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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:
30 Nov - 29 Dec 2016
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)
Version / remarks:
17 Jul 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany (24 Sep 2013)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: sewage treatment plant Rossdorf, Germany
- Storage conditions: Washed sludge suspension (3.5 g dry material/L) were mixed with test water and aerated until use.
- Storage length: The washed activated sludge will be pre-conditioned in test water for a maximum of 2 d.
- Preparation of inoculum for exposure: The activated sludge was washed three times by centrifugation, decanting the supernatant liquid phase and resuspending the solid material in test water.
- Concentration of sludge: The washed sludge was adjusted to 3.5 g dry material per litre (± 10%) (stock suspension).
- Initial cell/biomass concentration: 28.7 mg sludge/L (final sludge concentration in test flasks)
Duration of test (contact time):
28 d
Initial conc.:
101.6 mg/L
Based on:
test mat.
Initial conc.:
177.9 mg/L
Based on:
ThOD
Remarks:
ThODNH4
Initial conc.:
230.7 mg/L
Based on:
ThOD
Remarks:
ThODNO3
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Reconstituted test water, according to guideline
- Test temperature: 22 ± 1 °C
- pH: 7.4 - 7.6 (at the start of the test), 6.8 - 8.3 (at the end of the test)
- Continuous darkness: Yes
- Other: The closed test flasks were incubated in a climatised chamber under continuous stirring.
- Other: pH-values were measured in the procedure control, a separately prepared test flask with test item (to prevent loss of test item in the test flasks) and a separately prepared test flask without test item (control) at test start and in all flasks at the end of the test, except in the abiotic and toxicity control, using a pH-electrode WTW pH 340i.

TEST SYSTEM
- Culturing apparatus: BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany containing a volume of approximately 500 mL.
- Number of culture flasks/concentration: 2
- Measuring equipment: BSB/BOD-Sensor-System, Aqualtyic Dortmund, Germany
- Test performed in open system: The test flasks were closed gas-tight by a measuring head.
- Details of trap for CO2 and volatile organics if used: Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.
- Other: The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.
- Preparation of test flasks: The amounts of test item and reference item were directly weighed into the test flasks.

SAMPLING
- Sampling frequency: Continuous
- Sampling method: The change of pressure in the test flask was measured by means of a manometric method (BSB/BOD-Sensor-System, Aqualytic Dortmund, Germany).
- Sterility check if applicable: The oxygen demand in the abiotic control containing test item, CuSO4 and test water without activated sludge was measured in parallel.
- Other: Determination of nitrification was performed in samples of a control and a test item treated flask, which was sampled at test start and stored deep frozen (-20 ± 5 °C) until nitrate determination was done. At test end, control and test item treated groups were sampled and nitrification was photometrically determined using an AA3 Continuous Flow Analyzer and equipment (with potassium nitrate and sodium nitrite as reference items).

CONTROL AND BLANK SYSTEM
- Inoculum control: 2 flasks, containing 2 mL activated sludge (stock suspension of 3.5 g/L dry matter) and 242 mL test water.
- Abiotic sterile control: 1 flask, containing 25.3 mg test item/flask, 5 mL CuSO4 (stock solution of 1 g/L) and 239 mL test water.
- Toxicity control: 1 flask, containing 25.0 mg test item/flask, 25.2 mg reference item/flask, 2 mL activated sludge (stock suspension of 3.5 g/L dry matter) and 242 mL test water.
- Procedure control: 1 flask, containing 24.8 mg reference item (sodium benzoate)/flask, 2 mL activated sludge (stock suspension of 3.5 g/L dry matter) and 242 mL test water.
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
74.6
Sampling time:
28 d
Remarks on result:
other:
Remarks:
Corrected biodegradation based on ThOD under consideration of nitrification on Day 28
Details on results:
The test item reached at least 10% biodegradation on Day 4. At the end of the 10-d window on Day 14, the biodegradation was 100% and 77% based on ThODNH4 and ThODNO3, respectively. Therefore, the 10-d window describing the period between reaching at least 10% and 60% degradation was met.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 81% after 14 d and to 84% after 28 d of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

BIODEGRADATION OF TEST ITEM & NITRIFICATION

The nitrate-N formation in the controls after 28 d of incubation was 1.769 mg Nitrate-N/L (mean). The nitrate concentration in the test item treated vessels was 12.653 mg/L Nitrate-N (mean), indicating nitrification occurred. Therefore, biodegradation was corrected for nitrification (Table 3).

TOXICITY CONTROL

In the toxicity control containing both the test item and the reference item sodium benzoate, 73% (ThODNH4) biodegradation was noted within 14 d and 72% (ThODNH4) biodegradation after 28 d of incubation (64% and 62% based on ThODNO3) (Table 1 and Table 2). Therefore, the test item is not inhibitory to aerobic activated sludge microorganisms (degradation > 25% within 14 d).

ABIOTIC CONTROL

The oxygen demand in the abiotic control was 0 mg/L throughout the test duration. There was no need to correct the degradation of the test item and toxicity control.

Table 1. Percentage Biodegradation of Test item, Reference Item (Sodium Benzoate) and of the Toxicity Control based on ThODNH4.

 

Time

[d]

Percentage biodegradation1

Test item

Sodium Benzoate2

Toxicity Control1, 2

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

0

0

30

16

2

0

0

38

23

3

6

6

47

48

4

34

36

65

64

5

62

65

68

72

6

76

76

71

75

7

84

84

74

75

8

89

88

75

75

9

89

94

78

75

10

92

97

78

75

11

94

100

81

75

12

94

100

81

73

13

97

100

81

73

14

97

102

81

73

15

97

102

84

73

16

97

102

84

73

17

100

105

84

73

18

99

104

83

72

19

99

104

83

74

20

99

104

83

74

21

101

107

86

74

22

101

107

86

74

23

100

105

84

73

24

103

105

84

73

25

103

105

84

73

26

101

107

86

72

27

100

105

84

72

28

100

105

84

72

1ThODNH4 of test item: 1.754 mg O2/mg test item

2ThODNH4 of Sodium Benzoate: 1.666 mg O2/mg reference item

 

 

Table 1. Percentage Biodegradation of Test item and of the Toxicity Control based on ThODNO3, and of the Reference Item (Sodium Benzoate) based on ThODNH4

 

Time

[d]

Percentage biodegradation1

Test item

Sodium Benzoate2

Toxicity Control1, 2

Flask 1 [%]

Flask 2 [%]

Flask 5 [%]

Flask 7 [%]

1

0

0

30

14

2

0

0

38

20

3

4

4

47

42

4

26

28

65

56

5

48

50

68

63

6

59

58

71

65

7

65

65

74

65

8

68

68

75

65

9

68

72

78

65

10

71

75

78

65

11

73

77

81

65

12

73

77

81

64

13

75

77

81

64

14

75

79

81

64

15

75

79

84

64

16

75

79

84

64

17

77

81

84

64

18

76

80

83

63

19

76

80

83

64

20

76

80

83

64

21

78

82

86

64

22

78

82

86

64

23

77

81

84

64

24

79

81

84

64

25

79

81

84

64

26

78

82

86

63

27

77

81

84

62

28

77

81

84

62

1ThODNO3 of test item: 2.274 mg O2/mg test item

2ThODNH4 of Sodium Benzoate: 1.666 mg O2/mg reference item

 

Table 3. Correction of Percentage Biodegradation of the Test Item based on ThOD under consideration of nitrification on day 28.

 

Nitrite-N

Nitrate-N

Total3

Measured4

 

Corrected5

Day 28

Day 0

Day 28

∆ Nitrite-N1

O2 Equi.2

 

Day 0

Day 28

∆ Nitrate-N1

O2 Equi.2

 

O2 Equi.

ThODNH4

ThODNH4

Degradation6

mg NO2-N/L

mg

mg NO3-N/L

mg

mg

mg

mg

%

Test item rate 1

0.000

0.000

0.000

0.000

0.022

12.625

12.603

12.081

12.081

43.310

31.229

72.1

Test item rate 2

0.000

0.000

0.000

0.000

0.022

12.724

12.702

12.191

12.191

45.750

33.559

77.1

Control (mean)

0.000

0.000

0.000

0.000

0.000

1.769

1.769

---

---

---

---

---

Test item (mean)

0.000

0.000

0.000

0.000

0.022

12.675

12.653

12.136

12.136

44.530

32.394

74.6

--: not applicable

1: ∆ nitrite-N/∆ nitrate-N: Difference of Nitrite-N / Nitrate-N between day 28 and day 0.

2: ∆ nitrite-N/∆ nitrate-N: multiplied by empirically determined conversion factor (NH4-N to NO2-N:3.43; NH4-N to NO3-N: 4.57), corrected by test volume (0.244 L) and concentration of control

3: Sum of nitrite-N O2-equivalent and nitrate-N O2-equivalent

4: BSB value test item day 28 [mg/L] – BSB value control day 28 [mg/L] multiplied with test volume [0.244 L]

5: Corrected ThODNH4 = ThODNH4 – O2-Equivalent

6: Degradation = Corrected ThODNH4/Calculated ThODNH4 * 100

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable

Description of key information

Readily biodegradable: 74.6% after 28 d (O2 consumption corrected for nitrification, 301 F)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

There is one study available investigating the ready biodegradability of N-Acetyl-DL-tryptophan (CAS 87-32-1) according to OECD guidelines 301 F and GLP.

In the manometric respirometry test 101.6 mg/L test item, corresponding to about 177.9 mg/L ThODNH4 and 230.7 mg/L ThODNO3, was inoculated with 28.7 mg domestic, aerobic activated sludge/L for 28 d. A procedural, inoculum, toxicity and abiotic control were run in parallel. Biodegradation was followed by the oxygen uptake of microorganisms and the degradation rate of the test item was calculated by the oxygen consumption after 28 d. Since the test item contains nitrogen, the evaluation of biodegradation was based on ThODNH4 (total theoretical oxygen demand for complete chemical oxidation) and ThODNO3 (total theoretical oxygen demand with nitrification for complete chemical oxidation).

The measured nitrate concentration in the test vessels indicated that nitrification occurred. After 28 d the mean biodegradation of the test item, corrected for nitrification, was 74.6%. The substance reached at least 10% biodegradation on day 4. On day 14 biodegradation was 100% (based on ThODNH4) and 77% (based on ThODNO3), thus meeting the 10-d criterion. The substance was not inhibitory to activated sludge microorganisms since biodegradation in the toxicity control was > 25% within 14 d. The procedural control confirmed the suitability of the activated sludge inoculum.

Therefore, the test item is readily biodegradable according to the criteria of the OECD guideline 301 F.