Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-07-22 to 2009-08-19
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): p-Nitrobenzoylaminobenzamid trocken
- Molecular formula: C14H11N3O4
- Molecular weight: 285.26 g/mol
- Physical state: Solid, white powder
- Expiration date of the lot/batch: 2010-10-01
- Storage condition of test material: Room temperature, protected from moisture and light.

Study design

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): Activated sludge from the municipal sewage treatment plant, 31137 Hildesheim, Germany
- Preparation of inoculum for exposure: The activated sludge was washed twice with autoclaved tap water. After the second washing the settled sludge was filled up with mineral salts medium and was maintained in an aerobic condition by aeration for 4 hours. Thereafter the sludge was homogenized with a blender. The supernatant was decanted and maintained in an aerobic condition by aeration with CO2-free air for 5 days.
10 mL/L were used to initiate inoculation.
- Initial cell/biomass concentration: 1.1 x 10e9 CFU/L
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
55 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301F
- Additional substrate: No
- Test temperature: 20.5-22.0 °C
- pH:
pH-Values at Test Start and Test End

pH-Value
Start End
Inoculum Control 7.53 1) 7.69
2) 7.64
Functional Control 7.55 7.60
Test Item 7.47 1) 7.43
2) 7.49
Toxicity Control 7.46 7.40

- pH adjusted: no
- Dispersion treatment: Continuous stirring
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Closed bottles with a rubber sleeve with soda lime
- Measuring equipment: The bottles were closed with OxiTOP measuring heads.
The oxygen consumption was determined continuously from the pressure in the incubation vessels by OxiTop measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period.



SAMPLING
- Sampling frequency: 360 measuring points (every 112 minutes) during the 28 d incubation period.
- Sampling method: Pressure measurements directly from the test vessels



CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and reference item
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentrations



STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD) of the test item and functional control.
Concerning the OECD-guideline 301 the index of the atoms of the item CcHhClclNnNanaOoPpSs was used for the calculation of the ThOD.
The oxygen depletion of the test and reference item was calculated out of the difference of the test and inoculum control replicates.
The biodegradation of the test and reference item was calculated out of the BOD in relation to the ThOD.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Test performance:
Based on the calculated oxygen demand the test concentration of 55 mg/L corresponding to an oxygen demand of 75.4 mg O2/L in the test vessel was selected.

The test solutions were prepared in measuring flasks and given into brown glass bottles as incubation vessels (inoculum control, functional control) or were prepared directly in brown glass bottles (test item, toxicity control):

• two for the test item (P1, P2)
• one for the functional control (R1)
• one for the toxicity control (T1)
• two for the inoculum control (C1, C2)

Separate replicates of test item and toxicity control were prepared for measurement of the pH-value at test start:

• one for the test item (P1)
• one for the toxicity control (T1)

The test item was weighed out and was transferred directly into the brown glass bottle with 250 mL test medium (consisting of the appropriate volumes of mineral medium stock solutions, deminer-alised water and 10 mL/L inoculum).
Accordingly, the test and reference item for the toxicity control were weighed out and transferred directly into the brown glass
bottle.
For the functional control the reference item was weighed out and transferred into a 250 mL measuring flask with demineralised water. The appropriate volumes of mineral medium stock solutions and 10 mL/L inoculum were added, then the flask was filled up with demineralised water. This test solution was given into the brown glass bottle.
The inoculum control, consisting of mineral medium stock solutions, demineralised water and inoculum, was prepared in a
500 mL measuring flask. This test solution was divided using a 250 mL measuring flask before being filled into the brown glass bottles.
A rubber sleeve with soda lime was hung into the opening of the bottles. The bottles were closed with OxiTop measuring heads and the measuring system was activated.
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
29
Sampling time:
28 d
Details on results:
Results of the Functional Control
The pass level of a biodegradation > 60 % was reached after 2 days. The validity criterion that the degradation should be > 60 % after 14 d was fulfilled.
Results of the Toxicity Control
In the toxicity control the biodegradation achieved 49 % after 14 days. After 28 days the biodegradation rate came to 68 %. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.
In case of the functional control, the adaptation phase changed to degradation phase after 1 day (degradation > 10 %). The pass level > 60 % was reached after 2 days. The biodegradation rate reached a maximum of 100 % on day 13
The 1st test item replicate reached the 10 % level (beginning of biodegradation) after 17 days and came to a biodegradation rate of 36 % after 28 days. The 2nd test item replicate reached the 10 % level after 20 days and came to a biodegradation rate of 22 % after 28 days.
Biodegradation [%] of the Test Item p-Nitrobenzoylaminobenzamid trocken in
Comparison to the Functional Control and Toxicity Control

Biodegradation [%]
Date [d] Functional
Control
45 mg/L Test Item

55 mg/L
Toxicity Control
55 mg/L Test
Item + 45 mg/L
Reference Item
R1 P1 P2 T1
22.07.09 0 0 0 0 0
23.07.09 1 49 0 0 18
24.07.09 2 67 0 0 29
25.07.09 3 77 0 0 34
26.07.09 4 81 2 0 37
27.07.09 5 81 2 0 41
28.07.09 6 79 6 4 46
29.07.09 7 90 6 2 43
30.07.09 8 90 6 2 44
31.07.09 9 92 6 4 45
01.08.09 10 94 6 2 45
02.08.09 11 96 6 2 46
03.08.09 12 96 7 4 47
04.08.09 13 100 9 6 50
05.08.09 14 99 7 6 49
06.08.09 15 99 9 7 48
07.08.09 16 99 9 7 49
08.08.09 17 100 11 7 49
09.08.09 18 100 13 7 50
10.08.09 19 100 15 9 52
11.08.09 20 100 19 11 51
12.08.09 21 100 20 14 55
13.08.09 22 100 22 15 56
14.08.09 23 100 23 16 57
15.08.09 24 100 25 16 58
16.08.09 25 100 29 20 60
17.08.09 26 100 31 18 62
18.08.09 27 100 32 19 65
19.08.09 28 100 36 22 68

BOD5 / COD results

Results with reference substance:
In the toxicity control the biodegradation achieved 49 % after 14 days. After 28 days the biodegradation rate came to 68 %. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.

Any other information on results incl. tables

Biological Oxygen Demand (BOD) of Inoculum Control, Functional Control,
Test Item p-Nitrobenzoylaminobenzamid trocken and Toxicity Control

BOD [mg O2/L]

BOD [mg O2/L]

Date

[d]

Inoculum
Control

Functional
Control
45 mg/L

Test Item
55 mg/L

Toxicity Control
55 mg/L Test
Item+ 45 mg/L
Reference Item

R1

P1

P2

T1

mean

gross

net

gross

net

gross

net

gross

net

23.07.09

1

0.0

36.6

36.6

0.0

0.0

0.0

0.0

26.8

26.8

24.07.09

2

0.0

50.7

50.7

0.0

0.0

0.0

0.0

43.7

43.7

25.07.09

3

0.0

57.8

57.8

0.0

0.0

0.0

0.0

50.7

50.7

26.07.09

4

0.0

60.6

60.6

1.4

1.4

0.0

0.0

56.4

56.4

27.07.09

5

0.0

60.6

60.6

1.4

1.4

0.0

0.0

62.0

62.0

28.07.09

6

0.0

59.2

59.2

4.2

4.2

2.8

2.8

69.0

69.0

29.07.09

7

0.0

67.6

67.6

4.2

4.2

1.4

1.4

64.8

64.8

30.07.09

8

0.0

67.6

67.6

4.2

4.2

1.4

1.4

66.2

66.2

31.07.09

9

0.0

69.0

69.0

4.2

4.2

2.8

2.8

67.6

67.6

01.08.09

10

0.0

70.5

70.5

4.2

4.2

1.4

1.4

67.6

67.6

02.08.09

11

0.0

71.9

71.9

4.2

4.2

1.4

1.4

69.0

69.0

03.08.09

12

0.0

71.9

71.9

5.6

5.6

2.8

2.8

70.5

70.5

04.08.09

13

0.0

76.1

76.1

7.0

7.0

4.2

4.2

74.7

74.7

05.08.09

14

0.0

74.7

74.7

5.6

5.6

4.2

4.2

73.3

73.3

06.08.09

15

0.0

74.7

74.7

7.0

7.0

5.6

5.6

71.9

71.9

07.08.09

16

0.0

74.7

74.7

7.0

7.0

5.6

5.6

73.3

73.3

08.08.09

17

0.0

76.1

76.1

8.5

8.5

5.6

5.6

73.3

73.3

09.08.09

18

0.0

77.5

77.5

9.9

9.9

5.6

5.6

74.7

74.7

10.08.09

19

0.0

77.5

77.5

11.3

11.3

7.0

7.0

78.9

78.9

11.08.09

20

0.0

77.5

77.5

14.1

14.1

8.5

8.5

77.5

77.5

12.08.09

21

2.1

78.9

76.8

16.9

14.8

12.7

10.6

84.5

82.4

13.08.09

22

1.4

78.9

77.5

18.3

16.9

12.7

11.3

86.0

84.6

14.08.09

23

2.1

78.9

76.8

19.7

17.6

14.1

12.0

87.4

85.3

15.08.09

24

2.1

80.3

78.2

21.1

19.0

14.1

12.0

88.8

86.7

16.08.09

25

0.7

81.7

81.0

22.5

21.8

15.5

14.8

91.6

90.9

17.08.09

26

0.7

80.3

79.6

24.0

23.3

14.1

13.4

94.4

93.7

18.08.09

27

1.4

81.7

80.3

25.4

24.0

15.5

14.1

98.6

97.2

19.08.09

28

0.0

78.9

78.9

26.8

26.8

16.9

16.9

103.0

103.0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test item must be regarded as not readily biodegradable in the 10-d-window and after
28 days. The mean biodegradation rate after 28 days was 29%.
Executive summary:

The ready biodegradability of the test item p-Nitrobenzoylaminobenzamid trocken was determined with a non adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted according to OECD guideline 301 F. The test item concentration selected as appropriate was 55 mg/L, corresponding to a ThOD of 75.4 mg O2/L per test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day.

The mean oxygen depletion in the inoculum control was 0.0 mg O2/L on day 28.

In order to check the activity of the test system sodium benzoate was used as functional control. The pass level > 60 % was reached after 2 days. The biodegradation rate reached a maximum of 100 % on day 13.

In the toxicity control containing both test and reference item 49 % degradation occurred within 14 days. After 28 days the biodegradation rate came to 68 %. The degradation of the reference item was not inhibited by the test item.

The biodegradation of the test item is shown in Table 1. The 1st test item replicate reached the 10 % level (beginning of biodegradation) after 17 days and came to a biodegradation rate of 36 % after 28 days. The 2nd test item replicate reached the 10 % level after 20 days and came to a biodegradation rate of 22 % after 28 days. The mean biodegradation rate after 28 days was 29%.

The validity criteria of the guideline are fulfilled.

The test item must be regarded as

not readily biodegradable

in the 10-d-window and after 28 days

Mean Biodegradation of the Test Item p-Nitrobenzoylaminobenzamid trocken in Comparison to the Functional Control and Toxicity Control

Biodegradation [%]

Study Day [d]

Replicate

7

14

21

28

Test Item
55 mg/L

1

6

7

20

36

2

2

6

14

22

Functional Control
45 mg/L

1

90

99

100

100

Toxicity Control
55 mg/L Test Item +
45 mg/L Reference Item

1

43

49

55

68