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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:
2018-06-26 to 2018-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry Test", adopted July 17, 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
Commission Regulation 440/2008/EC, Method C.4-D of May 30, 2008: Manometric Respirometry Test (EEC Publication No. L 142/496, May 2008)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Name: Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene
Synonym: Kalama® Vetimoss
CAS No.: 21112-37-8
Lot No.: A180118C
Purity: Vetimoss 99.78%
Aggregate State at Room Temperature: Liquid
Color: Clear, pale yellow (according to data sheet), Clear, almost colorless (according to CoA)
Molecular Formula / Molecular Weight: C12H18O2 / 194.27 g/mol
Theoretical Oxygen Demand: ThODNH4: 2.553 mg oxygen per mg test item
Expiry / Retest Date: June 06, 2019
Storage Conditions at Test Facility: At 20°C ± 5°C, in the dark under inert gas (Nitrogen)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Species/Origin: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.
- Conditioning: The aerobic activated sludge used for this study was deposited for 60 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.
Duration of test (contact time):
28 d
Initial conc.:
ca. 25 other: mg/flask
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Surrounding Type: Climatised chamber
- Temperature: 22°C ± 1°C
- Light Conditions: Darkness
- pH value of Test Solutions: 7.4 (measured at the start of the test); 7.4 to 7.6 (measured at the end of the test)
- Test vessel preparation: Test item and/or reference item were weighed directly into test flasks and 2.4 mL silicon oil AR 20 per flask was added for better distribution.
- Incubation: Closed test flasks were incubated in a climatised chamber under continuous stirring.

TEST WATER
Reconstituted Test Water: Analytical grade salts were added to pure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; The pH-value was 7.4.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume.
In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage. 50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water. The pH was adjusted to pH 7.4 with NaOH solution.

REFERENCE ITEM
- Source of information: The information concerning the reference item was provided by the Manufacturer
- Name: Sodium Benzoate
- Batch No.: SLBW2610
- Recommended Retest Date: November 2021
- Purity: 100.0%
- Aggregate State at Room Temperature: Solid
- Colour: White
- Quality Release Date: November 08, 2017
- Empirical Formula / Molecular Weight: C7H5O2Na / 144.10 g/mol
- Theoretical Oxygen Demand: ThODNH4: 1.666 mg oxygen per mg sodium benzoate
- Storage Conditions at Test Facility: At 20°C ± 5°C, in the desiccator in the dark

VALIDITY CRITERIA
- Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 15 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.
- pH value: The pH value of the test item flasks at the end of the test was 7.5 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.
- Reference Item: The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline. The reference item sodium benzoate was degraded to more than 60% after 4 days of incubation.
- Test Item: The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window was less than 20%. The difference of duplicate values at day 28 differed by 0%.
- Toxicity Control: If in a toxicity test, containing both the test item and a reference item less than 25% biodegradation (based on total ThOD) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 30% at day 14; the test item was not inhibitory.
Reference substance:
benzoic acid, sodium salt
Test performance:
- Dispersant: The silicone oil added to the test vessels achieved its purpose. No clotting of the silicone oil or any other inhibiting effects on the dispersion of the test substances were recorded.
- Percentage biodegradation in the toxicity control: Vessel containing both the test item and the reference item sodium benzoate, 30% (ThODNH4) biodegradation was noted within 14 days and 33% (ThODNH4) biodegradation after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.
- Abiotic Control: The oxygen demand in the abiotic control was 0 mg/L during the test duration. There was no need to correct the degradation of the test item and toxicity control.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
- Percentage biodegradation in test vessels: The criterion for ready biodegradability under the conditions of a manometric respirometry test is the degradation of the test item of at least 60%, reached within a 10-day window; the 10-day window starts when the degradation of the test item reaches at least 10% degradation. Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene never reached 10% biodegradation. The mean biodegradation percentage at the end of the 28-day exposure period was 0% (ThODNH4).
- Conclusion: The degradation rate of Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene never reached 10%. Therefore, Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene is considered to be not readily biodegradable.
Results with reference substance:
- Percentage biodegradation of reference item: The reference item sodium benzoate was sufficiently degraded to 72% after 14 days and to 75% after 28 days of incubation. The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks during the Test Period of 28 Days

Time

Flask No.

(days)

1

2

3

4

5

6

7

 

1

5

5

0

0

55

0

40

 

2

5

5

5

5

80

0

80

 

3

5

5

5

5

90

0

105

 

4

5

5

10

5

115

0

120

 

5

10

5

10

5

120

0

125

 

6

10

10

10

10

125

0

130

 

7

10

10

10

10

130

0

135

 

8

10

10

10

10

130

0

135

 

9

10

10

10

10

130

0

135

 

10

10

10

10

10

135

0

140

 

11

10

10

15

10

135

0

140

 

12

10

10

15

15

135

0

145

 

13

10

10

15

15

135

0

145

 

14

10

10

15

15

140

0

145

 

15

10

10

15

15

140

0

150

 

16

10

10

15

15

140

0

150

 

17

10

10

15

15

140

0

150

 

18

10

10

15

15

140

0

150

 

19

10

10

15

15

140

0

155

 

20

10

10

15

15

140

0

155

 

21

10

10

15

15

140

0

155

 

22

10

10

15

15

140

0

155

 

23

10

10

15

15

140

0

160

 

24

10

10

15

15

145

0

160

 

25

10

10

15

15

145

0

160

 

26

10

10

15

15

145

0

160

 

27

10

10

15

15

145

0

160

 

28

10

10

15

15

145

0

160

 

Flasks 1 and 2: Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene

Flasks 3 and 4: inoculum control

Flask 5: reference (procedure control)

Flask 6: abiotic control

Flask 7: toxicity control

Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThODNH4

Time

Percentage Biodegradation

(Days)

Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene1

Sodium Benzoate2

Toxicity
control1, 2

 

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

2

2

32

9

2

0

0

43

17

3

0

0

49

23

4

0

0

62

26

5

1

0

65

27

6

0

0

67

27

7

0

0

69

29

8

0

0

69

29

9

0

0

69

29

10

0

0

72

30

11

0

0

71

29

12

0

0

69

30

13

0

0

69

30

14

0

0

72

30

15

0

0

72

31

16

0

0

72

31

17

0

0

72

31

18

0

0

72

31

19

0

0

72

32

20

0

0

72

32

21

0

0

72

32

22

0

0

72

32

23

0

0

72

33

24

0

0

75

33

25

0

0

75

33

26

0

0

75

33

27

0

0

75

33

28 0 0 75 33

1ThODNH4of Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene: 2.553 mg O2/mg test item
2ThODNH4of sodium benzoate: 1.666 mg O2/mg reference item

pH-Values at the End of the Test

Flask No.

Treatment

pH-value

1

Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene

7.5

2

Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene

7.5

3

Inoculum control

7.4

4

Inoculum control

7.5

5

Reference item (procedure control)

7.6

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The degradation rate of Vetimoss never reached 10%. Therefore, Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene is considered to be not readily biodegradable.
Executive summary:

The ready biodegradation potential of Vetimoss was determined in a Manometric Respirometry Test according to OECD Technical Guideline 301F. The degradation rate of Vetimoss never reached 10%. Therefore, Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene is considered to be not readily biodegradable.

Description of key information

The ready biodegradation potential of Vetimoss was determined in a Manometric Respirometry Test according to OECD Technical Guideline 301F. The degradation rate of Vetimoss never reached 10%. Therefore, Vetimoss: 2-tert-butyl-1,4-dimethoxybenzene is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information