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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 2018-08-10 to 2018-11-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
None.
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Species/Origin:Aerobic activated sludge (microorganisms from a domestic waste¬water treatment plant) was supplied by the sewage treatment plant of Bensheim, Germany.
- Conditioning:The aerobic activated sludge used for this study was deposited for 15 min, washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in test water and centrifuged again. This procedure was done three times. 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 3.5 g dry material per litre were mixed with test water and aerated overnight. This suspension was used for the experiment.
Duration of test (contact time):
28 d
Initial conc.:
102.7 mg/L
Based on:
test mat.
Remarks:
Test Item Loading Rate (initial concentration in medium C0)
Initial conc.:
304.4 mg/L
Based on:
ThOD
Remarks:
ThOD (NH4)
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Solubilising agent (type and concentration if used): Silicon oil AR20 (1%V/V)
- Temperature: 22°C ± 1°C
- Light Conditions: Darkness
- pH-Value of Test Solutions: 7.5 (measured at the start of the test)
- 7.3 to 7.6 (measured at the end of the test)
- Recording: Test conditions (temperature) were recorded continuously with suitable instruments, documented in the raw data and reported in the final report. (Short-term deviations (< 2 hours) from the recommended temperature range do normally not result in major disturbances of the test performance and were not reported.)
- Comopsition of medium :Aliquots of washed sludge suspension were mixed with reconstitued test water.
- Reconstituted Test Water: Analytical grade salts were added to pure water to prepare the following stock solutions:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H2O, 0.5 g NH4Cl filled up with pure water to 1000 mL volume; The pH-value was 7.3.
b) 11.25 g MgSO4 x 7 H2O filled up with pure water to 500 mL volume
c) 18.2 g CaCl2 x 2 H2O filled up with pure water to 500 mL volume
d) 0.125 g FeCl3 x 6 H2O filled up with pure water to 500 mL volume
In order to avoid precipitation of iron hydroxide in the stock solution d), one drop of concentrated HCl per litre was added before storage.
50 mL of stock solution a) and 5 mL of the stock solutions b) to d) were combined and filled up to a final volume of 5000 mL with pure water. The pH of the solution was 7.4.


TEST SYSTEM
- Culturing apparatus: The closed test flasks were incubated in a climatised chamber under continuous stirring.
- Number of culture flasks/concentration: 2
- The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide.
- Measuring equipment: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany

SAMPLING
O2 consumed daily by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 (activated sludge + silicone oil + test water)
- Abiotic sterile control: 1
- Toxicity control: 1
- Other: Procedure control : (Ref. Item + activated sludge + silicone oil + test water)
Reference substance:
benzoic acid, sodium salt
Preliminary study:
None.
Test performance:
No data.
Parameter:
% degradation (O2 consumption)
Remarks:
ThOD (NH4)
Value:
62
Sampling time:
28 d
Details on results:
Test item :The 10-day windows began on day 3 after application, the mean value was calculated to be 11% biodegradation (ThODNH4). Therefore, the end of the 10-day window was day 13. The mean biodegradation percentage based on ThODNH4 at the end of the 10-day window was 40%; the criterion of the 10 day window was no passed. The mean biodegradation percentage at the end of the 28-day exposure period was 62% (ThODNH4).

Toxicity control: In the toxicity control containing both, the test item and the reference item sodium benzoate, 43% (ThODNH4) biodegradation was noted within 14 days and 58% (ThODNH4) biodegradation after 28 days of incubation.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 81% after 14 days and to 88% after 28 days of incubation.

Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks during the Test Period of 28 Days

Time

Flask No.

(days)

1

2

3

4

5

6

7

 

1

5

5

5

5

45

0

55

 

2

15

5

5

5

75

0

80

 

3

45

40

10

10

110

0

120

 

4

60

50

10

10

120

0

130

 

5

70

65

15

15

125

0

140

 

6

80

75

15

15

130

0

150

 

7

90

85

15

15

135

0

155

 

8

100

95

15

15

135

0

165

 

9

110

105

15

15

140

0

175

 

10

120

115

15

15

145

0

180

 

11

130

125

15

20

150

0

190

 

12

135

130

20

20

150

0

200

 

13

140

140

20

20

155

0

215

 

14

150

145

20

20

160

0

225

 

15

155

150

20

25

160

0

230

 

16

165

155

20

25

165

0

240

 

17

170

160

20

25

165

0

250

 

18

175

165

20

25

165

0

260

 

19

180

175

20

25

165

0

265

 

20

180

180

20

25

165

0

275

 

21

185

185

20

25

170

0

280

 

22

185

185

20

25

170

0

285

 

23

190

190

20

25

170

0

290

 

24

195

195

20

25

170

0

295

 

25

200

200

20

25

175

0

300

 

26

205

205

20

25

175

0

300

 

27

210

210

20

25

175

0

300

 

28

210

210

20

25

175

0

300

 

Flasks 1 and 2: Thuja Oil; Flasks 3 and 4: inoculum control

Flask5: reference (procedure control);        

Flask 6: abiotic control

 Flask 7: toxicity control

Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThODNH4

Time

Percentage Biodegradation

(Days)

Thuja Oil1

Sodium Benzoate2

Toxicity
control1, 2

 

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

0

0

23

10

2

3

0

40

16

3

11

10

58

23

4

16

13

63

25

5

18

16

63

26

6

21

20

66

28

7

25

23

69

29

8

28

26

69

31

9

31

30

72

33

10

34

33

75

34

11

37

35

76

36

12

38

36

75

37

13

39

40

78

41

14

43

41

81

43

15

43

42

79

43

16

47

44

82

45

17

48

45

82

47

18

50

47

82

49

19

52

50

82

50

20

52

52

82

53

21

53

53

85

54

22

53

53

85

55

23

55

55

85

56

24

57

57

85

57

25

58

58

88

58

26

60

60

88

58

27

61

62

88

58

28

61

62

88

58

1ThODNH4of Thuja Oil: 2.965 mg O2/mg test item
2ThODNH4of sodium benzoate: 1.666 mg O2/mg reference item

Validity Criteria of the Study

Inoculum Control:

The oxygen demand of the inoculum control (medium and inoculum) was 22.5 mg O2/L and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.

pH-Value:

The pH-value of the test item flasks at the end of the test was 7.4 and therefore within the range of pH 6.0 to 8.5 as required by the test guideline.

Reference Item:

The percentage degradation of the reference item should reach the level for ready biodegradability (>60%) within 14 days as required by the test guideline.

The reference item sodium benzoate was degraded to more than 60% after 4 days of incubation.

Test Item:

The difference of duplicate values for the degradation of the test item at the plateau, at the end of the test and at the end of the 10-day window was less than 20%. The difference of duplicate values at day 28 differed by 1%.

The validity criterion was fulfilled.

Toxicity Control:

If in a toxicity test, containing both the test item and a reference item less than 25% biodegradation (based on total ThOD) occurred within 14 days, the test item can be assumed to be inhibitory. The biodegradation was 43% at day 14; the test item was not inhibitory.

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The degradation rate of Thuja Oil reached 60% after 28 days. Therefore, Thuja Oil is considered to be readily biodegradable.
Executive summary:

The test item Thuja Oil was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.

At day 28, the mean biodegradation was 62%.The test item is considered as readily biodegradable based on ThODNH4 after 28 days.

The reference item sodium benzoate was sufficiently degraded to 81% after 14 days and to 88% after 28 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

In the toxicity control containing both, the test item and the reference item sodium benzoate, 43% biodegradation was noted within 14 days and 58% biodegradation after 28 days of incubation.According to the test guidelines, the test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms because degradation was >25% within 14 days.

The oxygen demand of the inoculum control (medium and inoculum) was not greater than 60 mg O2/L within 28 days as required by the test guideline.

pH values were in the range required in the guideline. Therefore, all validity criteria of the study were fulfilled.

Description of key information

The degradation rate of Thuja Oil reached 60% after 28 days. Therefore, Thuja Oil is considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

For that endpoint, an experimental study on the registered substance was available.

The test item was investigated for its ready biodegradability in a manometric respirometry test over a period of 28 days,according to the OECD 301F guideline.

The degradation rate of Thuja Oil reached 60% after 28 days. Therefore, Thuja Oil is considered to be readily biodegradable.