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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:
05-07-2013 - 06-11-2013
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)
Deviations:
yes
Remarks:
Ammonium chloride was omitted from the medium to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).
Qualifier:
according to
Guideline:
ISO 10707 Water quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of biochemical oxygen demand (closed bottle test)
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic/anaerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
Secondary activated sludge (04-07-2013) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating 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.
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Initial conc.:
1.8 mg/L
Initial conc.:
6.7 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles containing only inoculum, 10 bottles containing only inoculum in bottles treated before with DCM (added and evaporated), 10 bottles containing inoculum and test substance, 10 bottles containing inoculum and dibutyl maleate, and 6 bottles containing sodium acetate and inoculum. The concentrations of the test substance, dibutyl maleate and sodium acetate in the bottles were 3.0, 1.8, and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

Nutrients, stocks and administration:
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. Accurate administration of the water-insoluble test substance and dibutyl maleate was accomplished by preparing stock solutions of 1.0 and 0.6 g/L in DCM, respectively. Volumes of 0.9 mL of the test substance and dibutyl maleate in DCM were added to the bottles. The solvent was allowed to evaporate for approximately 2 hours in a ventilated hood. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L in water.

Test bottles:
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.
Reference substance:
acetic acid, sodium salt
Remarks:
Purity >99% Batch/lot number 050M0213V
Test performance:
The calculated theoretical oxygen demand (ThOD) of active substance (OO-tert-butyl monoperoxymaleate) and dibutyl maleate are 0.72 and 2.11 mg/mg, respectively. The ThOD of test substance containing OO-tert-butyl monoperoxymaleate, dibutyl maleate, maleic acid, tert butyl hydroperoxide and proprietary impurities is 1.55 mg/mg. This ThOD was calculated by including maleic acid, tert butyl hydroperoxide and proprietary impurities in the dibutyl maleate fraction of the test substance. The ThOD of sodium acetate is 0.78 mg/mg.
Key result
Parameter:
% degradation (O2 consumption)
Value:
68
Sampling time:
28 d
Remarks on result:
other:
Remarks:
28 day in the Closed Bottle test
Details on results:
OO-tert-butyl monoperoxymaleate (test substance) is biodegraded by 68% at day 28 in the Closed Bottle test. The pass level of 60% was not reached within 14 days upon achieving 10% biodegradation. The reason for failing the 14-day window is the poor water solubility of the test substance and the test substance being a mixture of chemicals.
Poor water solubility of OO-tert-butyl monoperoxymaleate and dibutyl maleate is expected to affect biodegradation kinetics because slow desorption and dissolution rates of substances present at high concentrations may limit the biodegradation in ready biodegradability tests. The biodegradation curves of substances with a poor water solubility and limited bioavailability are usually linear instead of the anticipated S-shaped curve. Poor water solubility is an important reason for not applying the time window.
The time window concept assumes that biodegradation of a single organic compound takes place in a ready biodegradability test. The test substance is a mixture of chemicals. The biodegradation kinetics of the individual compounds in a mixture are not necessarily the same. The biodegradation of a mixture is therefore an addition of different biodegradation curves. It is thus possible that individual compounds meet the time window criterion whereas the biodegradability curve of the mixture suggests that the test substance is not readily biodegradable (Battersby, 2000, Richterich and Steber, 2001).
OO-tert-butyl monoperoxymaleate (test substance) is therefore classified as readily biodegradable only based on the biodegradation percentage of 68% reached at day 28.
Dibutyl maleate is biodegraded by 66% at day 28 in the Closed Bottle test and is therefore classified as readily biodegradable. Biodegradation percentages in excess of 60 at day 28 were therefore found with both the test substance and butyl maleate. The biodegradability of the active substance OO-tert-butyl monoperoxymaleate can be calculated by subtracting the oxygen consumption caused by dibutyl maleate from the oxygen consumption due to degradation of the test substance. This gives an oxygen consumption (BOD) of 0.8 mg/L for the active substance. The biodegradation percentage is calculated by dividing the BOD by the concentration of the active material in the bottles i.e. 1.2 mg/L. This results in a biodegradation percentage of 66 for the active substance. This biodegradation percentage is however inaccurate due to the high content of readily biodegradable dibutyl maleate in the test substance.

Table 1: Dissolved oxygen concentrations (mg/L) in the closed bottles.

Time (days)

Oxygen concentration (mg/L)

Ocs

Ot

Odbm

Oc

Oa

0

8.8

8.8

8.8

8.8

8.8

8.8

8.8

8.8

8.8

8.8

Mean (M)

8.8

8.8

8.8

8.8

8.8

7

8.1

7

7.1

8.2

4.4

8.2

6.9

7.2

8.1

4.5

Mean (M)

8.2

7.0

7.2

8.2

4.5

14

8

6.3

6.5

8

3.8

8.1

6.1

6.7

8

3.7

Mean (M)

8.1

6.2

6.6

8.0

3.8

28

7.9

4.8

5.4

7.9

7.9

4.5

5.6

7.8

Mean (M)

7.9

4.7

5.5

7.9

Ocs Mineral nutrient solution with only inoculum in bottles treated with DCM.
Ot Mineral nutrient solution with test material (3.0 mg/L), and inoculum.
Odbm Mineral nutrient solution with dibutyl maleate (1.8 mg/L), and inoculum.
Oc Mineral nutrient solution with only inoculum.
Oa Mineral nutrient solution with sodium acetate (6.7 mg/L) and with inoculum.

Table 2: Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, OO-tert-butyl monoperoxymaleate (BOD/ThOD), dibutyl maleate (BOD/ThOD), and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time
(days)

Oxygen consumption (mg/L)

Biodegradation (%)

 

Test
substance

Dibutyl
maleate

Acetate

Test
substance

Dibutyl
maleate

Acetate

0

0

0

0

0

0

0

7

1.2

1

3.7

26

26

71

14

1.9

1.5

4.2

40

39

81

21

2.5

2.4

53

63

28

3.2

2.4

68

66

Validity criteria fulfilled:
yes
Remarks:
The validity of the test by an endogenous respiration is of 0.9 mg/L at day 28. The differences of the replicate at day 28 were less than 20%.The validity of the test by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
OO-tert-butyl monoperoxymaleate (test substance) is biodegraded by 68% (mean) at day 28 in the Closed Bottle test. The pass level of 60% was not reached within 14 days upon achieving 10% biodegradation. The reason for failing the 14-day window is the poor water solubility of the test substance and the test substance being a mixture of chemicals.
Executive summary:

The ready biodegradability of OO-tert-butyl monoperoxymaleate was determined in the Closed Bottle test performed according to OECD 301 D (OECD 1992), EU Method C.4-E (1992) and ISO Test Guideline 10707 (1994) under GLP.

Degradation was followed by analysis of dissolved oxygen over a 28-d period. The amount of oxygen uptake by the microbial population during biodegradation of the test substance, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of ThOD.

The test material is a phlegmatized peroxide, therefore the test substance OO-tert-butyl monoperoxymaleate was tested in mixture with the stabilizer dibutyl maleate. The biodegradability of the active substance OO-tert-butyl monoperoxymaleate was calculated by subtracting the oxygen consumption caused by dibutyl maleate from the oxygen consumption due to degradation of the test item.

Use was made of 10 bottles containing only inoculum, 10 bottles containing only inoculum in bottles treated before with DCM (dichloromethane; added and evaporated), 10 bottles containing inoculum and test substance, 10 bottles containing inoculum and dibutyl maleate, and 6 bottles containing sodium acetate and inoculum. The concentrations of the test substance, dibutyl maleate and the reference substance sodium acetate in the bottles were 3.0, 1.8, and 6.7 mg/L, respectively.

OO-tert-butyl monoperoxymaleate (test substance) is biodegraded by 68% at day 28 in the Closed Bottle test. The pass level of 60% was not reached within 14 days upon achieving 10% biodegradation. The reason for failing the 14-day window is the poor water solubility of the test substance and the test substance being a mixture of chemicals. Poor water solubility of OO-tert-butyl monoperoxymaleate and dibutyl maleate is expected to affect biodegradation kinetics because slow desorption and dissolution rates of substances present at high concentrations may limit the biodegradation in ready biodegradability tests. The biodegradation curves of substances with a poor water solubility and limited bioavailability are usually linear instead of the anticipated S-shaped curve. Poor water solubility is an important reason for not applying the time window. The time window concept assumes that biodegradation of a single organic compound takes place in a ready biodegradability test. The test substance is a mixture of chemicals. The biodegradation kinetics of the individual compounds in a mixture are not necessarily the same. The biodegradation of a mixture is therefore an addition of different biodegradation curves. It is thus possible that individual compounds meet the time window criterion whereas the biodegradability curve of the mixture suggests that the test substance is not readily biodegradable.

OO-tert-butyl monoperoxymaleate phlegmatized with dibutyl maleate is therefore classified as readily biodegradable only based on the biodegradation percentage of 68% reached at day 28.

The active substance OO-tert-butyl monoperoxymaleate is biodegraded by 66% at day 28 in the Closed Bottle test and is therefore classified as readily biodegradable.

Description of key information

The active substance OO-tert-butyl monoperoxymaleate is biodegraded by 66% at day 28 in the Closed Bottle test. The pass level of 60% was not reached within 14 days upon achieving 10% biodegradation due to the poor water solubility of the test substance and the test substance being a mixture of chemicals.

(> The biodegradation of OO-tert-butyl monoperoxymaleate phlegmatized with dibutyl maleate was 68 % after 28 days. The phlegmatizer dibutyl maleate was degraded by 66 % within 28 days.)

Key value for chemical safety assessment

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

Additional information