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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:
from 1998-10-14 to 1999-02-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to international standard guidelines in compliance with GLP and with all validity criteria fulfilled.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Minor deviations: Instead of an effluent/ extract/ mixture, activated sludge was used as an inoculum; The test was prolonged because the pass level was not reached at day 28; Ammoniumchloride was omitted in the test medium to avoid nitrification.
Qualifier:
according to
Guideline:
EU Method C.6 (Degradation: Chemical Oxygen Demand)
Deviations:
yes
Remarks:
See above
Qualifier:
according to
Guideline:
other: ISO/TC 147/SC 5WG 4N152
Deviations:
yes
Remarks:
See above
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
from 1999.03.31
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Secondary activated sludge was obtained from the WWTP Nleuwgraaf in Duiven, The Netherlands (1998-10-22). The WWTP Nieuwgraaf is an activated sludge plant treating predominantly domestic waste water. A minor deviation of the test procedures described in the guidelines was introduced: instead of an effluent/extract mixture, activated sludge was used as an inoculum. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 200 mg Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted to a concentration of 2 mg DW/L in the BOD bottles.
Duration of test (contact time):
112 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
The nutrient medium of the close bottle test contained per liter of deionised water: 8.5 mg/l KH2PO4, 21.75 mg K2HPO4, 4 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H2O, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O.
The test substance is poorly soluble and was therefore first dissolved in dichloromethane (1g/l). The test substance in dichloromethane (0.53 mL) was added to 2 g silica gel (100-200 mesh) weighed in a glass petri dish. The solvent was allowed to evaporate by placing the petri dish in a ventilated hood for 2 hours, and the entire contents were then transferred to the BOD bottle. The test was performed in 250 to 300 ml BOD (biological oxygen demand) bottles with glass stoppers.

Use was made of 10 bottles containing silica gel and inoculum, 10 bottles containing test substance, silicagel and inoculum, 6 bottles containing only inoculum, and 6 bottles containing sodium acetate (at 6.7 mg/l) and inoculum.

Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7,14,21, and 28.
Reference substance:
acetic acid, sodium salt
Remarks:
Sodium acetate trihydrate (Lot No. D 33284) was purchased from J.T. Baker BV, Deventer, The Netherlands.
Preliminary study:
Not applicable
Test performance:
The test is valid as shown by an endogenous respiration of 1.2 mglL at day 28 and by the total mineralization of the reference compound, sodium
acetate. Sodium acetate was degraded 76% 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. The pH of the media was 7.1 at the start of the test. The pH of the medium at day 28
was 6.7. Temperatures, ranged from 19 to 21°C.
Parameter:
% degradation (O2 consumption)
Value:
37
Sampling time:
112 d
Parameter:
% degradation (O2 consumption)
Value:
2
Sampling time:
28 d
Details on results:
Suggests inherent Biodegradability
Results with reference substance:
Sodium acetate was degraded 76% of its theoretical oxygen demand after 14 days.

Table 1- Dissolved oxygen concentrations (mg/l) in the close bottles

Time (Days)

Oxygen concentration (mg/l)

 

Ocs

Ots

Oc

Oa

0

8.7

8.7

8.7

8.7

8.7

8.7

8.7

8.7

Mean

8.7

8.7

8.7

8.7

7

8.3

8.3

8.3

4.5

8.3

8.1

8.4

4.5

Mean

8.3

8.2

8.4

4.5

14

8.0

8.0

8.1

4.0

8.0

7.9

8.0

4.0

Mean

8.0

8.0

8.1

4.0

21

7.7

7.8

 

 

7.8

7.6

 

 

Mean

7.8

7.7

 

 

28

7.6

7.6

 

 

7.5

7.4

 

 

Mean

7.6

7.5

 

 

42

7.4

7.2

 

 

7.4

6.6

 

 

Mean

7.4

6.9

 

 

56

7.2

5.9

 

 

7.3

5.8

 

 

Mean

7.3

5.9

 

 

84

6.9

5.2

 

 

7.2

5.4

 

 

Mean

7.1

5.3

 

 

112

6.8

5.0

 

 

7.2

5.2

 

 

Mean

7.0

5.1

 

 

Ocs=Mineral nutrient solution without test material but with inoculum and evaporated silica gel.

Ots=Mineral nutrient solution with test material (2 mg/l), inoculum and silica gel.

Oc=Mineral nutrient solution without test material but with inoculum.

Oa=Mineral nutrient solution with sodium acetate (6.7 mg/l) and inoculum.

Table2 -Oxygen consumption (mg/l) and the percentages biodegradation of 1,1 -di-(tert-Butylperoxy)-3,3,5-tri-methylcyclohexane (BOD/ThOD) and sodium acetate (BOD/ThOD) in the close bottle test.

Time (days)

Oxygen consumption (mg/l)

Biodegradation (%)

 

Test

Acetate

Test

Acetate

0

0.0

0.0

0

0

7

0.1

3.9

2

72

14

0.0

4.1

0

76

21

0.1

 

2

 

28

0.1

 

2

 

42

0.5

 

10

 

56

1.4

 

27

 

84

1.8

 

35

 

112

1.9

 

37

 

Validity criteria fulfilled:
yes
Remarks:
Endogenous respiration of 1,1 mg/l at day 28, the total mineralisation of the reference compound, sodium acetate (degraded 76% of its theoretical oxygen demand after 14 days) and oxygen concentrations > 0.5 mg/l in all bottles during the test period.
Interpretation of results:
inherently biodegradable
Conclusions:
Test substance was tested in the Closed Bottle in the presence of silica gel and was biodegraded 2% at day 28. Silica gel was added to permit an accurate administration of the test compound. In the prolonged Closed Bottle test with silica gel the biodegradation percentage reached 37% at day 112. Triganox 29 should therefore be classified as inherently biodegradable.
Executive summary:

The biotic degradation of the test material containing 90% of 1,1 -di-(tert-Butylperoxy)-3,3,5-tri-methylcyclohexane and 10% of isododecane was assessed performing a Closed Bottle Test according to slightly modified OECD, EEC and ISO test guidelines and in compliance with the OECD principles of GLP. It is not clear whether any of the isododecane was included in the test due to the procedure used. The test was prolonged to 112 days because the pass level was not reached at day 28. Secondary activated sludge was obtained from the WWTP Nieuwgraaf in Duiven, The Netherlands (1998-10-22). The inoculum was collected at the WWTP Nieuwgraaf activated sludge plant treating predominantly domestic waste water. The activated sludge was aerated for one week and was diluted to a concentration of 2 mg DW/L in the BOD bottles. The pH of the media was 7.1 and 6.7 at the start of the test and at day 28, respectively. Temperatures, ranged from 19 to 21°C. Ammonium chloride was omitted from the test medium to avoid nitrification.

Because 1,1-di-(tert-Butylperoxy)-3,3,5-tri-methylcyclohexane is poorly soluble in water, it was first dissolved in dichloromethane and then adsorbed onto silica gel particles to enable accurate administration. The initial test concentrations were 2 mg/l for the test material and 6.7 mg/l for the reference substance (sodium acetate). Use was made of 10 bottles containing silica gel and inoculum, 10 bottles containing test substance, silicagel and inoculum, 6 bottles containing only inoculum, and 6 bottles containing sodium acetate and inoculum. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21 and 28. The biodegradation was calculated as the ratio of the Biochemical Oxygen Demand (BOD) to the Theoretical Oxygen Demand (ThOD). The BOD of the test compound 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. The dissolved oxygen concentrations were determined electrochemicaliy using an oxygen electrode and meter.

2% of the test substance was biodegraded at day 28 and 37% at day 112. The test is valid as shown by endogenous respiration of 1,1 mg/l at day 28, the total mineralisation of the reference compound, sodium acetate (degraded 76% of its theoretical oxygen demand after 14 days) and oxygen concentrations > 0.5 mg/l in all bottles during the test period.

In conclusion the test material is not readily biodegradable under the conditions of this study but it should be classified as inherently biodegradable.

Description of key information

A Closed Bottle Test according to a slightly modified version of OECD 310 D test guideline (use of silca gel to increase bioavailability) and according to GLP was performed. 2% biodegradation was determined at day 28 and 37% at day 112. A MITI-I study was also available indicating 8-12 % biodegradation..  

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable, not fulfilling specific criteria
Type of water:
freshwater

Additional information

Two ready biodegradability studies were located for this substance: A MITI-I(OECD TG 301C) test was performed with 1,1 -bis(tert-butylperoxy)-3,3,5 -trimethylcyclohexane at 100 ppm and activated sludge at 30 ppm for 4 weeks. Indirect analysis of BOD at 0% was determined; direct analysis shows a GC at 12% and UV-VIS at 8% suggesting that some degradation had occurred after 28 days. The judgment for ultimate degradability was non-biodegradability under the conditions of this study.

In a second study the biotic degradation of the test material containing 90% pure test substance and 10% of isododecane was assessed performing a Closed Bottle Test according to slightly modified OECD 301D test guidelines and according to GLP. The test was prolonged to 112 days because the pass level was not reached at day 28. Use was made of silica gel in order to increase the bioavailability of the test substance. Analyses of the dissolved oxygen concentration were performed on days 7, 14, 21, and 28. Ultimate biodegradation of 2% was determined at day 28 and 37% at day 112. The test meets the validity criteria of the Guideline method.

In conclusion the test material is not readily biodegradable under the strict conditions of these studies. There are indications from the extended test that degradation is possible and may occur under more favourable conditions such as in the CAS test for example.