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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:
October 26, 2017 to November 23, 2017
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Name: 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol
Batch No.: C036K0111
Synonym: LOWINOX® TBP-6
Assay: 99.8%
Aggregate State at Room Temperature: Solid
Colour: White
Molecular Formula / Molecular Weight: C22H30O2S / 358.54 g/mol
Theoretical Oxygen Demand: ThODNH4: 2.678 mg oxygen per mg test item
Expiry (Retest) Date: March 29, 2018
Storage Conditions at Test Facility: At 20 +/-5 °C, in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
other: Aerobic activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the sewage treatment plant Rossdorf, Germany.
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:
In the aerobic activated sludge used for this study, coarse particles were removed by settling for a short period (30 minutes). Afterwards the sludge was 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 (see 6.5) and aerated overnight. This suspension was used for the experiment.

Test Units
Type and Size: Manometric Test System with test flasks containing a volume of approximately 500 mL.
Apparatus: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany
Principle: The test flasks prepare were incubated at 22°C +/- 1°C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 28 days using the BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.
Identification: Each test unit was uniquely identified with the study number, treatment and replicate number.

Test Conditions
Surrounding Type: Climatised chamber
Temperature: 22°C ± 1°C
Light Conditions: Darkness
pH-Value of Test Solutions: 7.2 to 7.7 (measured at the start of the test)
7.2 to 7.5 (adjusted at test start)
7.2 to 7.4 (measured at the end of the test)

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.

Course of the Test
Preparation of Test Flasks: The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used.
Incubation: The closed test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.
Duration of test (contact time):
28 d
Initial conc.:
103.3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not applicable
Test performance:
Validity Criteria of the Study
Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 30 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 7.4 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 7 days of incubation.
Test Item: The difference of duplicate values for the degradation of the test item at the end of the test 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 substance and a reference compound less than 25% biodegradation (based on ThOD) occurred within 14 days, the test substance can be assumed to be inhibitory. The biodegradation was 30% at day 14; the test item was not inhibitory.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Remarks on result:
other: Not readily biodegradable
Details on results:
Biodegradation of Test Item
Percentage Biodegradation:
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.
6,6’-di-tert-butyl-2,2’-thiodi-p-cresol did never reach a biodegradation of 10% or higher during the incubation time of 28 days. The mean biodegradation at test end after 28 days was 0% (ThODNH4).

The degradation rate of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol did never reach 60% within the test duration of 28 days. The mean biodegradation (ThODNH4) for the test substance after 28 days was 0%.
Therefore, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol is considered to be not readily biodegradable.

Biodegradation in the Toxicity Control
Percentage Biodegradation: In the toxicity control containing both, the test item and the reference item sodium benzoate, 30% biodegradation was noted within 14 days and 31% bio-degradation 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.
Conclusion: 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
Oxygen Demand:The oxygen demand in the abiotic control was 0 mg/L during the test duration (see Table 2). There was no use to correct the degradation of the test item and toxicity control.
Key result
Parameter:
ThOD
Value:
0 mg O2/g test mat.
Remarks on result:
other: Mean ThODNH4
Results with reference substance:
Biodegradation of Reference Item Sodium Benzoate
The reference item sodium benzoate was sufficiently degraded to 69% after 14 days and to 67% after 28 days of incubation The percentage biodegradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

Biodegradation in the Toxicity Control
the test item and the reference item sodium benzoate, 30% biodegradation was noted within 14 days and 31% 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.

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

0

0

0

5

45

0

45

 

2

5

5

5

5

70

0

65

 

3

5

10

5

5

80

0

75

 

4

5

15

5

5

90

0

90

 

5

5

15

5

5

105

0

105

 

6

5

15

10

10

110

0

125

 

7

10

15

10

10

120

0

135

 

8

10

15

10

10

125

0

145

 

9

10

15

10

10

125

0

150

 

10

10

20

10

15

130

0

150

 

11

15

20

15

15

130

0

150

 

12

15

20

15

15

130

0

150

 

13

15

20

15

15

130

0

150

 

14

15

20

15

15

135

0

150

 

15

15

20

15

15

135

0

155

 

16

20

20

15

20

135

0

155

 

17

20

25

20

20

135

0

155

 

18

20

25

20

20

140

0

155

 

19

20

25

20

20

140

0

160

 

20

20

25

20

20

140

0

160

 

21

20

25

20

20

140

0

160

 

22

25

25

25

25

140

0

165

 

23

25

25

25

25

140

0

165

 

24

25

25

25

25

140

0

165

 

25

25

25

25

25

140

0

165

 

26

25

25

25

25

140

0

165

 

27

30

25

30

30

145

0

170

 

28

30

25

30

30

145

0

170

 

            Flasks 1 and 2: 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

            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 Biodegradation1

(Days)

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol1

Sodium Benzoate2

Toxicity
control1, 2

 

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

0

0

25

10

2

0

0

38

13

3

0

2

43

16

4

0

4

49

19

5

0

4

58

22

6

0

2

58

26

7

0

2

64

28

8

0

2

67

30

9

0

2

67

31

10

0

3

68

31

11

0

2

67

30

12

0

2

67

30

13

0

2

67

30

14

0

2

69

30

15

0

2

69

31

16

1

1

68

31

17

0

2

67

30

18

0

2

69

30

19

0

2

69

31

20

0

2

69

31

21

0

2

69

31

22

0

0

67

31

23

0

0

67

31

24

0

0

67

31

25

0

0

67

31

26

0

0

67

31

27

0

0

67

31

28

0

0

67

31

            1ThODNH4of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol: 2.678 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

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

7.4

2

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

7.4

3

Inoculum control

7.2

4

Inoculum control

7.2

5

Reference item (procedure control)

7.2

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
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. 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol did never reach a biodegradation of 10% or higher during the incubation time of 28 days. The mean biodegradation at test end after 28 days was 0% (ThODNH4).
Therefore, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol is considered to be not readily biodegradable based on the mean biodegradation (ThODNH4) after 28 days was 0%.
Executive summary:

The purpose of this study was to determine the ready biodegradability of the test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol. The test item was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure con-trol. This type of study is recognized by the international test guidelines and should provide a rational basis to assess the ready biodegradation properties of the test item when incubated with aerobic activated sludge.

This study was designed to comply with the follow-ing methods:

-       Commission Regulation 440/2008/EC, Method C.4-D of May 30, 2008: Manometric Respirome-try Test (EEC Publication No. L 142/496, May 2008)

-       OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respi-rometry Test", adopted July 17, 1992

The reference item sodium benzoate was sufficiently degraded to 69% after 14 days and to 67% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 30% biodegradation was noted within 14 days and 31% 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

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. 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol did never reach a biodegradation of 10% or higher during the incubation time of 28 days. The mean biodegradation at test end after 28 days was 0% (ThODNH4).

Therefore, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol is considered to be not readily biodegradable based on the mean biodegradation (ThODNH4) after 28 days was 0%.

Description of key information

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol is not readily biodegradable. The mean biodegradation (ThODNH4) after 28 days was 0%

Key value for chemical safety assessment

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

Additional information

The purpose of this study was to determine the ready biodegradability of the test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol. The test item was exposed to aerobic activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure con-trol. This type of study is recognized by the international test guidelines and should provide a rational basis to assess the ready biodegradation properties of the test item when incubated with aerobic activated sludge.

This study was designed to comply with the follow-ing methods:

-       Commission Regulation 440/2008/EC, Method C.4-D of May 30, 2008: Manometric Respirome-try Test (EEC Publication No. L 142/496, May 2008)

-       OECD Guideline for Testing of Chemicals No. 301 F: "Ready Biodegradability: Manometric Respirometry Test", adopted July 17, 1992

The reference item sodium benzoate was sufficiently degraded to 69% after 14 days and to 67% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 30% biodegradation was noted within 14 days and 31% 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

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. 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol did never reach a biodegradation of 10% or higher during the incubation time of 28 days. The mean biodegradation at test end after 28 days was 0% (ThODNH4).

Therefore, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol is considered to be not readily biodegradable based on the mean biodegradation (ThODNH4) after 28 days was 0%.

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