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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:
2004-12-02 to 2004-12-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
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 sampled from municipal sewage treatment plant (Hildesheim, Germany) which treated mostly municipal sewage and hardly industrial chemical waste
- Preparation of inoculum for exposure: the activated sludge was maintained in aerobic condition by aeration for four hours and was then homogenized with a mixer; the sludge was filtered and the filtrate (30 mL) was subsequently used for inoculation
- Pretreatment: no
- Concentration of sludge: 30 mL
- Initial cell/biomass concentration: 10E5-10E6 CFU/L in the test vessels
- Water filtered: not reported
Duration of test (contact time):
28 d
Initial conc.:
45 mg/L
Based on:
test mat.
Initial conc.:
11.7 mg/L
Based on:
other: carbon content in test vessel
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: mineral nutrient solution acc. to OECD guideline 301B
- Additional substrate: not reported
- Solubilising agent (type and concentration if used): no
- Test temperature: 20-24°C
- pH: initial pH not reported; at the end of the study the pH was measured in all bottles: 7.51-7.59 (control), 7.86 (functional control), 7.59 (test item), and 7.96 (toxicity control)
- pH adjusted: not reported
- Aeration of dilution water: not reported
- Suspended solids concentration: filtrate of activated sludge was used as inoculum (30 mL), 10E5-10E6 CFU/L in the test vessels
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: brown glass bottles
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: aeration
- Measuring equipment: pH-Meter, Multilab 340i, WTW, Thermohygrograph, type 3.015/3 K, fabr.-no. 9003146, Thermohygrograph, LUFFT, Flow meter, KROHNE DUISBURG TYP DK 800 PV
- Test performed in closed vessels: yes
- Details of trap for CO2 and volatile organics if used: after aeration of the test vessels with CO2 free air for 24 h the CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 3 gas wash bottles containing Ba(OH)2

SAMPLING
- Sampling frequency: CO2 production was determined on day 1,4,6,8,11,14,18,21,25,28,and 29* (*last two gas wash bottles)
- Sampling method: CO2 was determined in the adsorption units

CONTROL AND BLANK SYSTEM
- Inoculum blank: performed
- Toxicity control: performed
- Other: functional control (reference substance) performed
Reference substance:
acetic acid, sodium salt
Test performance:
The study was conducted according to OECD Guideline 301B 'CO2 evolution test'.
Key result
Parameter:
% degradation (CO2 evolution)
Value:
68
Sampling time:
28 d
Results with reference substance:
Results with the reference substance sodium acetate
study day biodegradation (%)
day 1: 8
day 8: 55
day 14: 68
day 21: 74
day 28: 82

In a study conducted according to OECD Guideline 301 B 'Ready Biodegradability: CO2 Evolution Test' the following results were obtained:

a. CO2 production and biodegradation in the control and test item samples

Study day

Control (mg CO2/3L); mean value

Test item (45 mg/L; 11.7 mg C/L)

Replicate 1

Replicate 2

Gross (mg CO2/3L)

Net (mg CO2/3L)

Degradation (%)

Gross (mg CO2/3L)

Net (mg CO2/3L)

Degradation (%)

1

4.0

3.2

-0.8

0

2.9

-1.1

0

4

21.4

17.7

-3.7

0

28.3

6.9

5

6

35.3

50.9

15.6

12

53.4

18.1

14

8

48.4

79.4

31.0

24

78.4

30.0

23

11

62.1

108.1

46.0

35

108.7

46.6

36

14

79.0

134.2

55.2

43

135.7

56.7

44

18

99.9

163.7

63.8

49

161.2

61.3

47

21

119.3

190.5

71.2

55

186.4

67.1

52

25

139.6

220.4

80.8

62

214.3

74.7

58

28

154.8

245.0

90.2

70

241.7

86.9

67

29*

168.1

260.6

92.5

71

256.3

88.2

68

*results of the last two gas wash bottles determined on day 29 (after 28 days the aeration was continued for further 24 h and the quantity of CO2 released in the last two gas wash bottles was measured)

Based on the results, the test item can be regarded as readily biodegradable, but failing the 10-day window (the 10% level was reached after 6 days and the pass level of 60% on day 25). In the control a gross CO2 production of 168.1 mg CO2/3L (=56.0 mg CO2/L) was measured. According to guideline the CO2 evolution in the control should not normally exceed 40 mg/L, but values up to 70 mg/l were acceptable.

b. CO2 production and biodegradation in the control, functional control and toxicity control samples

Functional control (35 mg/L)

Toxicity control (35 mg/L reference item and 45 mg/L test item)

Study day

Control (mg CO2/3L); mean value

Gross (mg CO2/3L)

Net (mg CO2/3L)

Degradation (%)

Gross (mg CO2/3L)

Net (mg CO2/3L)

Degradation (%)

1

4.0

13.4

9.4

8

3.9

-0.1

0

4

21.4

54.1

32.7

29

58.5

37.1

15

6

35.3

88.1

52.8

47

113.0

77.7

32

8

48.4

109.9

61.5

55

154.6

106.2

44

11

62.1

133.7

71.6

64

187.5

125.4

52

14

79.0

155.3

76.3

68

215.7

136.7

56

18

99.9

179.2

79.3

71

244.7

144.8

60

21

119.3

202.1

82.8

74

264.5

145.2

60

25

139.6

228.0

88.4

79

292.9

153.3

63

28

154.8

247.1

92.3

82

315.7

160.9

67

29*

168.1

269.2

101.1

90

345.4

177.3

73

Based on the results of toxicity testing, it can be concluded that the test item was not inhibitory to the inoculum (>35% biodegradation after 14 d).

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
In a study conducted according to OECD Guideline 301 B 'Ready Biodegradability: CO2 Evolution Test' the test item proved to be readily biodegradable but failing the 10-day window criterion (the 10% level was reached after 6 days and the pass level of 60% on day 25).
Biodegradation after
1 day: 0%
8 days: 23-24%
14 days: 43-44%
21 days: 52-55%
28 days: 67-70%
29 days: 68-71%

Description of key information

Key study: Test method according to OECD 301B. GLP study. The test substance is considered as readily biodegradable (but failing 10 day window) after 28 days with a biodegradation percentage of 68%.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window

Additional information

The ready biodegradability was studied in a number of biodegradation studies. Two modified Sturm OECD 301B CO2 evolution tests are available, both conducted under GLP (Noack, 2005 and Lebertz, 2005). The results showed that the test substance is considered as readily biodegradable (but failing 10-day window) after 28 days with a biodegradation percentage of 68% and 75% respectively. These results are supported by other ready biodegradability studies. An OECD 301C modified MITI study (Ishihara, 2000) is available for a structural analogue, N-(3-N,N-dimetylaminooxidetrimetylene)alkylamide, the substance showed 85% degradation after 28 days.

In a study conducted according to EU method C.4 -B (determination of the "Ready Biodegradability - Modified OECD Screening Test, 1984) and similar to OECD Guideline 301 E (Ready biodegradability) (Wörmann, 1989), the test substance proved not to be readily biodegradable as the pass level of 70% was not reached within the test period (biodegradation after 28 d: 66%). A biodegradation study according to OECD guideline 306 was also conducted with a structural analogue (N-[3-(dimethylamino)propyl]myristamide N-oxide + N-[3-(dimethylamino)propyl]dodecanamide N-oxide). The substance was degraded 52% after 28 days. Based on this, the test substance cannot be characterised as readily biodegradable in the marine environment according to the OECD guideline 306. Hence, it can be concluded that the test substance C12 -C18 AAAO is readily biodegradable but does not meet the 10-day window.