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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

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 study according to protocol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Ammonium chloride was omitted from the medium to prevent nitrification.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
- Chemical name: dihexadecyl peroxodicarbonate (dicetylperoxydicarbonate)
- Purity: 93.3%
- Batch/lot No.: 1009530211
- Appearance: white flakes
- Solubility in water: insoluble in water
- Storage: in the freezer (<-20°C) in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Activated sludge (17-06-2011) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This activated sludge plant treats predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 400 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the BOD bottles (van Ginkel and Stroo, 1992).
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The nutrient medium of the Closed Bottle test contained per liter of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.3 mg Na2HPO4·2H2O, 22.5 mg MgSO4·7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3·6H2O. Ammonium chloride was omitted from the medium to prevent nitrification. Sodium acetate was added to the bottles using aqueous stock solution of 1.0 g/L.
- Additional substrate: no
- Solubilising agent (type and concentration if used): Accurate administration of water-insoluble dihexadecyl peroxodicarbonate was accomplished by dissolving the test substance in dichloromethane (1.0 g/L). dichloromethane (0.6 mL) was directly added to bottles. Dichloromethane (0.6
mL) was added to a series of control bottles. The solvent was allowed to evaporate by placing the bottles on a roller bank in a ventilated hood for 24 hours to obtain an even distribution of the test substance on the walls of the bottles.
- Test temperature: 22 to 24°C
- pH: 7.0 to 7.2
- pH adjusted: no
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 0.30 L BOD (biological oxygen demand) bottles with glass stoppers
- Number of culture flasks/concentration: Use was made of 10 bottles containing only inoculum (not agitated), 6 bottles containing inoculum and sodium acetate (not agitated), 10 bottles containing inoculum and test substance (agitated), and 10 bottles treated with dichloromethane (added and evaporated) only containing inoculum (agitated).
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: no


CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: no


STATISTICAL METHODS:
Calculation of endogenous respiration The endogenous respiration (oxygen depletion in the control) was calculated as follows;
Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc (day 28)
Mc is the mean oxygen level in the control bottles with inoculum.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of dihexadecyl peroxodicarbonate and sodium acetate were calculated from their molecular formulae and molecular weights as follows:
ThOD(mgO2/mg) = 16(2C +1/2(H-Cl-3N)+3S+2 1/2P + 1/2Na-O)/MW

Calculation of the biochemical oxygen demand (BOD)
Provided that the oxygen concentrations in all bottles at the start of the test were equal, the amounts of oxygen consumed in test and reference compound bottles were calculated as follows:
Oxygen consumptionn (mg/L) by test substance = Mcs - Mt
Oxygen consumptionn (mg/L) by reference compound = Mc - Ma
Mc and cs is the mean oxygen level in the control bottles (not agitated) (c) and control bottles treated with dichloromethane (agitated) n days after the start of the test.
Mt or a is the mean oxygen concentration in the bottles containing the test substance (agitated) (t) or the reference compound, sodium acetate (a), n-days after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test substance and sodium acetate was calculated by dividing the oxygen consumption by the concentration of the test substance and sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages
The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD).
Reference substance
Reference substance:
acetic acid, sodium salt

Results and discussion

% Degradation
Parameter:
% degradation (O2 consumption)
Value:
68
Sampling time:
28 d
Details on results:
Theoretical oxygen demand (ThOD)
The calculated theoretical oxygen demand (ThOD) of dihexadecyl peroxodicarbonate is 2.65 mg/mg. The ThOD of sodium acetate is 0.78 mg/mg.

Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test compound in the Closed Bottle test was not determined
because possible toxicity of dihexadecyl peroxodicarbonate to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance tested was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial concentration of the test
compound is expected.

Validity of the test
The validity of the test is demonstrated by an endogenous respiration of 0.8 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%. The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 85. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.

The Closed Bottle test
Dihexadecyl peroxodicarbonate is biodegraded by 68% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a
period of less than 12 days immediately following the attainment of 10% biodegradation. The 14-day window of the Closed Bottle test was therefore met. Dihexadecyl peroxodicarbonate should therefore be classified as readily biodegradable.

BOD5 / COD results

Results with reference substance:
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 85.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
This study is reliable without restrictions. It is performed according to OECD guideline 301D under GLP and all critical validity criteria were fulfilled.
The test result is adequate for C&L and risk assessment purposes.
Executive summary:

In order to assess the biotic degradation, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD (OECD TG 301), EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

Dihexadecyl peroxodicarbonate did not cause a reduction in the endogenous respiration. The test substance is therefore considered to be non-inhibitory to the inoculum. Dihexadecyl peroxodicarbonate was biodegraded by 68% at day 28 in the Closed Bottle test. Over 60% biodegradation was achieved in a period of less than 12 days immediately following the attainment of 10% biodegradation. Hence this substance should be classified as readily biodegradable.

The test is valid as shown by an endogenous respiration of 0.8 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 85% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.