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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 November 2002 to 8 April 2003
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:
not specified
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
not specified
GLP compliance:
yes
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 oblained from a municipal sewage treatment plant: Waterschap de Maaskant','s-Hertogenbosch, the Netherlands.
Treatment: The sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 3.9 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (30-90 minutes) and the liquid decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
Reason for selection: The test has been accepted internationally (EEC, OECD) for determining the 'ready' biodegradability of test substances under aerobic conditions.
Duration of test (contact time):
28 d
Initial conc.:
35 mg/L
Based on:
ThIC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
PREPARATION OF TEST SOLUTIONS
The batch of HATCOL 3331 tested was a clear colourless liquid and hardly soluble in water. Based on the Total Carbon content (TC) of HATCOL 3331 (68.90%) the test substance was tested in duplicate at 35 mg per 2 litres, corresponding to 12 mg TC/l.
Weighed amounts of HATCOL 3331 (test substance bottle A and B: 35.1 mg and toxicity control bottle: 35.5 mg) were mixed with 10 ml of milli-RO water. After vigorous shaking the resulting suspension was added quantitatively to the test medium containing the microbial organisms. The test solutions were continuously stirred during the test, to ensure optimal contact between the lest substance and the test organisms.

TEST PROCEDURE AND CONDITIONS
Test duration: 28 days (last CO2-measurement on the 29th day). During the test period aeration and stirring took place.
Test vessels: 2 litre all-glass brown coloured bottles.
Milli-RO / Milli-Q water: Tap-water purified by reverse osmosis (MiIli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Mill-Q) (Millipore Corp.,
Bedford, Mass., USA).
Stock solutions of mineral components:
A) 8.50 g KH2PO4; 21.75 g K2HPO4; 67.20 g Na2HPO4.12H2O; 0.50 g NH4CI; dissolved in 1 I Mill-Q water, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H20 dissolved in 1 I Milli-Q water.
C) 36.40 g CaCI2.2H2O dissolved in 1 l Milli-Q water.
D) 0.25 g FeCl3.6H2O dissolved in 1 l MiIli-Q water.
Mineral medium: 11 mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and MilIi-RO water.
Barium hydroxide: 0.0125 M, stored in a sealed vessel to prevent absorption of CO2 from the air.
CO2-free air: A mixture of oxygen (21%) and nitrogen (79%) was led 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 CO2-free air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second (ca. 30-100 ml/min).
Test concentration: The test substance was tested in duplicate al 35 mg per 2 litres, corresponding to 12 mg TC/l. The carbon content was based on TC-analysis.

Preparation of bottles:
Pre-incubation medium: Mineral components, Milli-RO water (ca. 80% total volume) and inoculum (1 % final volume) were added to each bottle. This mixture was aerated with CO2-free 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 lest substance, reference substance and inoculum (1 bottle).
Preparation: The test substance and positive control were added to the bottles.
The volumes of suspensions were made up to 2 litres with MiIli-RO water, resulting in the mineral medium described before. Three CO2-absorbers (bottles filled with 100 ml 0.0125 M Ba(OH2) 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 HCI.
Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. Each lime 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 was used as pH-indicator. On the 28th day, the pH of the lest suspensions was measured and 1 ml of concentrated HCI was added to each bottle. 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: Because the theoretical calculation of the CO2 production was not possible and the test substance was insoluble in water, a sample of pure test substance was taken for determination of TC. TC analysis was performed at the Chemical Laboratory "Dr. A. Verwey", Rotterdam, the Netherlands using a Vario-analyzer. (Method: Vario EI AV/29.007, 1999). TC analysis was not performed under GLP conditions.
Reference substance:
other: sodium acetate
Key result
Parameter:
% degradation (CO2 evolution)
Value:
>= 18 - <= 22
Sampling time:
28 d
Details on results:
Theoretical CO2 production
The Total Carbon content (TC) of HATCOL 3331 was determined to be 68.90%.
Based on the TC content the Theoretical CO2 production (ThC02) of HATCOL 3331 was calculated to be 2.53 mg CO2/mg.
The concentration was 35.1 mg (for both bottles) HATCOL 3331 in 211tres lest medium. Hence, the theoretical CO2 production following complete degradation was 88.8 mg per 2 litres for both bottles.

Biodegradation
The relative degradation values calculated from the measurements performed during the test period revealed 22 and 18% degradation of HATCOL 3331 for bottle A and B respectively.
Since biodegradation of HATCOL 3331 of at least 60% was not reached within 10 days after exceeding 10%, the criterion for ready biodegradability was not met.
Results with reference substance:
Theoretical CO2 production
The positive control contained 80.0 mg sodium acetate (ThCO2= 1.07 mg CO2/mg) resulting in a theoretical CO2 production following complete degradation of 85.6 mg per 2 litres.
The toxicity control contained 80.0 mg sodium acetate and 35.5 mg HATCOL 3331 in 2 litres of test medium. Hence, the theoretical CO2 production following complete degradation of HATCOL 3331 plus sodium acetate was 175.4 mg per 2 litres.

Biodegradation
In the toxicity control more than 25% degradation occurred within 14 days (38%, based on ThCO2). Therefore, the test substance was assumed to be not inhibitory on microbial activity.

Monitoring of temperature and pH

The temperature recorded in a vessel with water in the same room varied between 21.6 and 23.4°C

The pH values of the different test media were:

 

Just before the start of the test:

On day 28:

Blank control (A)

7.5

7.3

Blank control (B)

7.5

7.3

Positive control

7.4

7.6

HATCOL 3331 (A)

7.4

7.3

HATCOL 3331 (B)

7.3

7.3

Toxicity control

7.4

7.6

 

Table 1: 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)

Degradation1)(%)

Blank (mean)

Positive control

0

-

-

-

-

-

0

2

43.47

40.29

3.18

3.5

3.5

4

5

43.77

20.96

22.81

25.1

28.6

33

7

45.27

33.74

11.53

12.7

41.3

48

9

46.38

38.63

7.75

8.5

49.8

58

14

45.36

32.80

12.56

13.8

63.6

74

19

44.58

38.35

6.23

6.9

70.4

82

23

43.11

39.22

3.89

4.3

74.7

87

27

42.63

39.69

2.94

3.2

78.0

91

29

43.77

40.95

2.82

3.1

81.1

95

29

45.00

44.32

0.68

0.7

81.8

96

29

45.22

44.75

0.47

0.5

82.3

96

1)Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 85.6 mg CO2/2l

 

Table 2a: 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)

Degradation1)(%)

Blank (mean)

Positive control

0

-

-

-

-

-

0

2

43.47

39.89

3.58

3.9

3.9

4

5

43.77

39.57

4.20

4.6

8.6

10

7

45.27

41.92

3.35

3.7

12.2

14

9

46.38

44.62

1.76

1.9

14.2

16

14

45.36

43.13

2.23

2.4

16.6

19

19

44.58

43.41

1.17

1.3

17.9

20

23

43.11

43.35

0.00

0.0

17.9

20

27

42.63

42.53

0.10

0.1

18.0

20

29

43.77

42.69

1.08

1.2

19.2

22

29

45.00

44.34

0.66

0.7

19.9

22

29

45.22

49.28

0.00

0.0

19.9

22

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

 

Table 2b: 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)

Degradation1)(%)

Blank (mean)

Positive control

0

-

-

-

-

-

0

2

43.47

45.03

0.00

0.0

0.0

0

5

43.77

41.95

1.82

2.0

2.0

2

7

45.27

44.61

0.66

0.7

2.7

3

9

46.38

45.11

1.27

1.4

4.1

5

14

45.36

40.23

5.13

5.6

9.7

11

19

44.58

41.78

2.80

3.1

12.8

14

23

43.11

42.02

1.09

1.2

14.0

16

27

42.63

41.49

1.14

1.3

15.3

17

29

43.77

43.26

0.51

0.6

15.8

18

29

45.00

44.81

0.19

0.2

16.0

18

29

45.22

49.50

0.00

0.0

16.0

18

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

 

Table 2c: Comparison of biodegradation of the test substance in bottles A and B

Day

Biodegradation (%)

Bottle A

Bottle B

Mean A and B

∆ A-B1)

0

0

0

0

0

2

4

0

2

4

5

10

2

6

7

7

14

3

8

11

9

16

5

10

11

14

19

11

15

8

19

20

14

17

6

23

20

16

18

4

27

20

17

19

3

29

22

18

20

4

29

22

18

20

4

29

22

18

20

4

1)Absolute difference in biodegradation between bottle A and B

 

Table 3: 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)

Degradation1)(%)

Blank (mean)

Toxicity control

0

-

-

-

-

-

0

2

43.47

47.99

0.00

0.0

0.0

0

5

43.77

19.16

24.61

27.1

27.1

15

7

45.27

33.29

11.98

13.2

40.2

23

9

46.38

36.18

10.20

11.2

51.5

29

14

45.36

31.89

13.47

14.8

66.3

38

19

44.58

37.74

6.84

7.5

73.8

42

23

43.11

40.46

2.65

2.9

76.7

44

27

42.63

40.78

1.85

2.0

78.7

45

29

43.77

39.99

3.78

4.2

82.9

47

29

45.00

44.98

0.02

0.0

82.9

47

29

45.22

49.88

0.00

0.0

82.9

47

1)Calculated as the ratio between CO2 produced (cumulative) and the sum of the ThCO2 of the test substance and positive control: 175.4 mg CO2/2l; ThCO2 test substance: 89.8 mg CO2/2l; ThCO2 sodium acetate: 85.6 mg CO2/2l

 

Table 4: CO2produced 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)

0

-

-

-

-

0.0

2

49.44

43.47

5.97

6.6

6.6

5

49.17

43.77

5.40

5.9

12.5

7

49.43

45.27

4.16

4.6

17.1

9

49.23

46.38

2.85

3.1

20.2

14

49.37

45.36

4.01

4.4

24.6

19

49.66

44.58

5.08

5.6

30.2

23

48.80

43.11

5.69

6.3

36.5

27

47.82

42.63

5.19

5.7

42.2

29

48.55

43.77

4.79

5.3

47.4

29

44.96

45.00

0.00

0.0

47.4

29

46.64

45.22

1.43

1.6

49.0

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

Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily biodegradable
Conclusions:
HATCOL 3331 was not readily biodegradable under the conditions of the modified Sturm test presently performed.
Executive summary:

Determination of 'ready' biodegradability: carbon dioxide (C02) evolution test (modified Sturmtest) with HATCOL 3331.

The study procedure was based on EEC directive 92/69, C4-C, December 1992, and OECD guideline No. 301 B July 17, 1992.

The batch of HATCOL 3331 tested was a clear colourless liquid and hardly soluble in water.

The Total Carbon content (TC) of HATCOL 3331 was determined to be 68.90%. Based on the TC content the Theoretical CO2 production (ThCO2) of HATCOL 3331 was calculated to be 2.53 mg CO2/mg. HATCOL 3331 was tested for its ready biodegradability at 35 mg per 2 litres, corresponding to 12 mg TC/I.

The study consisted of six bottles:

2 blank controls (no test material), 2 test bottles (HATCOL 3331, 17.5 mg/l), 1 positive control (sodium acetate, 40 mg/l) and 1 toxicity control (HATCOL 3331, 17.5 mg/l; plus sodium acetate, 40 mg/l).

Weighed amounts of HATCOL 3331 were mixed with 10 ml of milli-RO water and after vigorous shaking the resulting suspension was added quantitatively to the test medium containing the microbial organisms. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

The relative degradation values calculated from the measurements performed during the test period revealed 22 and 18% degradation of HATCOL 3331 for bottle A and B respectively.

Since biodegradation of HATCOL 3331 of at least 60% was not reached within 10 days after exceeding 10%, the criterion for ready biodegradability was not met.

In the toxicity control HATCOL 3331 was found not to inhibit microbial activity.

Since all acceptability criteria prescribed by the protocol were met, this study was considered to be valid.

In conclusion, HATCOL 3331 was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 November 2002 to 08 April 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
not specified
GLP compliance:
yes
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 de Maaskant, 's-Hertogenbosch, the Netherlands.
Treatment: The sludge was kept under continuous aeration until further treatment. The concentration of suspended solids was 3.9 g/l in the concentrated sludge (information obtained from the municipal sewage treatment plant). Before use, the sludge was allowed to settle (30-90 minutes) and the liquid decanted for use as inoculum at the amount of 10 ml/l of mineral medium.
Reason for selection: The test has been accepted internationally (EEC, OECD) for determining the 'ready' biodegradability of test substances under aerobic conditions.
Duration of test (contact time):
28 d
Initial conc.:
35 - 36 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST PROCEDURE AND CONDITIONS
Test duration: 28 days (last CO2-measurement on the 29th day). During the test period aeration and stirring took place.
Test vessels: 2 litre all-glass brown coloured bottles.
Milli-RO/I Milli-Q water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion exchange cartridges (Mill-Q) (Millipore Corp., Bedford, Mass., USA).
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 1l MilIi-Q water, pH 7.4 ± 0.2
B) 22.50 g MgSO4.7H2O dissolved in 1I Milli-Q water.
C) 36.40 g CaCI2.2H2O dissolved in 1I Milli-Q water.
D) 0.25 g FeCI3.6H2O dissolved in 1I Milli-Q water.
Mineral medium: 1I mineral medium contains: 10 ml of solution (A), 1 ml of solutions (B) to (D) and MiIli-RO water
Barium hydroxide: 0.0125 M, stored in a sealed vessel to prevent absorption of CO2 from the air.
CO2-free air: A mixture of oxygen (21%) and nitrogen (79%) was led through a bottle, containing 0.5 -1litre 0.0125 M Ba(OH)2 solution to trap CO2 which might be present in small amounts. The CO2-free air was sparged through the scrubbing solutions at a rate of approximately 1-2 bubbles per second
(ca. 30-100 ml/min).
Test concentration: The test substance was tested in duplicate at 35-36 mg per 2 litres, corresponding to 12 mg TC/l. The carbon content was based on TC-analysis.

Preparation of bottles:
Pre-incubation medium: Mineral components. MilIi-RO water (ca. 80% total volume) and inoculum (1% final volume) were added to each bottle. This mixture was aerated with CO2-free 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: The test substance and positive control were added to the bottles. The volumes of suspensions were made up to 2 litres with MilIi-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.
Measurements: Titrations were made every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day. 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 was used as pH-indicator. On the 28th day, the pH of the test suspensions was measured and 1 ml of concentrated HCI was added to each bottle. 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: Because the theoretical calculation of the CO2 production was not possible and the test substance was insoluble in water, a sample of pure test substance was taken for determination of TC. TC analysis was performed at the Chemical Laboratory "Dr. A. Verwey", Rotterdam, the Netherlands using a Varia-analyzer. (Method: Varia EI AV/29.007, 1999). TC analysis was not performed under GLP conditions.
Reference substance:
acetic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
61 - 67
Sampling time:
28 d
Remarks on result:
other: 60% not reached within the 10day window
Details on results:
Theoretical CO2 production
The Total Carbon content (TC) of HATCOL 5236 was determined to be 65.97%.
Based on the TC content the Theoretical CO2 production (ThCO2) of HATCOL 5236 was calculated to be 2.42 mg CO2/mg.
The concentration was 35.8 (A) and 35.0 mg (B) HATCOL 5236 in 2 litres test medium. Hence, the theoretical CO2 production following complete degradation was 86.6 mg per 2 litres for A and 84.7 mg per 2litres for B.
The positive control contained 80.0 mg sodium acetate (ThCO2= 1.07 mg CO2/mg) resulting in a theoretical CO2 production following complete degradation of 85.6 mg per 2 litres.
The toxicity control contained 80.0 mg sodium acetate and 36.4 mg HATCOL 5236 in 2 litres of test medium. Hence, the theoretical CO2 production following complete degradation of HATCOL 5236 plus sodium acetate was 173.7 mg per 2litres.

Biodegradation
The relative degradation values calculated from the measurements performed during the test period revealed 67 and 61 % degradation of HATCOL 5236 for bottle A and B respectively.
However, biodegradation of HATCOL 5236 of at least 60% was not reached within the 10-day window. Thus, the criterion for ready biodegradability was not met.

Monitoring of temperature
The temperature recorded in a vessel with water in the same room varied between 21.6 and 23.4°C.
Results with reference substance:
In the toxicity control more than 25% degradation occurred within 14 days (46%, based on ThCO2). Therefore, the test substance was assumed to be not inhibitory on microbial activity.

Monitoring of pH

The pH values of the different test media were:

 

Just before the start of the test:

On day 28:

Blank control (A)

7.5

7.3

Blank control (B)

7.5

7.3

Positive control

7.4

7.6

HATCOL 5236 (A)

7.5

7.3

HATCOL 5236 (B)

7.5

7.3

Toxicity control

7.4

7.7

 

Note: All calculations were performed without rounding off.

 

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)

Degradation1)(%)

Blank (mean)

Positive control

0

-

-

-

-

-

0

2

43.47

40.29

3.18

3.5

3.5

4

5

43.77

20.96

22.81

25.1

28.6

33

7

45.27

33.74

11.53

12.7

41.3

48

9

46.38

38.63

7.75

8.5

49.8

58

14

45.36

32.80

12.56

13.8

63.6

74

19

44.58

38.35

6.23

6.9

70.4

82

23

43.11

39.22

3.89

4.3

74.7

87

27

42.63

39.69

2.94

3.2

78.0

91

29

43.77

40.95

2.82

3.1

81.1

95

29

45.00

44.32

0.68

0.7

81.8

96

29

45.22

44.75

0.47

0.5

82.3

96

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of sodium acetate: 85.6 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)

Degradation1)(%)

Blank (mean)

Positive control

0

-

-

-

-

-

0

2

43.47

41.23

2.34

2.6

2.6

3

5

43.77

32.88

10.89

12.0

14.5

17

7

45.27

34.83

10.44

11.5

26.0

30

9

46.38

35.94

10.44

11.5

37.5

43

14

45.36

34.65

10.71

11.8

49.3

57

19

44.58

39.80

4.78

5.3

54.5

65

23

43.11

41.46

1.65

1.8

56.4

65

27

42.63

42.02

0.61

0.7

57.0

66

29

43.77

43.15

0.62

0.7

57.7

67

29

45.00

45.22

0.00

0.0

57.7

67

29

45.22

49.63

0.00

0.0

57.7

67

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 86.6 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)

Degradation1)(%)

Blank (mean)

Positive control

0

-

-

-

-

-

0

2

43.47

43.03

0.44

0.5

0.5

1

5

43.77

37.45

6.32

6.9

7.4

9

7

45.27

39.15

6.12

6.7

14.2

17

9

46.38

38.26

8.12

8.9

23.1

27

14

45.36

32.34

13.02

14.3

37.4

44

19

44.58

39.26

5.32

5.9

48.3

51

23

43.11

41.70

1.41

1.6

44.8

53

27

42.63

42.48

0.15

0.2

45.0

53

29

43.77

37.57

6.20

6.8

51.8

61

29

45.00

45.24

0.00

0.0

51.8

61

29

45.22

50.00

0.00

0.0

51.8

61

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance: 84.7 mg CO2/2l

 

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

Day

Biodegradation (%)

 

Bottle A

Bottle B

Mean A and B

∆ A-B1)

0

0

0

0

0

2

3

1

2

2

5

17

9

13

8

7

30

17

23

13

9

43

27

35

16

14

57

44

51

13

19

63

51

57

12

23

65

53

59

12

27

66

53

59

13

29

67

61

64

5

29

67

61

64

5

29

67

61

64

5

1): Absolute difference in biodegradation between bottles A and B.

 

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)

Degradation1)(%)

Blank (mean)

Positive control

0

-

-

-

-

-

0

2

43.47

41.24

2.23

2.5

2.5

1

5

43.77

21.48

22.29

24.5

27.0

18

7

45.27

29.25

16.02

17.6

44.6

26

9

46.38

30.39

15.99

17.6

62.2

35

14

45.36

28.96

16.40

18.0

80.2

46

19

44.58

31.47

13.11

14.4

94.6

64

23

43.11

31.60

11.51

12.7

107.3

62

27

42.63

37.32

5.31

5.8

113.1

65

29

43.77

42.74

1.03

1.1

114.3

66

29

45.00

43.72

1.28

1.4

115.7

67

29

45.22

48.36

0.00

0.0

115.7

67

1): Calculated as the ratio between CO2produced (cumulative) and the ThCO2of the test substance and positive control: 173.7 mg CO2/2l

ThCO2test substance: 88.1 mg CO2/2l

ThCO2sodium acetate: 85.6 mg CO2/2l

 

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)

0

-

-

-

-

0.0

2

49.44

43.47

5.97

6.6

6.6

5

49.17

43.77

5.40

5.9

12.5

7

49.43

45.27

4.16

4.6

17.1

9

49.23

46.38

2.85

3.1

20.2

14

49.37

45.36

4.01

4.4

24.6

19

49.66

44.58

5.08

5.6

30.2

23

48.80

43.11

5.69

6.3

36.5

27

47.82

42.63

5.19

5.7

42.2

29

48.55

43.77

4.79

5.3

47.4

29

44.95

45.00

0.00

0.0

47.4

29

46.64

45.22

1.43

1.6

49.0

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

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
HATCOL 5236 was degraded significantly (67 and 61 %) during the test period. However, since at least 60% biodegradation was not reached within the 10-day window, the criterion for ready biodegradability was not met. Thus, under the conditions of this test HATCOL 5236 was not readily biodegradable.
Executive summary:

Determination of 'ready' biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with HATCOL 5236.

The study procedure was based on EEC directive 92/69, C.4-C, December 1992, and OECD guideline No. 301 B July 17, 1992.

The batch of HATCOL 5236 tested was a clear pale yellow liquid and was considered hardly soluble in water. The Total Carbon content (TC) of HATCOL 5236 was determined to be 65.97%. Based on this TC content the Theoretical CO2 production (ThCO2) of HATCOL 5236 was calculated to be 2.42 mg CO2/mg.

HATCOL 5236 was tested for its ready biodegradability at 35-36 mg per 21itres, corresponding to 12 mg TC/l.

 

The study consisted of six bottles:

2 blank controls (no test material),

2 test bottles (HATCOL 5236, 18 mg/l),

1 positive control (sodium acetate. 40 mg/l) and

1 toxicity control (sodium acetate, 40 mg/l); plus HATCOL 5236, 18 mg/l.

 

Weighed amounts of HATCOL 5236 (test substance bottle A: 35.8 mg; test substance bottle B: 35.0 mg and toxicitycontrol bottle: 36.4 mg) were mixed with 10 ml of milil-RO water. After vigorous shaking the resulting suspension was added quantitatively to the test medium containing the microbial organisms. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

 

The relative degradation values calculated from the measurements performed during the test period revealed 67 and 61% degradation of HATCOL 5236 for bottle A and B respectively. However, biodegradation of HATCOL 5236 of at least 60% was not reached within the 10 day window. Thus, the criterion for ready biodegradability was not met. In the toxicity control HATCOL 5236 was found not to inhibit microbial activity.

 

Since all acceptability criteria prescribed by the protocol were met, this study was considered to be valid.

 

In conclusion, HATCOL 5236 was not readily biodegradable under the conditions of the modified Sturm test presently performed.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (no purity stated)
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
not specified
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge from the aeration basin of the domestic sewage treatment plant in Lachen-Speyerdorf, Germany (2008-05-08)- Preparation of inoculum for exposure: The sludge was aerated continually, washed with and re-suspended in mineral medium.
Duration of test (contact time):
29 d
Initial conc.:
29.7 mg/L
Based on:
test mat.
Initial conc.:
31.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS- Composition of medium: Mineral medium according to OECD guideline- Test temperature: 22 ± 1°C- Aeration of dilution water: Before adding test and reference item, all flasks were aerated for > 24 h with purified, CO2-free, moistened air.- Suspended solids concentration: 25 mg/LTEST SYSTEM- Culturing apparatus: 2000 mL-SCHOTT-flasks containing 1500 mL- Number of culture flasks/concentration: 2- Method used to create aerobic conditions: Aeration with purified, CO2-scribbed, moistened air. The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5-m-NaOH.- Measuring equipment: TOC multi N/C 2100S, Analytik Jena for IC measurement- Details of trap for CO2 and volatile organics if used: The emitted CO2 was trapped in 0.25-m-NaOH. Two scrubber containing 100 mL each were connected in series to the test vessels. SAMPLING- Sampling frequency: on day 0, 2, 4, 7, 9, 11, 14, 17, 23 and 29- Sampling method: On day 28, 5 mL HCl 2-m were added to all flasks in order to end the test and to drive off dissolved CO2 into the absorber flasks. Analyses of the emitted CO2 were made by IC measurement of samples from the front scrubber flask. Sample volume was 1 mL. Each sample was measured at least in duplicate. On day 29 samples from both absorber bottles were taken. CONTROL AND BLANK SYSTEM- Inoculum blank: Yes, 2 replicates- Abiotic control: Yes, 1 replicate- Toxicity control: Yes, 1 replicate
Reference substance:
aniline
Remarks:
26.8 mg/L
Key result
Parameter:
% degradation (CO2 evolution)
Value:
85.3
Sampling time:
29 d
Details on results:
Within the 10-day time window the 60% level was passed. Therefore, the test substance is regarded as readily biodegradable.
Results with reference substance:
Degradation: 83.5% after 7 days, 60% pass level was reached.

Table 1: Degradation (%) of reference substance, test substance, abiotic control and toxicity control

Day

Reference 1

Reference 2

Reference Mean

Test 1

Test 2

Test Mean

Abiotoc control

Toxicity control

2

-0.1

-0.1

-0.1

1.5

2.0

1.7

0.2

1.3

4

56.0

45.3

50.6

24.6

21.4

23.0

0.2

41.8

7

86.5

80.5

83.5

45.0

51.9

48.5

0.5

65.9

9

89.8

82.5

86.2

57.9

56.2

57.0

-0.2

70.4

11

89.5

89.2

89.4

64.0

61.2

62.6

-0.7

74.4

14

88.9

91.0

89.9

67.9

63.8

65.8

0.3

80.4

17

88.4

86.5

87.4

74.8

65.8

70.3

-0.7

77.2

23

90.1

88.1

89.1

79.7

77.1

78.4

-2.5

84.3

29

89.3

90.6

89.9

84.1

86.4

85.3

-1.1

87.9

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Jan - 20 Feb 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study. For read-across, maximum reliability score is 2.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge from the aeration tank of a municipal waste water treatment plant, ARA Werdhölzli, 8048 Zürich, Switzerland (22 January 2007)- Pretreatment: The sludge was washed twice with tap water.
Duration of test (contact time):
28 d
Initial conc.:
20.7 - 20.9 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS- Composition of medium: Aerobic mineral salts medium prepared with deionised water- Test temperature: 22 ± 0.5 °C- pH adjusted: Yes, the pH-value was checked at the beginning and at the end of the test and adjusted to pH 7.4 (± 0.2) with NaOH or HCL.- Suspended solids concentration: 30 mg/L- Continuous darkness: Yes, temperature-controlled dark roomTEST SYSTEM- Culturing apparatus: 2500 mL closed glass bottle containing a total volume of test solution of 2000 mL- Number of culture flasks/concentration: 2/concentration- Method used to create aerobic conditions: Aerated with synthetic CO2-free air for a maximum test period of 28 days.- Measuring equipment: Shimadzu 5050 TOC-Analyzer using the NPOC-mode for DOC-determination- Details of trap for CO2: The air leaving the individual vessels was passed through gas-absorption bottles filled with NaOH.SAMPLING- Sampling frequency: on day 0, 1, 3, 8, 10, 14, 17, 21, 24 and 28- Sampling method: Samples were centrifuged (15 min at 4500g) and acidified to pH<2. Prior to analysis the samples were sparged with CO2-free high purity air for 10 min to remove inorganic carbon.CONTROL AND BLANK SYSTEM- Inoculum blank: 2
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (CO2 evolution)
Value:
72
Sampling time:
28 d
Results with reference substance:
The reference substance was degraded to 90.6% at day 14 and to 87.9% at day 28.

The test substance is readily biodegradable under conditions tested. The biodegradation was > 60% after 28 days and the

10 -day-window was fulfilled, because the biodegradation reached 61% at the end of the 10 -day-window.

Table 1: Percentage biodegradation (mean of two replicates) of test and reference substance

Day

Test

substance

(% degradation)

Reference substance

(% degradation)

0

0.0

0.0

1

1.9

11.2

3

4.7

48.8

8

39.1

75.1

10

48.5

78.2

14

61.7

90.6

17

67.7

90.1

21

74.7

88.1

24

72.8

88.4

28

72.0

87.9

 

Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
06.06.-04.07.2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: From the aeration tank of a municipal sewage treatment plant, ARA Werdhölzli, 8048 Zürich, Switzerland
- Pretreatment: No
Duration of test (contact time):
28 d
Initial conc.:
22.1 mg/L
Based on:
other: TOC
Initial conc.:
21.8 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Aerobic mineral salts medium, prepared with deionised water
- Test temperature: 22.0 ± 0.5 °C
- pH: 7.4 ± 0.2
- pH adjusted: yes with NaOH or H2SO4
- Aeration of dilution water: with CO2-free air
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: 2500 mL glass bottles containing 2000 mL test solution;
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aerated with CO2-free air and fitted to gas-absorbtion bottles containing 125 ml of 0.15 M NaOH
- Measuring equipment: Shimadzu 5050 TOC-Analyzer using the NPOC-mode

SAMPLING
- Sampling frequency: Day 0, 1, 3, 7, 10, 14, 17, 21, 24 and 28

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
Reference substance:
benzoic acid, sodium salt
Remarks:
20.0 mg/L (TOC)
Key result
Parameter:
% degradation (CO2 evolution)
Value:
96
Sampling time:
28 d
Results with reference substance:
The reference substance was degraded to 96.9% by 14 day.

The test substance is readily biodegradable under conditions tested. The biodegradation was > 60% within 28 days and reached 84% at the end of the 10 -day-window.

Table 1: Percentage biodegradation of test and reference substance

Day

Test

substance

(% degradation)

Reference substance

(% degradation)

0

 0.0

0.0

1

0.6

4.6

3

5.3

45.2

7

52.1

81.2

10

71.5

92.8

14

86.6

96.7

17

91.7

97.6

21

93.1

98.0

24

96.3

97.8

28

96.0

96.9

 

Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
03.11.-01.12.2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge from the aeration basin of the domestic sewage treatment plant in 67435 Neustadt/Weinstraße, Lachen-Speyerdorf, Germany
- Preparation of inoculum for exposure: aerated continually, washed with and re-suspended in mineral medium
Duration of test (contact time):
28 d
Initial conc.:
25.9 mg/L
Based on:
test mat.
Initial conc.:
24.8 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium as described in OECD guideline
- Test temperature: 22 ± 1°C
- Suspended solids concentration: 25,1 mg/l

TEST SYSTEM
- Culturing apparatus: 2000 mL Schott flasks
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aerated with purified CO2 scrubbed, moistened air (bubbling the purified air through a flask containing 1.5M NaOH)
- Details of trap for CO2 and volatile organics if used: trapped in 0.25 M NaOH (scrubber containing 100 mL)

SAMPLING
- Sampling frequency: Day 0, 2, 4, 7, 9, 11, 14, 18, 23 and 29
- Sampling method: Analyses of the emitted CO2 were made by IC measurement of samples from the front scrubber flask using a TOC multi N/C
2100S, Analytik Jena. Sample volume was 1 ml. On day 29, samples from both absorber bottles were taken.
- Other: On day 28, 5 mL HCl2 were added to all flasks to drive off dissolved CO2 into the absorber flask.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 replicates
- Toxicity control: 1 replicate
- other: Abiotic control: 1 poisoned flask containing test item without inoculum

Reference substance:
aniline
Remarks:
25.2 mg/l
Key result
Parameter:
% degradation (CO2 evolution)
Value:
78
Sampling time:
28 d
Results with reference substance:
The reference substance was degraded to 100% by day 14.

The test substance is readily biodegradable under conditions tested. The biodegradation was 78% after 28 days and the 10-day-window was fulfilled. The 10 -day-window began on day 3 and at its end 63% were reached.

The IC content of test item in medium was higher then demanded in the guideline. This due to the fact of the test item is very poorly soluble in water. But the measured total carbon content corresponds to 4,61% of nominal TC. Therefore the result is considered valid.

Table 1: Percentage biodegradation of test substance, positive control, abiotic control and toxicity control

Day

Test

substance

(% degradation)

Positive

control

(% degradation)

Abiotic control

(% degradation)

Toxicity control

(% degradation)

2

4.6

4.3

0.9

2.6

4

22.4

34.7

0.5

27.3

7

44.4

62.7

-0.6

43.4

9

49.7

75.5

-1.2

58.6

11

58.8

77.7

-1.9

62.4

14

68.2

91.4

-2.5

83.1

18

75.9

96.2

-3.7

82.7

23

71.9

83.9

-4.4

73.3

29

78.4

75.9

-4.3

80.7

Interpretation of results:
readily biodegradable

Description of key information

Key value determined in a modified Sturm test performed to OECD guideline 301B and EU test standard C4-C.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

HATCOL 3331

Determination of 'ready' biodegradability: carbon dioxide (C02) evolution test (modified Sturm test) with HATCOL 3331.

The batch of HATCOL 3331 tested was a clear colourless liquid and hardly soluble in water.

The Total Carbon content (TC) of HATCOL 3331 was determined to be 68.90%. Based on the TC content the Theoretical CO2production (ThCO2) of HATCOL 3331 was calculated to be 2.53 mg CO2/mg. HATCOL 3331 was tested for its ready biodegradability at 35 mg per 2 litres, corresponding to 12 mg TC/I.

The study consisted of six bottles:

2 blank controls (no test material), 2 test bottles (HATCOL 3331, 17.5 mg/l), 1 positive control (sodium acetate, 40 mg/l) and 1 toxicity control (HATCOL 3331, 17.5 mg/l; plus sodium acetate, 40 mg/l).

Weighed amounts of HATCOL 3331 were mixed with 10 ml of milli-RO water and after vigorous shaking the resulting suspension was added quantitatively to the test medium containing the microbial organisms. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

The relative degradation values calculated from the measurements performed during the test period revealed 22 and 18% degradation of HATCOL 3331 for bottle A and B respectively.

Since biodegradation of HATCOL 3331 of at least 60% was not reached within 10 days after exceeding 10%, the criterion for ready biodegradability was not met.

In the toxicity control HATCOL 3331 was found not to inhibit microbial activity.

Since all acceptability criteria prescribed by the protocol were met, this study was considered to be valid.

In conclusion, HATCOL 3331 was not readily biodegradable under the conditions of the modified Sturm test presently performed.

 

HATCOL 5236

Determination of 'ready' biodegradability: carbon dioxide (CO2) evolution test (modified Sturm test) with HATCOL 5236.

The batch of HATCOL 5236 tested was a clear pale yellow liquid and was considered hardly soluble in water. The Total Carbon content (TC) of HATCOL 5236 was determined to be 65.97%. Based on this TC content the Theoretical CO2production (ThCO2) of HATCOL 5236 was calculated to be 2.42 mg CO2/mg.

HATCOL 5236 was tested for its ready biodegradability at 35-36 mg per 21itres, corresponding to 12 mg TC/l.

The study consisted of six bottles:

2 blank controls (no test material); 2 test bottles (HATCOL 5236, 18 mg/l); 1 positive control (sodium acetate. 40 mg/l) and 1 toxicity control (sodium acetate, 40 mg/l); plus HATCOL 5236, 18 mg/l.

Weighed amounts of HATCOL 5236 (test substance bottle A: 35.8 mg; test substance bottle B: 35.0 mg and toxicity control bottle: 36.4 mg) were mixed with 10 ml of milli-RO water. After vigorous shaking the resulting suspension was added quantitatively to the test medium containing the microbial organisms. The test solutions were continuously stirred during the test, to ensure optimal contact between the test substance and the test organisms.

The relative degradation values calculated from the measurements performed during the test period revealed 67 and 61% degradation of HATCOL 5236 for bottle A and B respectively. However, biodegradation of HATCOL 5236 of at least 60% was not reached within the 10 day window. Thus, the criterion for ready biodegradability was not met. In the toxicity control HATCOL 5236 was found not to inhibit microbial activity.

Since all acceptability criteria prescribed by the protocol were met, this study was considered to be valid.

In conclusion, HATCOL 5236 was not readily biodegradable under the conditions of the modified Sturm test presently performed.

CAS 78 -16 -0

The study was conducted in accordance with OECD Guideline 301B (CO2 Evolution Test). Domestic activated sludge (non adapted) was used as inoculum and a mean degradation of 72% after 28 days was observed. The 60% pass level at the 10-day-window was reached therefore the test substance can be classified as readily biodegradable according to OECD criteria.

CAS 11138 -60 -6

The study was also conducted in accordance with OECD Guideline 301B (CO2 Evolution Test). Domestic activated sludge (non adapted) was used as inoculum and a mean degradation of 85.3% after 28 days was observed. The 60% pass level at the 10-day-window was reached therefore the test substance can be classified as readily biodegradable according to OECD criteria.

HATCOL 1765

Two studies, investigating the ready biodegradability of pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS-No. 68424-31-7) according to OECD 301B (CO2 Evolution Test), are available. In both studies domestic activated sludge (non adapted) was used as inoculum. In the key study (Häner, 2006) a mean degradation of 96% and in the supporting study (Muckle, 2009) of 78% could be observed at test termination after 28 days. In both studies the 10-day-window was reached. Therewith the test substance can be classified as readily biodegradable according to OECD criteria.

[Type of water: freshwater]