter:
% degradation (O2 consumption)
Value:
50
Sampling time:
21 d
Parameter:
% degradation (O2 consumption)
Value:
60.2
Sampling time:
28 d
Remarks on result:
other: The original report gave a result of 60%. As this result is identical to the pass level for ready biodegradation, a detailed investigation of the report was performed yielding the more precise result of 60.2%.
Parameter:
% degradation (O2 consumption)
Value:
72
Sampling time:
40 d
Details on results:
The pass level of biodegradation of 60% removal of ThOD was reached. The requirment for the 10 d window was not fulfilled. After 40 days a degradation of 72% was found.
Results with reference substance:
The reference compound sodium benzoate showed 87 % degradation after 14 days.
Kinetic of reference substance (degradation in %):
78 after 7 days
87 after 14 days
91 after 21 days
91 after 28 days
92 after 40 days
Validity criteria fulfilled:
yes
Remarks:
(Ready biodegradation of reference compound ≥ 60 % within 14 days. The toxicity control exhibited degradation rates > 25 % within 14 days. Replicates difference < 20 %. Oxygen uptake of the inoculum blank was ≤ 60 mg/L. No pH influence.)
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
Within 28 days, a degradation rate of 60.2 % was determined. 2,2-Dimethylpropane-1,3-diyl dibenzoate is considered to be "Readily Biodegradable, but failing the 10 day window". The reference compound sodium benzoate showed 87 % degradation after 14 days.
Executive summary:

In accordance with the Council Regulation (EC) No 440/2008, Method C.4-D “Manometric Respirometry Test“(2008) which is in all essential parts identical with OECD Guideline 301 F (adopted July 1992) a study to assess the ready biodegradability of 2,2-dimethylpropane-1,3-diyl dibenzoate was carried out.

A suspension of 100 mg/L test item in a mineral medium, equalling to 50-100 mg ThOD or COD/Litre as the nominal sole source of organic carbon, was stirred in a closed flask and inoculated at a constant temperature (22 ± 1 °C) for up to 40 days under aerobic conditions in the dark.The consumption of oxygen (BOD) was determined by measuring the drop in pressure in the automated respirometer flasks.Evolved carbon dioxide was absorbed in sodium hydroxide. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of theoretical oxygen demand (ThOD). The endogenous activity of the inoculum was checked running parallel blanks with inoculum, but without test item. A reference compound (sodium benzoate) was run in parallel to check the operation of the procedures. A toxicity control was run in parallel, to ensure that the chosen concentration of the test item was not inhibitory to microorganisms. Degradation was followed by the determination of oxygen uptake and measurements were taken at frequent intervals to allow the identification of the beginning and end of biodegradation and the slope of the biodegradation curve. The test lasted for 40 days.

Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item and inoculum blank were carried out in triplicate and of reference compound in duplicate. The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given by the manufacturer of the equipment. At the end of incubation, the pH was measured in the flasks. Within 28 days, a degradation rate of 60.2 % was determined. 2,2-Dimethylpropane-1,3-diyl dibenzoate is considered to be "Readily biodegradable, but failing the 10 day window". The reference compound sodium benzoate showed 87 % degradation after 14 days.

Description of key information

Within 28 days, a degradation rate of 60.2 % was determined. 2,2-Dimethylpropane-1,3-diyl dibenzoate is considered to be "Readily biodegradable, but failing the 10 day window". The reference compound sodium benzoate showed 87 % degradation after 14 days.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window

Additional information

The pass level of biodegradation of 60 % removal of ThOD after 28 days was reached, but the 10 day window was failed. 2,2-Dimethylpropane-1,3-diyl dibenzoate showed a degradation of 72 % after 40 days.