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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:
15 Feb - 04 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 310 (Ready Biodegradability - CO2 in Sealed Vessels (Headspace Test)
Version / remarks:
March 2006
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany (03.01.2017)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Municipal sewage treatment plant, Hildesheim, Germany
- Storage conditions: Aerobic conditions (aeration with CO2-free air)
- Storage length: 7 d
- Pretreatment: The activated sludge was washed twice with chlorine free tap water. After the second washing, the settled sludge was resuspended in mineral salts medium and maintained for 2 h in aerobic conditions. Then, the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in aerobic conditions by aeration with CO2-free air for 7 d (1st definitive study)/5 d (second definitive study) until test start.
- Initial cell/biomass concentration (in test vessel): 1.73E07 CFU/L (1st study); 1.55E07 CFU/L (2nd study)
Duration of test (contact time):
28 d
Initial conc.:
26.4 mg/L
Based on:
test mat.
Initial conc.:
19.8 mg/L
Based on:
other: Carbon content: mg C/L
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
TIC analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium according to OECD 310
- Test temperature: 19.0 °C - 21.0 °C (1st study); 19.0 - 20.2 °C (2nd study)
- Continuous darkness: No, low light conditions
- Agitation: Shaker (150 - 200 rpm)

TEST SYSTEM
- Culturing apparatus: 120 mL headspace flasks
- Number of culture flasks/concentration: Triplicates and 5 replicates at test end
- Measuring equipment: Carbon analyser, Multi N/C 3100, Analytik Jena
- Test performed in closed vessels due to significant volatility of test substance: Yes
- Other: Headspace to liquid ratio = 1:2

SAMPLING
- Sampling frequency: 1st study; Day 1, 4, 7, 11, 14, 21, and 28; 2nd study: Day 1, 3, 7, 10, 14, 21, and 28
- Other: The TIC of the sodium hydroxide solution (blind value) was determined at each sampling time.

CONTROL AND BLANK SYSTEM
- Functional control: 30 mg/L reference item (17.5 mg C/L) + inoculum (triplicates and 5 replicates at test end)
- Inoculum blank: Mineral salts medium + inoculum (triplicates and 5 replicates at test end)
- Toxicity control: Test item + 30 mg/L reference item + inoculum (triplicates and 5 replicates at test end)
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
46
Sampling time:
28 d
Remarks on result:
other: 95% confidence interval: 39 - 53%
Remarks:
1st study
Key result
Parameter:
% degradation (CO2 evolution)
Value:
48
Sampling time:
28 d
Remarks on result:
other: 95% confidence interval: 44 - 52%
Remarks:
2nd study
Details on results:
FIRST DEFINITIVE STUDY

Test item
The biodegradation of the test item reached the 10% level (beginning of biodegradation) within 4 d. After 28 d biodegradation reached 46%. The 95% confidence interval on Day 28 was 39 - 53%.

Toxicity control
The toxicity control reached 59% biodegradation after 14 d and 37% after 28 d. Therefore, the biodegradation of the reference item was not inhibited by the test item. The 95% confidence interval on Day 28 was 27 - 46%.

SECOND DEFINITIVE STUDY

Test item
The 10% pass level was reached within 3 d. After 28 d biodegradation was 48%. The 95% confidence interval on Day 28 was 44 - 52%.

Toxicity control
Biodegradtion reached 55% on Day 14 and 62% on Day 28. Therefore, the test item did not inhibit the biodegradation of the reference item. The 95% confidence interval on Day 28 was 59 - 64%.
Results with reference substance:
FIRST STUDY
The functional control reached the degradation phase (≥ 10% level) within 4 d. The course of the degradation phase was rapid and the pass level of 60% was reached within 7 d. Biodegradation peaked after 7 d at 67%. Therefore, the validity criterion of > 60% was fulfilled. The 95% confidence interval on Day 28 was 22 - 65%.

SECOND STUDY
The functional control reached the 10% pass level within 3 d. The degradation phase was rapid and reached the pass level of 60% within 3 d. Biodegradation peaked at 81% on Day 21. Therefore, the validity criterion of > 60% after 14 d was fulfilled. The 95% confidence interval on Day 28 was 78 - 81%.

VALIDITY CRITERIA

Both definitive tests fulfilled the validity criteria defined by the guideline (Tables 1 and 2).

Table 1. Validity criteria (first study)

Criterion from the guideline

Outcome

Validity criterion fulfilled

The percentage degradation of the reference item must exceed the pass level of 60% by Day 14.

Biodegradation of the functional control peaked at 67% after 7 d.

Yes

The mean amount of TIC present in the inoculum controls at the end of the test is < 3 mg C/L.

The mean amount of TIC present in the inoculum controls on Day 28 is 1.77 mg C/L.

Yes

 

 

Table 2. Validity criteria (second study)

Criterion from the guideline

Outcome

Validity criterion fulfilled

The percentage degradation of the reference item must exceed the pass level of 60% by Day 14.

Biodegradation of the functional control was 61% after 3 d.

Yes

The mean amount of TIC present in the inoculum controls at the end of the test is < 3 mg C/L.

The mean amount of TIC present in the inoculum controls on Day 28 is 1.51 mg C/L.

Yes

 

RESULTS OF THE FIRST DEFINITIVE STUDY

Table 3. CO2 Production and Biodegradation in the test item samples of the first study.

 

CO2 production [mg C/L]

Day

P1

P2

P3

P4

P5

MP

Net MP

Degr. [%]

0

1.49

1.98

1.46

-

-

0.01

-

-

1

2.74

2.11

2.25

-

-

0.46

0.28

1

4

12.33

11.54

11.67

-

-

10.20

9.37

47

7

12.93

11.39

12.32

-

-

10.54

9.74

49

11

14.01

14.40

13.07

-

-

11.84

11.05

56

14

13.03

14.96

11.39

-

-

11.42

10.23

52

21

13.65

9.65

13.77

-

-

10.59

9.01

46

28

12.56

12.48

14.58

11.78

12.03

10.87

9.13

46

MP = mean values (NaOH corrected)

Net MP = netto mean production (corrected for inoculum control values)

Degr. = degradation

 

Table 4. Biodegradation and Confidence interval of the test item in comparison to the function control and the toxicity control of the first study

 

 

Replicates

 

Biodegradation [%] on Day 28

Confidence interval on Day 28

1

2

3

4

5

P = 95%

Test item

45

45

56

42

43

39 – 53

Function control

66

25

56

30

41

22 – 65

Toxicity control

26

35

39

47

37

27 - 46

 

RESULTS OF THE SECOND DEFINITIVE STUDY

Table 5. CO2 production and biodegradation in the test item samples of the second study.

 

CO2 production [mg C/L]

Day

P1

P2

P3

P4

P5

MP

Net MP

Degr. [%]

0

1.74

1.72

1.74

-

-

0.72

-

-

1

1.85

1.80

1.78

-

-

0.38

0.00

0

4

7.84

7.85

7.25

-

-

6.96

5.30

27

7

10.14

10.49

9.64

-

-

8.59

7.55

38

11

10.70

11.22

9.86

-

-

9.11

7.98

40

14

11.91

11.49

10.72

-

-

9.97

8.55

43

21

12.15

10.95

12.05

-

-

10.08

8.53

43

28

12.84

13.09

13.61

11.90

12.60

11.15

9.57

48

MP = mean values (NaOH corrected)

Net MP = netto mean production (corrected for inoculum control values)

Degr. = degradation

Table 6. Biodegradation and Confidence interval of the test item in comparison to the function control and the toxicity control of the second study

 

 

Replicates

 

Biodegradation [%] on Day 28

Confidence interval on Day 28

1

2

3

4

5

P = 95%

Test item

48

50

52

44

47

44 – 52

Function control

78

81

79

79

79

78 – 81

Toxicity control

64

59

61

62

62

59 - 64

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Interpretation of results:
not readily biodegradable
Conclusions:
The substance is not readily biodegradable according to the OECD 310 guideline criteria (48% within 28 d).

Description of key information

Not readily biodegradable (48% within 28 d, OECD 310)

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

There is one study available, in which the ready biodegradability of the substance was investigated according to OECD guideline 310 and GLP.

In the available study, two definitive studies were conducted due to the high confidence intervals measured in the replicates at the end of the (formally valid) first test. The second study confirmed the results of the first study and showed smaller confidence intervals, which is why it is considered more reliable.

In the conducted Headspace Test with a headspace to liquid ratio of 1:2, a nominal concentration of 26.4 mg/L test item (corresponding to a carbon content of 19.8 mg C/L) was inoculated with domestic activated sludge (1.73 E07 CFU/L) for 28 d. A functional, inoculum and toxicity control were run in parallel. The quantity of CO2 produced by the respiration of bacteria was followed by periodic analysis of total inorganic carbon (TIC).

After 28 d, biodegradation reached 46% in the first test and 48% in the second test. The functional controls in both tests confirmed the suitability of the inocula. Thus, it was concluded that the test item is not readily biodegradable according to guideline criteria. The toxicity controls in both studies showed that the test item did not inhibit the biodegradation of the test item at the tested concentrations.