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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:
16 March 2017 to 18 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at approximately 4°C (refrigerator) in the dark under N2 conditions.
- Stability under test conditions: N/A
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
A mixed population of activated sewage sludge micro-organisms (Test System) was obtained on March 20th, 2017 from the secondary treatment stage of the sewage treatment plant at Villette (STEP Villette, av. de Thônex 105, 1226 THONEX (Geneva, Switzerland)), which treats predominantly domestic sewage.

The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in culture medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and maintained on with pure oxygen at room temperature. Determination of dry weight is made to inoculate final solution with 30mg/L dry weight activated sludge.
Duration of test (contact time):
28 d
Initial conc.:
50 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The culture medium used in this study (see Appendix 1) was that recommended in the OECD Guideline N° 301F.
Solution A
(KH2PO4) 8.50 g/L
(K2HPO4) 21.75g/L
(Na2HPO4,2H2O) 33.40 g/L
(NH4Cl) 0.50 g/L
Solution B
(CaCl2,2H2O) 36.40 g/L
Solution C
(MgSO4,7H2O) 22.50 g/L
Solution D
(FeCl3,6H2O) 0.25 g/L
For 1 l of test medium, mix 10 ml of solution a) with 800 ml water and then add 1 ml each of solutions b), c) and d) and make up to 1 000 ml with water
- Additional substrate: N/A
- Solubilising agent (type and concentration if used): N/A
- Test temperature: 22°C +/- 1°C.
- pH: On day 28, all vessels were sampled for pH.
- pH adjusted: no
- CEC (meq/100 g): N/A
- Aeration of dilution water: N/A
- Suspended solids concentration: 30mg/L dry weight (activated sludge)
- Continuous darkness: no (diffuse light)
- Other:

TEST SYSTEM
- Culturing apparatus: The system consists of a sample flask sealed by a sensor head/CO2 trap in a temperature controlled incubator. The samples were stirred for the duration of the study with a magnetically coupled stirrer.
- Number of culture flasks/concentration: Blanks x 2, Test Item x 3, Reference x 1, Toxicity Control x 1, Abiotic Control x 1
- Method used to create aerobic conditions: Oxygen
- Method used to create anaerobic conditions: N/A
- Measuring equipment: WTW oxitopC
- Test performed in closed vessels due to significant volatility of test substance: No (sealed system as per OECD 301F)
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: NaOH
- Other:

SAMPLING
- Sampling frequency: Daily
- Sampling method: WTW oxitopC calculate automatically the consumption of oxygen.
- Sterility check if applicable: N/A
- Sample storage before analysis: N/A
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control:
- Other: N/A
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
59
St. dev.:
2.08
Sampling time:
28 d
Details on results:
The test item (test 1) attained 58% degradation after 28 days.
The test item (test 2) attained 61% degradation after 28 days and didn’t satisfy the 10-day window validation criterion.
The test item (test 3) attained 57% degradation after 28 days.
Results with reference substance:
Sodium Benzoate attained 81% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

The toxicity test attained 54% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatmentmicro-organismsused in the study.

Preparation of Test Solutions

 

flask

Test Item

reference

activated sludge 1.5g/L

medium

vol total

pH final

 

 

mg

mg

L

L

L

blank 1

1

xxxxxxxx

xxxxxxxx

0.0080

0.3920

0.4000

7.29

blank 2

2

xxxxxxxx

xxxxxxxx

0.0080

0.3920

0.4000

7.29

test 1

3

20.02

xxxxxxxx

0.0080

0.3920

0.4000

7.32

test 2

4

20.22

xxxxxxxx

0.0080

0.3920

0.4000

7.33

test 3

5

20

xxxxxxxx

0.0080

0.3920

0.4000

7.34

reference

6

xxxxxxxx

20.08

0.0080

0.3920

0.4000

7.47

toxicity

7

19.91

20.2

0.0080

0.3920

0.4000

7.48

*abiotic

8

19.96

xxxxxxxx

0.0080

0.3920

0.4000

7.4

 * NaN3 50 g/L

Cumulative Biochemical Oxygen Demand (mgO2/L) in test flask during the test period of 28 days

days

min

bl1

bl2

test1

test2

test3

reference

toxicity

abiotic

0

0

0

0

0

0

0

0

0

0

1

1456

2.5

4.9

4.9

2.5

2.5

9.9

22.2

2.5

2

2912

7.4

7.4

7.4

4.9

4.9

49.4

59.3

2.5

3

4368

9.9

9.9

7.4

7.4

7.4

61.8

64.2

2.5

4

5712

12.4

12.4

9.9

7.4

7.4

69.2

69.2

2.5

5

7168

12.4

14.8

9.9

9.9

9.9

71.7

71.7

2.5

6

8624

14.8

14.8

9.9

9.9

9.9

76.6

74.1

2.5

7

10080

14.8

17.3

12.4

9.9

9.9

79.1

76.6

2.5

8

11536

17.3

17.3

12.4

9.9

12.4

81.5

79.1

2.5

9

12992

17.3

19.8

12.4

12.4

9.9

84

86.5

2.5

10

14448

19.8

19.8

12.4

12.4

12.4

86.5

93.9

2.5

11

15792

19.8

22.2

14.8

12.4

12.4

89

98.8

2.5

12

17248

22.2

22.2

14.8

14.8

12.4

91.4

104

2.5

13

18704

22.2

22.2

14.8

14.8

14.8

91.4

119

4.9

14

20160

22.2

24.7

22.2

17.3

17.3

91.4

146

4.9

15

21616

22.2

24.7

32.1

19.8

24.7

93.9

161

4.9

16

23072

24.7

24.7

42

39.5

37.1

93.9

178

4.9

17

24528

24.7

24.7

54.4

64.2

54.4

96.4

185

4.9

18

25872

24.7

27.2

71.7

79.1

69.2

96.4

190

4.9

19

27328

24.7

27.2

84

91.4

81.5

96.4

195

4.9

20

28784

24.7

27.2

93.9

96.4

91.4

96.4

198

2.5

21

30240

24.7

27.2

98.8

101

93.9

96.4

200

2.5

22

31696

24.7

27.2

101

104

96.4

96.4

203

4.9

23

33152

27.2

27.2

106

106

98.8

98.8

205

4.9

24

34608

27.2

27.2

106

109

104

98.8

205

4.9

25

35952

27.2

29.7

109

111

104

98.8

208

2.5

26

37408

27.2

29.7

111

114

106

98.8

208

2.5

27

38864

27.2

29.7

111

116

109

98.8

210

2.5

28

40320

27.2

27.2

111

116

109

98.8

210

2.5

 

Percentage Biodegradation in Test 1

days

blank 1

blank 2

average

test1

BOD (mgO2)

BOD(mgO2/mg test)

% degradation

NH3

0

0

0

0

0

0.0

0.00

0

1

2.5

4.9

3.7

4.9

0.5

0.02

1

2

7.4

7.4

7.4

7.4

0.0

0.00

0

3

9.9

9.9

9.9

7.4

-1.0

-0.05

-2

4

12.4

12.4

12.4

9.9

-1.0

-0.05

-2

5

12.4

14.8

13.6

9.9

-1.5

-0.07

-3

6

14.8

14.8

14.8

9.9

-2.0

-0.10

-3

7

14.8

17.3

16.05

12.4

-1.5

-0.07

-3

8

17.3

17.3

17.3

12.4

-2.0

-0.10

-3

9

17.3

19.8

18.55

12.4

-2.5

-0.12

-4

10

19.8

19.8

19.8

12.4

-3.0

-0.15

-5

11

19.8

22.2

21

14.8

-2.5

-0.12

-4

12

22.2

22.2

22.2

14.8

-3.0

-0.15

-5

13

22.2

22.2

22.2

14.8

-3.0

-0.15

-5

14

22.2

24.7

23.45

22.2

-0.5

-0.02

-1

15

22.2

24.7

23.45

32.1

3.5

0.17

6

16

24.7

24.7

24.7

42

6.9

0.35

12

17

24.7

24.7

24.7

54.4

11.9

0.59

21

18

24.7

27.2

25.95

71.7

18.3

0.91

32

19

24.7

27.2

25.95

84

23.2

1.16

40

20

24.7

27.2

25.95

93.9

27.2

1.36

47

21

24.7

27.2

25.95

98.8

29.1

1.46

50

22

24.7

27.2

25.95

101

30.0

1.50

52

23

27.2

27.2

27.2

106

31.5

1.57

55

24

27.2

27.2

27.2

106

31.5

1.57

55

25

27.2

29.7

28.45

109

32.2

1.61

56

26

27.2

29.7

28.45

111

33.0

1.65

57

27

27.2

29.7

28.45

111

33.0

1.65

57

28

27.2

27.2

27.2

111

33.5

1.67

58

 

Percentage Biodegradation in Test 2

 

days

blank 1

blank 2

 average

test2

BOD (mgO2)

BOD(mgO2/mg test)

% degradation

NH3

0

0

0

0

0

0.0

0.00

0

1

2.5

4.9

3.7

2.5

-0.5

-0.02

-1

2

7.4

7.4

7.4

4.9

-1.0

-0.05

-2

3

9.9

9.9

9.9

7.4

-1.0

-0.05

-2

4

12.4

12.4

12.4

7.4

-2.0

-0.10

-3

5

12.4

14.8

13.6

9.9

-1.5

-0.07

-3

6

14.8

14.8

14.8

9.9

-2.0

-0.10

-3

7

14.8

17.3

16.05

9.9

-2.5

-0.12

-4

8

17.3

17.3

17.3

9.9

-3.0

-0.15

-5

9

17.3

19.8

18.55

12.4

-2.5

-0.12

-4

10

19.8

19.8

19.8

12.4

-3.0

-0.15

-5

11

19.8

22.2

21

12.4

-3.4

-0.17

-6

12

22.2

22.2

22.2

14.8

-3.0

-0.15

-5

13

22.2

22.2

22.2

14.8

-3.0

-0.15

-5

14

22.2

24.7

23.45

17.3

-2.5

-0.12

-4

15

22.2

24.7

23.45

19.8

-1.5

-0.07

-3

16

24.7

24.7

24.7

39.5

5.9

0.29

10

17

24.7

24.7

24.7

64.2

15.8

0.78

27

18

24.7

27.2

25.95

79.1

21.3

1.05

36

19

24.7

27.2

25.95

91.4

26.2

1.29

45

20

24.7

27.2

25.95

96.4

28.2

1.39

48

21

24.7

27.2

25.95

101

30.0

1.48

51

22

24.7

27.2

25.95

104

31.2

1.54

53

23

27.2

27.2

27.2

106

31.5

1.56

54

24

27.2

27.2

27.2

109

32.7

1.62

56

25

27.2

29.7

28.45

111

33.0

1.63

57

26

27.2

29.7

28.45

114

34.2

1.69

59

27

27.2

29.7

28.45

116

35.0

1.73

60

28

27.2

27.2

27.2

116

35.5

1.76

61

 
Percentage Biodegradation in Test 3

days

blank 1

blank 2

average

test3

BOD (mgO2)

BOD(mgO2/mg test)

% degradation

NH3

0

0

0

0

0

0.0

0.00

0

1

2.5

4.9

3.7

2.5

-0.5

-0.02

-1

2

7.4

7.4

7.4

4.9

-1.0

-0.05

-2

3

9.9

9.9

9.9

7.4

-1.0

-0.05

-2

4

12.4

12.4

12.4

7.4

-2.0

-0.10

-3

5

12.4

14.8

13.6

9.9

-1.5

-0.07

-3

6

14.8

14.8

14.8

9.9

-2.0

-0.10

-3

7

14.8

17.3

16.05

9.9

-2.5

-0.12

-4

8

17.3

17.3

17.3

12.4

-2.0

-0.10

-3

9

17.3

19.8

18.55

9.9

-3.5

-0.17

-6

10

19.8

19.8

19.8

12.4

-3.0

-0.15

-5

11

19.8

22.2

21

12.4

-3.4

-0.17

-6

12

22.2

22.2

22.2

12.4

-3.9

-0.20

-7

13

22.2

22.2

22.2

14.8

-3.0

-0.15

-5

14

22.2

24.7

23.45

17.3

-2.5

-0.12

-4

15

22.2

24.7

23.45

24.7

0.5

0.03

1

16

24.7

24.7

24.7

37.1

5.0

0.25

9

17

24.7

24.7

24.7

54.4

11.9

0.59

21

18

24.7

27.2

25.95

69.2

17.3

0.87

30

19

24.7

27.2

25.95

81.5

22.2

1.11

38

20

24.7

27.2

25.95

91.4

26.2

1.31

45

21

24.7

27.2

25.95

93.9

27.2

1.36

47

22

24.7

27.2

25.95

96.4

28.2

1.41

49

23

27.2

27.2

27.2

98.8

28.6

1.43

50

24

27.2

27.2

27.2

104

30.7

1.54

53

25

27.2

29.7

28.45

104

30.2

1.51

52

26

27.2

29.7

28.45

106

31.0

1.55

54

27

27.2

29.7

28.45

109

32.2

1.61

56

28

27.2

27.2

27.2

109

32.7

1.64

57

 

Percentage Biodegradation in Reference

 

days

blank 1

blank 2

average

ref

BOD (mgO2)

BOD(mgO2/mg test)

% degradation

NH3

0

0

0

0

0

0.0

0.00

0

1

2.5

4.9

3.7

9.9

2.5

0.12

7

2

7.4

7.4

7.4

49.4

16.8

0.84

50

3

9.9

9.9

9.9

61.8

20.8

1.03

62

4

12.4

12.4

12.4

69.2

22.7

1.13

68

5

12.4

14.8

13.6

71.7

23.2

1.16

69

6

14.8

14.8

14.8

76.6

24.7

1.23

74

7

14.8

17.3

16.05

79.1

25.2

1.26

75

8

17.3

17.3

17.3

81.5

25.7

1.28

77

9

17.3

19.8

18.55

84

26.2

1.30

78

10

19.8

19.8

19.8

86.5

26.7

1.33

80

11

19.8

22.2

21

89

27.2

1.35

81

12

22.2

22.2

22.2

91.4

27.7

1.38

83

13

22.2

22.2

22.2

91.4

27.7

1.38

83

14

22.2

24.7

23.45

91.4

27.2

1.35

81

15

22.2

24.7

23.45

93.9

28.2

1.40

84

16

24.7

24.7

24.7

93.9

27.7

1.38

83

17

24.7

24.7

24.7

96.4

28.7

1.43

86

18

24.7

27.2

25.95

96.4

28.2

1.40

84

19

24.7

27.2

25.95

96.4

28.2

1.40

84

20

24.7

27.2

25.95

96.4

28.2

1.40

84

21

24.7

27.2

25.95

96.4

28.2

1.40

84

22

24.7

27.2

25.95

96.4

28.2

1.40

84

23

27.2

27.2

27.2

98.8

28.6

1.43

86

24

27.2

27.2

27.2

98.8

28.6

1.43

86

25

27.2

29.7

28.45

98.8

28.1

1.40

84

26

27.2

29.7

28.45

98.8

28.1

1.40

84

27

27.2

29.7

28.45

98.8

28.1

1.40

84

28

27.2

27.2

27.2

98.8

28.6

1.43

86

 

Percentage Biodegradation in Toxicity

 

days

blanc 1

blanc 2

moy

toxicite

BOD (mgO2)

BOD(mgO2/mg test)

% degradation

NH3

0

0

0

0

0

0.0

0.00

0

1

2.5

4.9

3.7

22.2

7.4

0.37

8

2

7.4

7.4

7.4

59.3

20.8

1.03

23

3

9.9

9.9

9.9

64.2

21.7

1.08

24

4

12.4

12.4

12.4

69.2

22.7

1.12

25

5

12.4

14.8

13.6

71.7

23.2

1.15

26

6

14.8

14.8

14.8

74.1

23.7

1.17

26

7

14.8

17.3

16.05

76.6

24.2

1.20

27

8

17.3

17.3

17.3

79.1

24.7

1.22

27

9

17.3

19.8

18.55

86.5

27.2

1.35

30

10

19.8

19.8

19.8

93.9

29.6

1.47

33

11

19.8

22.2

21

98.8

31.1

1.54

34

12

22.2

22.2

22.2

104

32.7

1.62

36

13

22.2

22.2

22.2

119

38.7

1.92

42

14

22.2

24.7

23.45

146

49.0

2.43

54

15

22.2

24.7

23.45

161

55.0

2.72

60

16

24.7

24.7

24.7

178

61.3

3.04

67

17

24.7

24.7

24.7

185

64.1

3.17

70

18

24.7

27.2

25.95

190

65.6

3.25

72

19

24.7

27.2

25.95

195

67.6

3.35

74

20

24.7

27.2

25.95

198

68.8

3.41

76

21

24.7

27.2

25.95

200

69.6

3.45

76

22

24.7

27.2

25.95

203

70.8

3.51

78

23

27.2

27.2

27.2

205

71.1

3.52

78

24

27.2

27.2

27.2

205

71.1

3.52

78

25

27.2

29.7

28.45

208

71.8

3.56

79

26

27.2

29.7

28.45

208

71.8

3.56

79

27

27.2

29.7

28.45

210

72.6

3.60

80

28

27.2

27.2

27.2

210

73.1

3.62

80

 

Percentage Biodegradation in Abiotic Test

 

blanc 1

blanc 2

moy

abiotic

BOD (mgO2)

BOD(mgO2/mg test)

% degradation

NH3

0

0

0

0

0

0.0

0.00

0

1

2.5

4.9

3.7

2.5

-0.5

-0.02

-1

2

7.4

7.4

7.4

2.5

-2.0

-0.10

-3

3

9.9

9.9

9.9

2.5

-3.0

-0.15

-5

4

12.4

12.4

12.4

2.5

-4.0

-0.20

-7

5

12.4

14.8

13.6

2.5

-4.4

-0.22

-8

6

14.8

14.8

14.8

2.5

-4.9

-0.25

-9

7

14.8

17.3

16.05

2.5

-5.4

-0.27

-9

8

17.3

17.3

17.3

2.5

-5.9

-0.30

-10

9

17.3

19.8

18.55

2.5

-6.4

-0.32

-11

10

19.8

19.8

19.8

2.5

-6.9

-0.35

-12

11

19.8

22.2

21

2.5

-7.4

-0.37

-13

12

22.2

22.2

22.2

2.5

-7.9

-0.39

-14

13

22.2

22.2

22.2

4.9

-6.9

-0.35

-12

14

22.2

24.7

23.45

4.9

-7.4

-0.37

-13

15

22.2

24.7

23.45

4.9

-7.4

-0.37

-13

16

24.7

24.7

24.7

4.9

-7.9

-0.40

-14

17

24.7

24.7

24.7

4.9

-7.9

-0.40

-14

18

24.7

27.2

25.95

4.9

-8.4

-0.42

-15

19

24.7

27.2

25.95

4.9

-8.4

-0.42

-15

20

24.7

27.2

25.95

2.5

-9.4

-0.47

-16

21

24.7

27.2

25.95

2.5

-9.4

-0.47

-16

22

24.7

27.2

25.95

4.9

-8.4

-0.42

-15

23

27.2

27.2

27.2

4.9

-8.9

-0.45

-15

24

27.2

27.2

27.2

4.9

-8.9

-0.45

-15

25

27.2

29.7

28.45

2.5

-10.4

-0.52

-18

26

27.2

29.7

28.45

2.5

-10.4

-0.52

-18

27

27.2

29.7

28.45

2.5

-10.4

-0.52

-18

28

27.2

27.2

27.2

2.5

-9.9

-0.49

-17

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item (test 1 and 3) attained respectively 58% and 57% degradation after 28 days. Under the conditions of the test, the test item cannot be considered to be readily biodegradable.
The test item (test 2) attained respectively 61% and didn’t satisfy the 10-day window validation criterion. Under the conditions of the test, the test item cannot be considered to be readily biodegradable.

Executive summary:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed that described in the OECD Guideline for the testing of Chemicals (1992) N°301 F, “Ready Biodegradability; Manometric Respirometry Test”.

In addition, the procedures were designed to meet the test method of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-D laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

The test item, at a concentration of 50.05mg/L in test 1, at a concentration of 50.55mg/L in test 2 and at a concentration of 50.00mg/L in test 3 was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 22°C ± 1°C for 28 days.

 

The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

 

The test item (test 1 and 3) attained respectively58% and57% degradation after 28 days. Under the conditions of the test, the test item cannot be considered to be readily biodegradable.

The test item (test 2) attained respectively61% anddidn’t satisfy the 10-day window validation criterion. Under the conditions of the test, the test item cannot be considered to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 March 2017 to 01 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criterua were met.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms (Test System) was obtained on May 3rd, 2017 from the secondary treatment stage of the sewage treatment plant at Villette (STEP Villette, av. de Thônex 105, 1226 THONEX (Geneva, Switzerland)), which treats predominantly domestic sewage.

The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in culture medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and maintained on with pure oxygen at room temperature. Determination of dry weight is made to inoculate final solution with 30mg/L dry weight activated sludge.
Duration of test (contact time):
28 d
Initial conc.:
>= 44.29 - <= 44.67 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: The culture medium used in this study (see Appendix 1) was that recommended in the OECD Guideline N° 301F.
Solution A
(KH2PO4) 8.50 g/L
(K2HPO4) 21.75g/L
(Na2HPO4,2H2O) 33.40 g/L
(NH4Cl) 0.50 g/L
Solution B
(CaCl2,2H2O) 36.40 g/L
Solution C
(MgSO4,7H2O) 22.50 g/L
Solution D
(FeCl3,6H2O) 0.25 g/L
For 1 L of test medium, mix 10 mL of solution a) with 800 mL water and then add 1 ml each of solutions b), c) and d) and make up to 1 000 mL with water
- Additional substrate: N/A
- Solubilising agent (type and concentration if used): N/A
- Test temperature: 22 °C +/- 1 °C.
- pH: On day 28, all vessels were sampled for pH.
- pH adjusted: no
- CEC (meq/100 g): N/A
- Aeration of dilution water: N/A
- Suspended solids concentration: 30mg/L dry weight (activated sludge)
- Continuous darkness: no (diffuse light)
- Other:

TEST SYSTEM
- Culturing apparatus: The system consists of a sample flask sealed by a sensor head/CO2 trap in a temperature controlled incubator. The samples were stirred for the duration of the study with a magnetically coupled stirrer.
- Number of culture flasks/concentration: Blanks x 2, Test Item x 3, Reference x 1, Toxicity Control x 1, Abiotic Control x 1
- Method used to create aerobic conditions: Oxygen
- Method used to create anaerobic conditions: N/A
- Measuring equipment: WTW oxitopC
- Test performed in closed vessels due to significant volatility of test substance: No (sealed system as per OECD 301F)
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: NaOH
- Other:

SAMPLING
- Sampling frequency: Daily
- Sampling method: WTW oxitopC calculate automatically the consumption of oxygen.
- Sterility check if applicable: N/A
- Sample storage before analysis: N/A
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes
- Toxicity control:
- Other: N/A
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
67
St. dev.:
5.2
Sampling time:
28 d
Details on results:
The test item (test 1) attained 64% degradation after 28 days and satisfied the 10-day window validation criterion.
The test item (test 2) attained 64% degradation after 28 days and satisfied the 10-day window validation criterion.
The test item (test 3) attained 73% degradation after 28 days and satisfied the 10-day window validation criterion.
The toxicity test attained 57% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatmentn micro-organismsused in the study.
Results with reference substance:
Sodium Benzoate attained 86% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.

Cumulative Biochemical Oxygen Demand (mgO2/L) in test flask during the test period of 28 days

days min bl1 bl2 test1 test2 test3 reference toxicity  abiotic
0 0 0 0 0 0 0 0 0 0
1 1456 4.1 6.2 4.1 4.1 4.1 10.3 18.5 0
2 2912 10.3 10.3 6.2 6.2 6.2 47.3 51.4 0
3 4368 12.3 12.3 8.2 8.2 8.2 57.6 55.5 0
4 5712 14.4 14.4 10.3 10.3 8.2 63.7 59.6 0
5 7168 16.4 16.4 10.3 10.3 8.2 69.9 63.7 0
6 8624 16.4 18.5 12.3 10.3 10.3 74 65.8 0
7 10080 18.5 18.5 12.3 12.3 10.3 76.1 69.9 0
8 11536 18.5 20.6 12.3 12.3 12.3 78.1 74 0
9 12992 20.6 20.6 14.4 12.3 12.3 80.2 80.2 0
10 14448 20.6 20.6 14.4 14.4 14.4 82.2 88.4 0
11 15792 20.6 20.6 14.4 14.4 14.4 84.3 96.6 0
12 17248 22.6 22.6 16.4 16.4 16.4 84.3 105 0
13 18704 22.6 22.6 20.6 20.6 20.6 84.3 119 0
14 20160 22.6 22.6 30.8 26.7 30.8 86.4 138 0
15 21616 24.7 22.6 53.5 39.1 55.5 86.4 152 0
16 23072 24.7 22.6 80.2 63.7 80.2 88.4 164 0
17 24528 24.7 22.6 86.4 80.2 88.4 88.4 171 0
18 25872 24.7 24.7 90.5 86.4 92.5 88.4 173 0
19 27328 24.7 24.7 94.6 92.5 98.7 90.5 177 0
20 28784 24.7 24.7 98.7 96.6 101 90.5 179 0
21 30240 24.7 24.7 103 101 103 90.5 183 0
22 31696 24.7 24.7 103 103 107 92.5 185 0
23 33152 26.7 24.7 105 105 109 92.5 185 0
24 34608 26.7 24.7 107 105 111 92.5 187 0
25 35952 26.7 26.7 107 105 115 92.5 189 0
26 37408 26.7 26.7 107 107 115 92.5 189 0
27 38864 26.7 24.7 109 107 119 94.6 191 0
28 40320 26.7 26.7 109 109 121 94.6 193 0

Percentage Biodegradation in Test 1

days blank 1 blank 2 average test1 BOD (mgO2) BOD(mgO2/mg test) % degradation
NH3
0 0 0 0 0 0 0 0
1 4.1 6.2 5.15 4.1 -0.5 -0.02 -1
2 10.3 10.3 10.3 6.2 -1.8 -0.09 -3
3 12.3 12.3 12.3 8.2 -1.8 -0.09 -3
4 14.4 14.4 14.4 10.3 -1.8 -0.09 -3
5 16.4 16.4 16.4 10.3 -2.7 -0.14 -5
6 16.4 18.5 17.45 12.3 -2.3 -0.12 -4
7 18.5 18.5 18.5 12.3 -2.8 -0.14 -5
8 18.5 20.6 19.55 12.3 -3.3 -0.16 -6
9 20.6 20.6 20.6 14.4 -2.8 -0.14 -5
10 20.6 20.6 20.6 14.4 -2.8 -0.14 -5
11 20.6 20.6 20.6 14.4 -2.8 -0.14 -5
12 22.6 22.6 22.6 16.4 -2.8 -0.14 -5
13 22.6 22.6 22.6 20.6 -0.9 -0.04 -2
14 22.6 22.6 22.6 30.8 3.7 0.18 6
15 24.7 22.6 23.65 53.5 13.4 0.67 23
16 24.7 22.6 23.65 80.2 25.4 1.27 44
17 24.7 22.6 23.65 86.4 28.2 1.41 49
18 24.7 24.7 24.7 90.5 29.6 1.48 51
19 24.7 24.7 24.7 94.6 31.5 1.57 54
20 24.7 24.7 24.7 98.7 33.3 1.66 57
21 24.7 24.7 24.7 103 35.2 1.76 61
22 24.7 24.7 24.7 103 35.2 1.76 61
23 26.7 24.7 25.7 105 35.7 1.78 62
24 26.7 24.7 25.7 107 36.6 1.82 63
25 26.7 26.7 26.7 107 36.1 1.8 62
26 26.7 26.7 26.7 107 36.1 1.8 62
27 26.7 24.7 25.7 109 37.5 1.87 65
28 26.7 26.7 26.7 109 37 1.85 64

Percentage Biodegradation in Test 2

days blank 1 blank 2 average test2 BOD (mgO2) BOD(mgO2/mg test) % degradation
NH3
0 0 0 0 0 0 0 0
1 4.1 6.2 5.15 4.1 -0.5 -0.02 -1
2 10.3 10.3 10.3 6.2 -1.8 -0.09 -3
3 12.3 12.3 12.3 8.2 -1.8 -0.09 -3
4 14.4 14.4 14.4 10.3 -1.8 -0.09 -3
5 16.4 16.4 16.4 10.3 -2.7 -0.14 -5
6 16.4 18.5 17.45 10.3 -3.2 -0.16 -6
7 18.5 18.5 18.5 12.3 -2.8 -0.14 -5
8 18.5 20.6 19.55 12.3 -3.3 -0.16 -6
9 20.6 20.6 20.6 12.3 -3.7 -0.19 -6
10 20.6 20.6 20.6 14.4 -2.8 -0.14 -5
11 20.6 20.6 20.6 14.4 -2.8 -0.14 -5
12 22.6 22.6 22.6 16.4 -2.8 -0.14 -5
13 22.6 22.6 22.6 20.6 -0.9 -0.05 -2
14 22.6 22.6 22.6 26.7 1.8 0.09 3
15 24.7 22.6 23.65 39.1 7 0.35 12
16 24.7 22.6 23.65 63.7 18 0.9 31
17 24.7 22.6 23.65 80.2 25.4 1.28 44
18 24.7 24.7 24.7 86.4 27.8 1.39 48
19 24.7 24.7 24.7 92.5 30.5 1.53 53
20 24.7 24.7 24.7 96.6 32.4 1.62 56
21 24.7 24.7 24.7 101 34.3 1.72 60
22 24.7 24.7 24.7 103 35.2 1.77 61
23 26.7 24.7 25.7 105 35.7 1.79 62
24 26.7 24.7 25.7 105 35.7 1.79 62
25 26.7 26.7 26.7 105 35.2 1.77 61
26 26.7 26.7 26.7 107 36.1 1.81 63
27 26.7 24.7 25.7 107 36.6 1.84 64
28 26.7 26.7 26.7 109 37 1.86 64

Percentage Biodegradation in Test 3

days blank 1 blank 2 average test3 BOD (mgO2) BOD(mgO2/mg test) % degradation
NH3
0 0 0 0 0 0 0 0
1 4.1 6.2 5.15 4.1 -0.5 -0.02 -1
2 10.3 10.3 10.3 6.2 -1.8 -0.09 -3
3 12.3 12.3 12.3 8.2 -1.8 -0.09 -3
4 14.4 14.4 14.4 8.2 -2.8 -0.14 -5
5 16.4 16.4 16.4 8.2 -3.7 -0.18 -6
6 16.4 18.5 17.45 10.3 -3.2 -0.16 -6
7 18.5 18.5 18.5 10.3 -3.7 -0.18 -6
8 18.5 20.6 19.55 12.3 -3.3 -0.16 -6
9 20.6 20.6 20.6 12.3 -3.7 -0.19 -6
10 20.6 20.6 20.6 14.4 -2.8 -0.14 -5
11 20.6 20.6 20.6 14.4 -2.8 -0.14 -5
12 22.6 22.6 22.6 16.4 -2.8 -0.14 -5
13 22.6 22.6 22.6 20.6 -0.9 -0.04 -2
14 22.6 22.6 22.6 30.8 3.7 0.18 6
15 24.7 22.6 23.65 55.5 14.3 0.71 25
16 24.7 22.6 23.65 80.2 25.4 1.27 44
17 24.7 22.6 23.65 88.4 29.1 1.45 50
18 24.7 24.7 24.7 92.5 30.5 1.52 53
19 24.7 24.7 24.7 98.7 33.3 1.66 57
20 24.7 24.7 24.7 101 34.3 1.71 59
21 24.7 24.7 24.7 103 35.2 1.75 61
22 24.7 24.7 24.7 107 37 1.84 64
23 26.7 24.7 25.7 109 37.5 1.86 65
24 26.7 24.7 25.7 111 38.4 1.91 66
25 26.7 26.7 26.7 115 39.7 1.98 68
26 26.7 26.7 26.7 115 39.7 1.98 68
27 26.7 24.7 25.7 119 42 2.09 72
28 26.7 26.7 26.7 121 42.4 2.11 73

Percentage Biodegradation in Reference

days blank 1 blank 2 average ref BOD (mgO2) BOD(mgO2/mg test) % degradation
NH3
0 0 0 0 0 0 0 0
1 4.1 6.2 5.15 10.3 2.3 0.12 7
2 10.3 10.3 10.3 47.3 16.7 0.83 50
3 12.3 12.3 12.3 57.6 20.4 1.01 61
4 14.4 14.4 14.4 63.7 22.2 1.1 66
5 16.4 16.4 16.4 69.9 24.1 1.2 72
6 16.4 18.5 17.45 74 25.4 1.27 76
7 18.5 18.5 18.5 76.1 25.9 1.29 77
8 18.5 20.6 19.55 78.1 26.3 1.31 79
9 20.6 20.6 20.6 80.2 26.8 1.33 80
10 20.6 20.6 20.6 82.2 27.7 1.38 83
11 20.6 20.6 20.6 84.3 28.7 1.43 86
12 22.6 22.6 22.6 84.3 27.8 1.38 83
13 22.6 22.6 22.6 84.3 27.8 1.38 83
14 22.6 22.6 22.6 86.4 28.7 1.43 86
15 24.7 22.6 23.65 86.4 28.2 1.4 84
16 24.7 22.6 23.65 88.4 29.1 1.45 87
17 24.7 22.6 23.65 88.4 29.1 1.45 87
18 24.7 24.7 24.7 88.4 28.7 1.43 86
19 24.7 24.7 24.7 90.5 29.6 1.47 88
20 24.7 24.7 24.7 90.5 29.6 1.47 88
21 24.7 24.7 24.7 90.5 29.6 1.47 88
22 24.7 24.7 24.7 92.5 30.5 1.52 91
23 26.7 24.7 25.7 92.5 30.1 1.5 90
24 26.7 24.7 25.7 92.5 30.1 1.5 90
25 26.7 26.7 26.7 92.5 29.6 1.47 88
26 26.7 26.7 26.7 92.5 29.6 1.47 88
27 26.7 24.7 25.7 94.6 31 1.54 93
28 26.7 26.7 26.7 94.6 30.6 1.52 91

Percentage Biodegradation in Toxicity Control

days blank 1 blank 2 average toxicity BOD (mgO2) BOD(mgO2/mg test) % degradation
NH3
0 0 0 0 0 0 0 0
1 4.1 6.2 5.15 18.5 6 0.3 7
2 10.3 10.3 10.3 51.4 18.5 0.93 20
3 12.3 12.3 12.3 55.5 19.4 0.97 21
4 14.4 14.4 14.4 59.6 20.3 1.02 22
5 16.4 16.4 16.4 63.7 21.3 1.06 23
6 16.4 18.5 17.45 65.8 21.8 1.09 24
7 18.5 18.5 18.5 69.9 23.1 1.16 25
8 18.5 20.6 19.55 74 24.5 1.23 27
9 20.6 20.6 20.6 80.2 26.8 1.34 30
10 20.6 20.6 20.6 88.4 30.5 1.53 34
11 20.6 20.6 20.6 96.6 34.2 1.71 38
12 22.6 22.6 22.6 105 37.1 1.85 41
13 22.6 22.6 22.6 119 43.4 2.17 48
14 22.6 22.6 22.6 138 51.9 2.6 57
15 24.7 22.6 23.65 152 57.8 2.89 64
16 24.7 22.6 23.65 164 63.2 3.16 70
17 24.7 22.6 23.65 171 66.3 3.32 73
18 24.7 24.7 24.7 173 66.7 3.34 74
19 24.7 24.7 24.7 177 68.5 3.43 76
20 24.7 24.7 24.7 179 69.4 3.47 76
21 24.7 24.7 24.7 183 71.2 3.56 78
22 24.7 24.7 24.7 185 72.1 3.61 79
23 26.7 24.7 25.7 185 71.7 3.59 79
24 26.7 24.7 25.7 187 72.6 3.63 80
25 26.7 26.7 26.7 189 73 3.65 80
26 26.7 26.7 26.7 189 73 3.65 80
27 26.7 24.7 25.7 191 74.4 3.72 82
28 26.7 26.7 26.7 193 74.8 3.74 82

Percentage Biodegradation in Abiotic Test

 days blank 1 blank 2 average abiotic BOD (mgO2) BOD(mgO2/mg test) % degradation
NH3
0 0 0 0 0 0 0 0
1 4.1 6.2 5.15 0 -2.3 -0.11 -4
2 10.3 10.3 10.3 0 -4.6 -0.23 -8
3 12.3 12.3 12.3 0 -5.5 -0.27 -10
4 14.4 14.4 14.4 0 -6.5 -0.32 -11
5 16.4 16.4 16.4 0 -7.4 -0.37 -13
6 16.4 18.5 17.45 0 -7.9 -0.39 -13
7 18.5 18.5 18.5 0 -8.3 -0.41 -14
8 18.5 20.6 19.55 0 -8.8 -0.44 -15
9 20.6 20.6 20.6 0 -9.3 -0.46 -16
10 20.6 20.6 20.6 0 -9.3 -0.46 -16
11 20.6 20.6 20.6 0 -9.3 -0.46 -16
12 22.6 22.6 22.6 0 -10.2 -0.5 -17
13 22.6 22.6 22.6 0 -10.2 -0.5 -17
14 22.6 22.6 22.6 0 -10.2 -0.5 -17
15 24.7 22.6 23.65 0 -10.6 -0.53 -18
16 24.7 22.6 23.65 0 -10.6 -0.53 -18
17 24.7 22.6 23.65 0 -10.6 -0.53 -18
18 24.7 24.7 24.7 0 -11.1 -0.55 -19
19 24.7 24.7 24.7 0 -11.1 -0.55 -19
20 24.7 24.7 24.7 0 -11.1 -0.55 -19
21 24.7 24.7 24.7 0 -11.1 -0.55 -19
22 24.7 24.7 24.7 0 -11.1 -0.55 -19
23 26.7 24.7 25.7 0 -11.6 -0.57 -20
24 26.7 24.7 25.7 0 -11.6 -0.57 -20
25 26.7 26.7 26.7 0 -12 -0.6 -21
26 26.7 26.7 26.7 0 -12 -0.6 -21
27 26.7 24.7 25.7 0 -11.6 -0.57 -20
28 26.7 26.7 26.7 0 -12 -0.6 -21
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item (replicates 1, 2 and 3) attained respectively 64%, 64% and 73% degradation after 28 days and satisfied the 10-day window. Under the conditions of the test, the test item can be considered to be readily biodegradable.
Executive summary:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed that described in the OECD Guideline for the testing of Chemicals (1992) N°301 F, “Ready Biodegradability; Manometric Respirometry Test”. In addition, the procedures were designed to meet the test method of the European Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-D laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

The test item, at a concentration of 44.60mg/L in test 1, at a concentration of 44.29mg/L in test 2 and at a concentration of 44.67mg/L in test 3 was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 22°C ± 1°C for 28 days.

The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test item (replicates 1, 2 and 3) attained respectively 64%, 64% and 73% degradation after 28 days and satisfied the 10-day window validation criterion. Under the conditions of the test, the test item can be considered to be readily biodegradable.

Description of key information

Biodegradation (28 -days): 67 % (meets 10 -day window criterion), readily biodegradable, OECD 301F, Anon, 2017b

Biodegradation (28 -days): 59 %, not readily biodegradable, OECD 301F, Anon., 2017a

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Two key studies were submitted for the biodegradation in water: screening test (Anon., 2017a and Anon., 2017b). Both studies were considered reliable with a reliability score of 1.

Anon., 2017a was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed that described in the OECD Guideline for the testing of Chemicals (1992) N°301 F, “Ready Biodegradability; Manometric Respirometry Test”. In addition, the procedures were designed to meet the test methods of the European Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-D laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The test item - at a concentration of 50.05 mg/L in test 1, at a concentration of 50.55 mg/L in test 2 and at a concentration of 50.00 mg/L in test 3 - was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffuse light at 22 °C ± 1 °C for 28 days. The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes. The test item (replicates 1 and 3) attained respectively 58% and 57% degradation after 28 days. Under the conditions of the test, the test item cannot be considered to be readily biodegradable.

The test item (replicate 2) attained 61% degradation and didn’t satisfy the 10-day window validation criterion. Under the conditions of this test, the test item cannot be considered to be readily biodegradable.

Anon., 2017b was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed that described in the OECD Guideline for the testing of Chemicals (1992) N°301 F, “Ready Biodegradability; Manometric Respirometry Test”. In addition, the procedures were designed to meet the test methods of the European Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-D laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The test item, at a concentration of 44.60 mg/L in test 1, at a concentration of 44.29 mg/L in test 2 and at a concentration of 44.67 mg/L in test 3 was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffuse light at 22 °C ± 1 °C for 28 days. The degradation of the test item was assessed by the measurement of daily oxygen consumption on days 0 and 28. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes. The test item (replicates 1, 2 and 3) attained 64%, 64% and 73% degradation, respectively, after 28 days and satisfied the 10-day window validation criterion. Under the conditions of the test, the test item can be considered to be readily biodegradable.

In accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7B (Version 4.0 - June 2017), it is recognised that ready biodegradability tests may sometimes fail to report a representative result because of the stringent test conditions, positive results (i.e. those showing ready biodegradabality) should supersede negative results. The only significant difference in the test system between the two studies was the addition rate (50 mg/L Anon., 2017a and 45 mg/L Anon., 2017b). The source of inoculum was also identical for both studies, indicating that this was not a factor affecting the two studies. Given that the only significant difference in test systems was based on addition rates of test item, both of which were within guideline specification, the positive result from Anon., 2017b is considered the most relevant for concluding on this endpoint.

The substance is considered readily biodegradable.