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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-06-20 to 2003-02-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992-07-17
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
September 1993
GLP compliance:
yes (incl. QA statement)
Remarks:
1999-05-25
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
at ambient temp., dark, dry and in original container

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 biological wastewater treatment plant of the city Neustadt (ESN, Im Altenschemel, 67435 NW-Lachen-Speyerdorf).
- Date of collection and batch No.: 2002-09-02 and 02092002
- Sampling and treatment of activated sludge: The activated sludge was filtered through a cotton cloth, 2 times washed with tap water, re-suspended in test medium and finally aerated for ≥ 12 h. The dry residue was 11680 mg solids / L.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
inorg. C analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: 1 L of test medium contains: 10 mL stock solution (A), 1 mL stock solution (B), 1 mL stock solution (C),1 mL stock solution (D). For details of the stock solutions see table 1.
- Pretreatment of medium: Aeration with cleaned, humidified, CO2-free air for > 12 h to purge the medium of CO2.
- Test temperature: 20 ± 2°C
- Illumination during test. No
- Suspended solids concentration: 11.68 g/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Woulff bottels (5 L)
- Number of culture flasks/concentration: The test batches were prepared in 3 L test medium supplemented with sufficient inoculum of the activated sludge to give a concentration of suspended solids of 24.9 mg/L. The Flasks ‘Test1’ and ‘Test2’ contained 103.5 mg and 109.0 mg (calculated carbon content of 20 mg/L) of the test substance respectively. Blank1 and Blank2 functioned as negative control containing just test medium and inoculum. ‘Ref1’ contained 73.6 mg aniline as positive control. ‘Abio’ contained 107.0 mg of the test substance but no inoculum as abiotic sterile control and ‘Tox’ contained 105.6 mg of the test substance plus 73.6 mg aniline as the toxicity control.
- Measuring equipment: A Shimadzu TOC 5050A analyzer, calibrated with a mix of diluted stock solution and a Na2CO3/NaHCO3 mix according to the manufacturer instructions.
- Details of trap for CO2: Two in series connected CO2-trap flasks filled with 0.25 M NaOH.
- CO2 determination: 0.5 mL samples were taken, and 10 times diluted before the CO2 evolution in the samples were determined by IC-analysis. The samples were taken 10 times (days 2, 5, 7, 9, 12, 14, 19, 23, 28 and 29). The samples of day 0 to 28 were taken from the first CO2-trap bottle connected to the test flasks, whereas the samples of day 29 were collected from both CO2-trap bottles.
Reference substance:
aniline
Key result
Parameter:
% degradation (CO2 evolution)
Value:
17
St. dev.:
14
Sampling time:
28 d
Details on results:
The test substance was degraded on average up to 16.5% within the 28-day test period. In the blank controls 4.26 mg/L carbon corresponding to 15.6 mg/L CO2 were produced on average at the end of the test period (28 days). The 10-d window started at day 14. After 24 days of testing a degradation of 15% was reached. The results are summarized in table 2-4.
Results with reference substance:
The reference substance (Ref-control) was degraded by 88.2% within 14 days and up to 87.9% at the end of the test (28 days). In combination with the test substance (Tox-control) the level of degradation was 39.5% within 28 days. The results are summarized in table 2-4.

Table 2: Biodegradation in %

Day

Ref

Test1

Test2

Average of the test substance

Abio

Tox

2

4.0

7.4

3.6

5.5

8.9

2.4

5

59.1

6.4

1.7

4.1

11.4

27.6

7

70.1

5.8

0.4

3.1

12.3

31.8

9

76.8

11.3

2.3

6.8

16.8

34.7

12

81.8

13.7

1.6

7.6

15.7

37.7

14

88.2

16.7

4.9

10.8

18.2

40.1

19

85.4

22.4

8.3

15.3

26.2

43.0

23

92.5

24.2

6.9

15.6

31.8

41.4

28

87.8

26.3

6.7

16.5

33.9

39.5

29

89.5

28.6

8.6

18.6

42.0

41.7

Table 3: Measured IC-Values in the NaOH samples

Day

Blank1

Blank2

Ref

Test1

Test2

Abio

Tox

0

5.47

5.47

5.47

5.47

5.47

5.47

5.47

2

31.37

53.85

66.43

89.65

66.72

63.80

72.53

5

75.95

63.46

421.30

110.70

81.16

81.16

413.60

7

121.80

84.48

503.60

121.70

87.03

87.09

483.50

9

96.48

93.03

556.50

167.50

110.40

117.70

532.00

12

85.68

78.35

576.10

171.00

92.73

110.90

559.80

14

88.52

89.75

624.60

198.30

122.90

128.40

599.50

19

115.30

115.90

636.40

262.30

173.00

183.50

665.90

23

129.00

127.60

695.70

288.20

176.50

222.50

660.50

28

136.50

133.10

676.40

309.30

181.90

237.70

645.20

29

137.65

141.57

694.12

330.40

199.69

295.09

681.09

 

Table 4: CO2 production in mg/L

Day

Blank1

Blank2

Ref

Test1

Test2

Abio

Tox

2

0.86

1.61

2.03

2.81

2.04

1.94

2.24

5

2.34

1.92

13.79

3.49

2.51

2.51

13.54

7

2.61

2.61

16.44

3.84

2.69

2.69

15.77

9

2.99

2.87

18.09

5.32

3.45

3.68

17.29

12

2.62

2.38

18.64

5.41

2.85

3.44

18.11

14

2.70

2.74

20.12

6.27

3.82

4.00

19.31

19

3.55

3.57

20.40

8.30

5.42

5.76

21.35

23

3.97

3.93

22.20

9.09

5.50

6.98

21.07

28

4.19

4.08

21.47

9.72

5.65

7.43

20.47

29

4.21

4.33

21.92

10.34

6.18

9.22

21.51

 

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance is readily not biodegradable since a 17% degradation was observed after 28 days (10-day window not passed). A toxicity and an abiotic control was performed.
Executive summary:

The substance was tested for its ready biodegradability in the carbon dioxide (CO2) evolution test (modified Sturm test) according to the OECD guideline No. 301 B. The test substance was exposed in an aerobic aqueous medium to activated sludge from a sewage plant. The degradability by microbial activity was determined by measurement of the produced CO2 over a test period of 28 days. Therefore, the produced CO2 was trapped in a 0.25 M NaOH solution and determined by IC-analysis. In addition to the test substance a positive control with aniline as reference substance, a toxic control with reference substance plus test substance, an abiotic control and a negative control without any substance were tested. At the end of the test period the test substance was degraded up to a maximum of 17%. Thus, the criterion for ready biodegradability, at least 60% within 28 days in a ten-day window, was not reached. The difference of values of the two replicate batches of the test substance was less than 20% (i.e., 14%). The degradation value of 40.1% at day 14 of the toxicity control passes the minimum requirement of 25% degradation within 14 days. Therefore, the test substance was assumed to be not inhibitory on microbial activity. The IC-content of the test substance in suspension at the start of the test was less than 5% and the total CO2 release in the blank control at the end of the period did not exceed 40 mg/L and. Since all validity criteria are met, the study can be considered as valid. In conclusion, the test substance was not readily biodegradable under the conditions of the test and showed no inhibitory effects. However, there is evidence that abiotic degradation occured (34% after 28d).

Description of key information

not readily biodegradable (17% after 28 days; 10-d window not passed)

Key value for chemical safety assessment

Biodegradation in water:
not biodegradable
Type of water:
freshwater

Additional information