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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:
June 13 2012 - August 13 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 301D study carried out under GLP
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
ammonium chloride was not added to prevent oxygen consumption due to nitrification (omission does not result in nitrogen limitation as shown by the biodegradation of the reference compound).
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
n/a
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
River water was sampled from the Rhine near Heveadorp, The Netherlands (07-06-2012). The nearest plant (Arnhem-Zuid) treating domestic wastewater biologically was 3 km upstream. The river water was aerated for 7 days before use to reduce the endogenous respiration (van Ginkel and Stroo, 1992). River water without particles was used as inoculum. The particles were removed by sedimentation after 1 day while moderately aerating.
Duration of test (contact time):
60 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

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 silicone oil was accomplished by preparing stock solutions in DCM. Both the test substance and silicone oil in DCM (1.2 mL) were added to 3.0 g of silica gel on watch glasses. The solvent was allowed to evaporate overnight in a ventilated hood. Next, entire quantities of 3 g of silica gel with the test substance or silicone oil were transferred to the bottles. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L in water.

Test procedures
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 con-taining only river water, 10 bottles containing river water and silica gel with silicone oil, 10 bottles containing river water and silica gel with test substance, 10 bottles containing river water and silica gel, and 6 bottles containing sodium acetate and river water. The concentrations of the test substance, silicone oil, and sodium acetate in the bottles were 4.0, 2.0 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. One extension from the protocol of the Closed Bottle test was introduced. The Closed Bottle test was prolonged by measuring the course of the oxygen decrease in the bottles of day 28 using a special funnel. This funnel fitted exactly in the BOD bottle. Subsequently, the oxygen electrode was inserted in the BOD bottle to measure the oxygen concentration. The medium dissipated by the electrode was collected in the funnel. After withdrawal of the oxygen electrode the medium collected flowed back into the BOD bottle, followed by removal of the funnel and closing of the BOD bottle (van Ginkel and Stroo 1992). This method allowed measurement at day 42 an 60.
Reference substance:
acetic acid, sodium salt
Remarks:
>99% pure
Test performance:
The validity of the test is demonstrated by an endogenous respiration of 1.0 mg/L at day 28 (Table I). 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 87. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Parameter:
% degradation (O2 consumption)
Value:
76
Sampling time:
7 d
Remarks on result:
other: test extended to 60 days
Details on results:
Theoretical oxygen demand (ThOD)
The calculated theoretical oxygen demand (ThOD) of bis(2,4-dichlorobenzyl) peroxide is 1.05 mg/mg. The ThOD of the test substance containing approximately 50% silicone oil is 0.53 mg/mg. Oxygen consumption due to the presence of silicone oil was not detected when comparing the oxygen concentrations in the bottles with silica gel and the bottle with silica gel and silicone oil (Table I). The ThOD of sodium acetate is 0.78 mg/mg
Results with reference substance:
see Table below

Oxygen consumption (mg/L) and the percentages biodegradation of the test substance, bis(2,4-dichlorobenzyl) peroxide (BOD/ThOD) and sodium acetate (BOD/ThOD) in the Closed Bottle test.

Time (days)

Oxygen consumption (mg/L)

Biodegradation (%)

Test substance

Acetate

Test substance

Acetate

 

 

 

 

 

0

0.0

0.0

0

0

7

-0.1

4.2

0

78

14

0.2

4.6

10

85

21

0.5

 

24

 

28

0.9

 

43

 

42

1.3

 

62

 

60

1.6

 

76

 

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
In the prolonged Closed Bottle test, bis(2,4-dichlorobenzyl) peroxide was biodegraded by 76% at day 60 (enhanced biodegradability testing), thus demonstrating that this substance is not persistent.
Executive summary:

ABSTRACT

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.

The presence of bis(2,4-dichlorobenzyl) peroxide did cause a slight reduction in the endogenous respiration at day 7. The biodegradation may as a consequence have been retarded slightly. Bis(2,4-dichlorobenzyl) peroxide was biodegraded by 43% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, bis(2,4-dichlorobenzyl) peroxide was biodegraded by 76% at day 60 (enhanced biodegradability testing), thus demonstrating that this substance is not persistent. The test is valid as shown by an endogenous respiration of 1.0 mg/L and by the total mineralization of the reference compound, sodium acetate. Sodium acetate was degraded by 87% 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.

Description of key information

An OECD 301D GLP study is available where, bis(2,4-dichlorobenzyl) peroxide is classified as inherently biodegradable

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

Bis(2,4-dichlorobenzyl) peroxide was biodegraded by 43% at day 28 in the Closed Bottle test. In the prolonged Closed Bottle test, bis(2,4-dichlorobenzyl) peroxide was biodegraded by 76% at day 60 (enhanced biodegradability testing), thus demonstrating that this substance is not persistent.