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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2000-08-01 to 2000-09-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: activated sludge, microorganisms from a domestic waste water treatment plant
- Origin: Sewage plant Groß-Zimmern, Germany
- Method of cultivation: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was resuspended in tap water and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 1.5 g dry material per litre were mixed with test water and then aerated until use.
Duration of test (contact time):
28 d
Initial conc.:
ca. 105 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline OECD 301 F (1992)
- Additional substrate: no
- Solubilising agent: no
- Test temperature: 21 - 22 °C
- pH: 7.6
- pH adjusted: none
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: The closed test flasks were incubated in a climatic chamber under continuous stirring.
- Number of culture flasks/concentration:
2 bottles containing the test item and inoculum
1 bottles containing the reference item aniline and inoculum (procedure control)
2 bottles containing only inoculum (inoculum control)
1 bottles containing the test item, reference item aniline and inoculum (toxicity control)
- Measuring equipment:The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic, 63231 Neu Isenburg, Germany) each day.
- Test performed in closed vessels due to significant volatility of test substance: no
- Test performed in open system: yes

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: yes
- Toxicity control: yes
- Positive control: yes
Reference substance:
aniline
Test performance:
The validity crietria were met:
- Inoculum Control: The oxygen demand of the inoculum control (medium and inoculum) was 10 mg O2/L and thus not greater than 60 mg O2/L within 28 days.
- Toxicity control: In the toxicity control containing both, the test item and the reference item aniline, 29 % biodegradation was noted within 14 days and 32 % biodegradation was determined after 28 days of incubation. According to the test guidelines the test item can be assumed to be not inhibitory on the activated sludge micro organisms because degradation was > 25 % within 14 days.
- pH-Value: The pH-value of the test item flasks at the end of the test was pH 7.6 and thus within the range of pH 6.0 - 8.5.
- Reference Item: The reference item aniline was sufficiently degraded to 77 % after 14 days, and to 92 % after 28 days of incubation, thus, confirming the suitability of the used activated sludge inoculum.
- Test Item: The difference of duplicate values for the degradation of the test item at any time during the test was less than 20 %.
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 2
Sampling time:
28 d
Details on results:
After correction of the mean biochemical oxygen demand of the inoculum controls at the end of the 28-day exposure period degradation rates of 2 % and 3 % were found. The test material was found to be not readily biodegradable under the test conditions within 28 days.
Results with reference substance:
The reference item aniline was sufficiently degraded to 77 % after 14 days, and to 92 % after 28 days of incubation.The biodegradation in the toxicity control containing both, the test item and the reference item Aniline was determined to be 29 % within 14 days and 32 % biodegradation was determined after 28 days of incubation.

Table 1. Percentage biodegradation (BOD/ThOD) of test item, Aniline and the toxicity control 

Time
(days)

Percentage BOD

Test item

Aniline

Toxicity control

Flask 1

Flask 2

Flask 5

Flask 7

1

0

0

0

0

2

0

0

0

0

3

-1

1

-1

0

4

1

1

-1

0

5

1

1

17

8

6

0

0

51

22

7

0

0

55

23

8

0

0

57

24

9

0

0

59

26

10

0

0

61

27

11

0

0

65

27

12

0

2

71

28

13

2

2

73

29

14

2

2

77

29

15

2

2

79

30

16

2

2

81

30

17

1

1

82

30

18

1

1

82

50

19

1

2

84

30

20

0

2

83

30

21

0

3

85

31

22

2

3

87

31

23

2

3

89

31

24

2

3

89

31

25

2

3

89

31

26

2

3

92

32

27

2

3

92

32

28

2

3

92

32

ThOD of the test item: 3.23 mg O2/mg test item

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The ready biodegradability of the test substance was determined according to OECD 301 F. Within the study period of 28 days, a degradation of 2 - 3 % was determined for the test substance. Therefore, the test substance is considered to be not readily biodegradable.
Executive summary:

The ready biodegradability of the test substance was determined according to OECD 301 F "Ready Biodegradability: Manometric Respirometry Test" under GLP conditions over a period of 28 days. The biodegradation was followed by the oxygen uptake of the micro-organisms during exposure. A measured volume of inoculated mineral medium, containing a known concentration of test substance ca. 105 mg/L as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature 22 °C for up to 28 days. The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test item. The closed test flasks were incubated in a climatic chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45 %) of potassium hydroxide. The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic) each day. The temperature was measured each working day in the climatic chamber. pH-values were measured in all flasks at the start and end of the test using a pH-electrode WTW pH 340i. Degradation was followed by the determination of oxygen uptake and measurements were taken at frequent intervals to allow the identification of the beginning and the end of biodegradation. The oxygen uptake was calculated from the readings taken at regular and frequent intervals.  All validity criteria of the test method were met. As a reference item aniline was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The reference item aniline was sufficiently degraded to 77 % after 14 days, and to 92 % after 28 days of incubation (ThODNH4). The results, thus, confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item aniline, 29 % biodegradation was noted within 14 days and 32 % biodegradation was determined after 28 days of incubation. Thus, the test item can be assumed not to be inhibitory on the activated sludge micro-organisms. Within the study period of 28 days, a degradation of 2 - 3 % was determined for the test substance. Therefore, the test substance is considered to be not ready biodegradable.

Description of key information

The ready biodegradability of the test substance was determined according to OECD 301 F. Within the study period of 28 days, a degradation of 2 - 3 % was determined for the test substance. Therefore, the test substance is considered to be not readily biodegradable.

Key value for chemical safety assessment

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

Additional information

The ready biodegradability of the test substance was determined according to OECD 301 F "Ready Biodegradability: Manometric Respirometry Test" under GLP conditions over a period of 28 days. The biodegradation was followed by the oxygen uptake of the micro-organisms during exposure. A measured volume of inoculated mineral medium, containing a known concentration of test substance ca. 105 mg/L as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature 22 °C for up to 28 days. The amounts of test item and reference item were directly weighed into the test flasks. No emulsifiers or solvents were used, but the solutions were dispersed by stirring to achieve a homogeneous solution of the test item. The closed test flasks were incubated in a climatic chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks. Evolved carbon dioxide was absorbed in an aqueous solution (45 %) of potassium hydroxide. The change of pressure in the test flasks was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic) each day. The temperature was measured each working day in the climatic chamber. pH-values were measured in all flasks at the start and end of the test using a pH-electrode WTW pH 340i. Degradation was followed by the determination of oxygen uptake and measurements were taken at frequent intervals to allow the identification of the beginning and the end of biodegradation. The oxygen uptake was calculated from the readings taken at regular and frequent intervals.  All validity criteria of the test method were met. As a reference item aniline was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The reference item aniline was sufficiently degraded to 77 % after 14 days, and to 92 % after 28 days of incubation (ThODNH4). The results, thus, confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item aniline, 29 % biodegradation was noted within 14 days and 32 % biodegradation was determined after 28 days of incubation. Thus, the test item can be assumed not to be inhibitory on the activated sludge micro-organisms. Within the study period of 28 days, a degradation of 2 - 3 % was determined for the test substance. Therefore, the test substance is considered to be not ready biodegradable (reference 5.2.1-1).