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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:
20 May 2015 to 18 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None specified
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Source: The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Treatment: The freshly obtained sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was determined to be 4.1 g/l in the concentrated sludge. Before use, the sludge was allowed to settle (31 minutes) and the supernatant liquid was used as inoculum at the amount of 10 ml/l of mineral medium.

Reason for selection: The test has been accepted internationally for determining the 'ready' biodegradability of test substances under aerobic conditions.
Duration of test (contact time):
28 d
Initial conc.:
17.4 mg/L
Based on:
test mat.
Initial conc.:
12 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Test concentration and preparation of test solutions
1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] was a white powder with a purity of 99.0%. The test substance was tested in duplicate at a target concentration of 17.4 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula.
Since the test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components (test substance bottle A: 34.6 mg; test substance bottle B: 35.1 mg and toxicity control bottle: 35.0 mg). To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test. Furthermore, the test medium was daily swirled around to ensure optimal contact between the test substance and test medium, since the test substance tended to float on the water surface.

Reference substance concentration and preparation of test solutions
A solution of sodium acetate was prepared by dissolving 400.9 mg in Milli-RO water and making this up to a total volume of 100 ml. Volumes of 20 ml from this stock solution were added to 2 litres of the test medium of the positive control bottle and the toxicity control bottle, resulting in a final concentration of 40 mg sodium acetate per litre (12 mg TOC/l).

Test procedure and conditions
Test duration: 28 days (last CO2 measurement on day 29). During the test period, the test media were aerated and stirred continuously.
Test vessels: 2 litre glass brown coloured bottles.
Milli-RO water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
Stock solutions of mineral components:
A) 8.50 g KH2PO4 ; 21.75 g K2HPO4 ; 67.20 g Na2HPO4.12H2O; 0.50 g NH4Cl - dissolved in Milli-RO water and made up to 1 litre, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in Milli-RO water and made up to 1 litre.
C) 36.40 g CaCl2.2H2O dissolved in Milli-RO water and made up to 1 litre.
D) 0.25 g FeCl3.6H2O dissolved in Milli-RO water and made up to 1 litre.
Mineral medium: 1 litre mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and Milli-RO water.
Barium hydroxide: 0.0125 M Ba(OH)2 (Boom, Meppel, The Netherlands), stored in a sealed vessel to prevent absorption of CO2 from the air.
Synthetic air (CO2 < 1 ppm): A mixture of oxygen (ca. 20%) and nitrogen (ca. 80%) was passed through a bottle, containing 0.5 - 1 litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The synthetic air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
Illumination: The test media were excluded from light.

Preparation of bottles
Pre-incubation medium: The day before the start of the test (day -1) mineral components, Milli-RO water (ca. 80% of final volume) and inoculum (1% of final volume) were added to each bottle. This mixture was aerated with synthetic air overnight to purge the system of CO2.
Type and number of bottles:
Test suspension: containing test substance and inoculum (2 bottles).
Inoculum blank: containing only inoculum (2 bottles)
Positive control: containing reference substance and inoculum (1 bottle).
Toxicity control: containing test substance, reference substance and inoculum (1 bottle).
Preparation: At the start of the test (day 0), test and reference substance were added to the bottles containing the microbial organisms and mineral components.
The volumes of suspensions were made up to 2 litres with Milli-RO water, resulting in the mineral medium described before.
Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

Determination of CO2
Experimental CO2 production: The CO2 produced in each test bottle reacted with the barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by titrating the remaining Ba(OH)2 with 0.05 M standardized HCl (1:20 dilution from 1 M HCl (Titrisol® ampoule), Merck, Darmstadt, Germany).
Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until day 28, for the inoculum blank and test suspension. Titrations for the positive and toxicity control were made over a period of at least 14 days.
Each time the CO2-absorber nearest to the test bottle was removed for titration; each of the remaining two absorbers was moved one position in the direction of the test bottle. A new CO2-absorber was placed at the far end of the series. Phenolphthalein (1% solution in ethanol, Merck) was used as pH-indicator.
On day 28, the pH of all test suspensions was measured and 1 ml of concentrated HCl (37%, Merck) was added to the bottles of the inoculum blank and test suspension. The bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.
Theoretical CO2 production: The theoretical CO2 production was calculated from the molecular formula.

Measurements and recording
pH: At the start of the test (day 0) and on day 28, before addition of concentrated HCl. The pH of the toxicity control was not measured on day 28.
Temperature of medium: Continuously in a vessel with Milli-RO water in the same room.

Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme "Rees Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature."
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
> 0 - < 2
Sampling time:
28 d
Details on results:
Theoretical CO2 production
The ThCO2 of 1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] was calculated to be 2.53 mg CO2/mg.
The ThCO2 of sodium acetate was calculated to be 1.07 mg CO2/mg.

Biodegradation
The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of 1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] (0% and 2%, based on ThCO2).
In the toxicity control, more than 25% biodegradation occurred within 14 days (38%, based on ThCO2 of the test and positive control substances combined). Therefore, the test substance was assumed not to inhibit microbial activity.
Functioning of the test system was checked by testing the reference substance sodium acetate, which showed a normal biodegradation curve.

pH values of different test media

Test medium:

At the start of the test:

On day 28:

Blank control (A)

7.8 →17.6

7.7

Blank control (B)

7.8 →17.6

7.7

Positive control

7.8 →17.6

8.0

Test substance (A)

7.8 →17.6

7.7

Test substance (B)

7.8 →17.6

7.7

Toxicity control

7.8 →17.6

N.D.

1 Adjusted using 1 M HCI (Merck, Darmstadt, Germany)

N.D. Not determined.

 

NOTES: Except for the percentages biodegradation, all calculations are performed without rounding off. Produced CO2: negative values are expressed as 0.00 ml HCI.

 

HCI titrated in duplicate blank bottles

Day

HCI (0.05 N) titrated (ml)

Blank A

Blank B

Mean Value

2

5

7

9

14

19

23

27

29

29

29

45.27

43.50

44.61

41.39

44.42

42.91

41.33

42.36

44.01

47.56

48.76

45.72

44.73

45.38

45.67

43.85

43.70

42.94

42.80

45.30

48.99

49.32

45.50

44.12

45.00

43.53

44.14

43.31

42.14

42.58

44.66

48.28

49.04

 

HCI titrated in Ba(OH)2solution (background bottles)

Day

HCI (0.05 N) titrated (ml)

Bottle A

Bottle B

Mean Value

2

5

7

9

14

19

23

27

29

29

29

47.51

47.75

47.36

46.41

47.99

48.08

48.63

49.55

50.55

51.81

49.80

47.91

48.28

46.71

48.41

48.01

46.78

49.05

50.24

51.43

51.29

50.33

47.71

48.02

47.04

47.41

48.00

47.43

48.84

49.90

50.99

51.55

50.07

 

CO2production in the blank

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2

(mg)

Cumulative CO2

(mg)

Ba(OH)21)

Blank (mean)

2

5

7

9

14

19

23

27

29

29

29

47.71

48.02

47.04

47.41

48.00

47.43

48.84

49.90

50.99

51.55

50.07

45.50

44.12

45.00

43.53

44.14

43.31

42.14

43.58

44.66

48.28

49.04

2.21

3.90

2.04

3.88

3.86

4.13

6.71

7.32

6.33

3.27

1.03

2.4

4.3

2.2

4.3

4.3

4.5

7.4

8.0

7.0

3.6

1.1

2.4

6.7

9.0

13.2

17.5

22.0

29.4

37.5

44.4

48.0

49.1

1)“Strength” of untreated 0.0125 M Ba(OH)2solution

 

CO2production and percentage biodegradation of the positive control substance

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)(%)

Blank (mean)

Positive control

2

5

7

9

14

45.50

44.12

45.00

43.53

44.14

35.31

23.06

36.30

41.42

36.62

10.19

21.06

8.70

2.11

7.52

11.2

23.2

9.6

2.3

8.3

11.2

34.4

43.9

46.2

54.5

13

40

51

54

64

1)Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 85.8 mg CO2/2l

 

CO2production and percentage biodegradation of the test substance (bottle A)

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)(%)

Blank (mean)

Bottle A

2

5

7

9

14

19

23

27

29

29

29

45.50

44.12

45.00

43.53

44.14

43.31

42.14

42.58

44.66

48.28

49.04

46.17

45.45

46.13

46.25

43.27

44.11

45.41

43.65

46.94

47.30

49.98

0.00

0.00

0.00

0.00

0.87

0.00

0.00

0.00

0.00

0.98

0.00

0.0

0.0

0.0

0.0

1.0

0.0

0.0

0.0

0.0

1.1

0.0

0.0

0.0

0.0

0.0

1.0

1.0

1.0

1.0

1.0

2.0

2.0

0

0

0

0

1

1

1

1

1

2

2

1)Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 87.5 mg CO2/2l

 

CO2production and percentage biodegradation of the test substance (bottle B)

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)(%)

Blank (mean)

Bottle B

2

5

7

9

14

19

23

27

29

29

29

45.50

44.12

45.00

43.53

44.14

43.31

42.14

42.58

44.66

48.28

49.04

46.19

45.46

46.10

44.69

44.29

44.90

43.76

43.87

46.18

48.36

48.66

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.38

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.4

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.4

0

0

0

0

0

0

0

0

0

0

0

1)Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 88.8 mg CO2/2l

 

CO2production and percentage biodegradation of the toxicity control

Day

HCI (0.05 N) titrated (ml)

Produced CO2

(ml HCI)

Produced CO2

(mg)

Cumulative CO2

(mg)

Biodegradation1)(%)

Blank (mean)

Toxicity control

2

5

7

9

14

45.50

44.12

45.00

43.53

44.14

35.31

18.06

32.15

39.00

37.38

10.19

26.06

12.85

4.53

6.76

11.2

28.7

14.1

5.0

7.4

11.2

39.9

54.0

59.0

66.4

6

23

31

34

38

1)Calculated as the ratio between CO2produced (cumulative) and the sum of the ThCO2of the test substance and positive control: 174.3 mg CO2/2l (ThCO2test substance: 88.6 mg CO2/2l + ThCO2sodium acetate: 85.8 mg CO2/2l)

 

Comparison of biodegradation of the test substance in bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B1)

2

5

7

9

14

19

23

27

29

29

29

0

0

0

0

1

1

1

1

1

2

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

1

1

1

1

1

0

0

0

0

1

1

1

1

1

2

2

1)Absolute difference in biodegradation between bottles A and B

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] was designated as not readily biodegradable.
Executive summary:

Determination of ‘ready’ biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) of 1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat].

 

The study procedures described in this report were based on the OECD guideline No. 301 B, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-C, ISO 9439, 1999 and ISO 10634, 1995.

 

The test substance was a white powder with a purity of 99.0%. The test substance was tested in duplicate at a target concentration of 17.4 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula. The Theoretical CO2production (ThCO2) of the test substance was calculated to be 2.53 mg CO2/mg.

 

The study consisted of six bottles:

- 2 inoculum blanks (no test substance),

- 2 test bottles (test substance),

- 1 positive control (sodium acetate) and

- 1 toxicity control (test substance plus sodium acetate).

 

Since the test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 days (last CO2-measurement on day 29).

 

The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of 1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] (0% and 2%, based on ThCO2).

 

In the toxicity control, the test substance was found not to inhibit microbial activity.

 

Since all criteria for acceptability of the test were met, this study was considered to be valid.

 

In conclusion, 1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] was designated as not readily biodegradable.

Description of key information

Key value determined in GLP laboratory study in accordance with OECD Guideline 301 B and EU Method C.4-C.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Determination of ‘ready’ biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) of 1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat].

The test substance was tested in duplicate at a target concentration of 17.4 mg/l, corresponding to 12 mg TOC/l. The organic carbon content was based on the molecular formula. The Theoretical CO2production (ThCO2) of the test substance was calculated to be 2.53 mg CO2/mg.

The study consisted of six bottles:

- 2 inoculum blanks (no test substance),

- 2 test bottles (test substance),

- 1 positive control (sodium acetate) and

- 1 toxicity control (test substance plus sodium acetate).

Since the test substance was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 1 g/l, weighed amounts were added to the 2-litres test bottles containing medium with microbial organisms and mineral components. To this end, 10 ml of Milli-RO water was added to each weighing bottle containing the test substance. After vigorous mixing (vortex) the resulting suspension was added quantitatively to the test medium. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms. Test duration was 28 days (last CO2-measurement on day 29).

The relative biodegradation values calculated from the measurements performed during the test period revealed no significant biodegradation of 1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] (0% and 2%, based on ThCO2).

In the toxicity control, the test substance was found not to inhibit microbial activity. 

In conclusion, 1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] was designated as not readily biodegradable.