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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Sep 2002 - 30 Jan 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Mixed population of aquatic microorganism (activated sludge) taken from secondary effluent of a domestic sewage treatment plant (Kläranlage Odenthal, Germany, collected: 06.08.1999).
- Laboratory culture: no
- Preparation of inoculum for exposure: aeration for 4 d
- Pretreatment: Separation of coarse particles by filtration.
- Amount of sludge used for inoculation: 5 mL/L
Duration of test (contact time):
28 d
Initial conc.:
0.84 mg/L
Based on:
test mat.
Initial conc.:
2 mg/L
Based on:
test mat.
Initial conc.:
2.5 mg/L
Based on:
ThOD
Initial conc.:
5.94 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline (prepared in ultrapure water)
- Test temperature: 19.3 - 19.7 °C
- pH: 6.2 - 7.1
- pH adjusted: no
- Continuous darkness: yes
- Other: To prevent leakage of gasses out of the BOD bottles the bottles were incubated up side down.

TEST SYSTEM
- Culturing apparatus: 300 mL BOD bottles
- Number of culture flasks/concentration: 4
- Method used to create aerobic conditions: The mineral medium was saturated with oxygen and afterwards placed in the BOD bottles.
- Measuring equipment: O2 electrode
- Test performed in closed vessels: yes

SAMPLING
- Sampling frequency: O2 consumption was measured on day 0, 7, 14, 21, 28 days for the test item. In the inoculum acitivity control and toxicity control the O2 consumption was measured on day 0, 7 and 14.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 4 replicates
- Abiotic sterile control: no
- Toxicity control: yes, 4 replicates
- Other: reference substance: yes, 4 replicates
Reference substance:
acetic acid, sodium salt
Remarks:
Concentration: 4.00 mg/L
Parameter:
% degradation (O2 consumption)
Value:
92
Sampling time:
28 d
Remarks on result:
other: at 0.84 mg/L
Parameter:
% degradation (O2 consumption)
Value:
79
Sampling time:
28 d
Remarks on result:
other: at 2.00 mg/L
Results with reference substance:
The reference substance was degraded to 86% after 7 d and 103% after 14 d.

The toxicity control containing both, test item (2 mg/L) and reference item (4 mg/L) was degraded to 60% after 7 d and 66% after 14 d. This result indicates that the test item is not inhibitory to the inoculum.

The oxygen depletion in the inoculum blank was 1.15 mg O2/L after 28 days of incubation. This is within the limit given in the guideline of 1.5 mg O2/L.

Table 1: Biodegradation of test item, reference substance and toxicity control

Flask

Biodegradability [%] after n-days

0

7

14

21

28

Test substance mean (0.84 mg/L)

0

64

90

93

92

Test substance min (0.84 mg/L)

61

87

91

91

Test substance max (0.84 mg/L)

65

94

97

93

Test substance mean (2.0 mg/L)

0

64

76

77

79

Test substance min (2.0 mg/L)

62

73

75

79

Test substance max (2.0 mg/L)

66

78

78

80

Reference substance mean

0

86

103

n.d.

n.d.

Reference substance min

83

99

n.d.

n.d.

Reference substance max

89

106

n.d.

n.d.

Toxicity control mean

0

60

66

n.d.

n.d.

Toxicity control min

58

66

n.d.

n.d.

Toxicity control max

61

67

n.d.

n.d.

 

Validity criteria
1. Difference in extremes between replicates was < 20%.

2. The percentage biodegradation in the toxicity control exceeded 25%.

3. O2 consumption the inoculum control was 1.16 mg O2/L after 28 d.

4. The residual oxygen concentration in the test bottles was >> 0.5mg O2/L at all time.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Test item was biodegraded for at least 79% within 28 days of incubation at the tested concentration of 2.00 mg/L. At lower concentration of 0.84 mg/L a maximum percentage of 93% biodegradation was reached after 21 days. Test item passed the criteria given in the guidelines (i.e. > 60% degradation reached within 10 day period counting from the day that the level of degradation exceeded 10%), and is therefore assessed to be readily biodegradable in this test.
Executive summary:

The objective of this study was to determine the biodegradability of test item in a ‘Closed Bottle Test’ as described in the OECD Guideline 301D under GLP using oxygen depletion as test criterion in a 28 day test. The test substance was added to the inoculated medium by means of a stock solution of test item in ultra-pure water. Aliquots of this stock solution were added to inoculated medium to obtain test substance concentrations of 0.84 and 2.00 mg/L, corresponding to a Theoretical Oxygen Demand (ThOD) of 2.50 and 5.94 mg O2/L, respectively. An inoculum was prepared from activated sludge taken from an oxidation ditch used to treat domestic sewage. The test fulfilled the conditions of validity given by the Guidelines. After 21 days a mean maximum biodegradability of 93% at a test concentration of 0.84 mg/L was determined. After 28 days a mean maximum biodegradability of 79% at a test concentration of 2.00 mg/Lwas determined. Test item is regarded to be ready biodegradable according to the guidelines.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
9 Oct - 7 Nov 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A sample of activated sludge was taken from an oxidation ditch situated in the municipality of Hazerswoude, the Netherlands. The oxidation ditch is used to treat domestic wastewater. The activated sludge was transported in a plastic bottle and aerated until use.
- Laboratory culture: no
- Storage length: 1 d
- Used supernatant: 5 mL/L
- Settling time of sludge: 50 min
Duration of test (contact time):
28 d
Initial conc.:
0.84 mg/L
Based on:
test mat.
Initial conc.:
2.01 mg/L
Based on:
test mat.
Initial conc.:
2.5 mg/L
Based on:
ThOD
Initial conc.:
5.97 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: according to guideline (prepared in ultrapure water)
- Test temperature: 19.3 - 19.7 °C
- pH: 6.2 - 7.1
- pH adjusted: no
- Continuous darkness: yes
- Other: To prevent leakage of gasses out of the BOD bottles the bottles were incubated up side down.

TEST SYSTEM
- Culturing apparatus: 300 mL BOD bottles
- Number of culture flasks/concentration: 4
- Method used to create aerobic conditions: The mineral medium was saturated with oxygen and afterwards placed in the BOD bottles.
- Measuring equipment: O2 electrode
- Test performed in closed vessels: yes

SAMPLING
- Sampling frequency: O2 consumption was measured on day 0, 7, 14, 21, 28 days for the test item. In the inoculum acitivity control and toxicity control the O2 consumption was measured on day 0, 7 and 14.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, 4 replicates
- Abiotic sterile control: no
- Toxicity control: yes, 4 replicates
- Other: reference substance: yes, 4 replicates
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
92
Sampling time:
28 d
Remarks on result:
other: at 0.84 mg/L
Parameter:
% degradation (O2 consumption)
Value:
76
Sampling time:
28 d
Remarks on result:
other: at 2.01 mg/L
Results with reference substance:
The reference substance was degraded to 89% after 7 d and 106% after 14 d.

The toxicity control containing both, test item (2 mg/L) and reference item (4 mg/L) was degraded to 53% after 7 d and 67% after 14 d. This result indicates that the test item is not inhibitory to the inoculum.

The oxygen depletion in the inoculum blank was 1.15 mg O2/L after 28 days of incubation. This is within the limit given in the guideline of 1.5 mg O2/L.

Table 1: Biodegradation of test item, reference substance and toxicity control

Flask

Biodegradability [%] after n-days

0

7

14

21

28

Test substance mean (0.84 mg/L)

0

64

92

90

92

Test substance min (0.84 mg/L)

61

88

88

90

Test substance max (0.84 mg/L)

68

96

93

95

Test substance mean (2.0 mg/L)

0

61

72

76

76

Test substance min (2.0 mg/L)

61

70

74

75

Test substance max (2.0 mg/L)

62

73

77

77

Reference substance mean

0

86

103

n.d.

n.d.

Reference substance min

83

99

n.d.

n.d.

Reference substance max

89

106

n.d.

n.d.

Toxicity control mean

0

53

67

n.d.

n.d.

Toxicity control min

53

65

n.d.

n.d.

Toxicity control max

54

68

n.d.

n.d.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Readily biodegradable: 76-92% degradation after 28 d.
Executive summary:

The objective of this study was to determine the biodegradability of test item in a ‘Closed Bottle Test’ as described in the OECD Guideline 301D and GLP, using oxygen depletion as test criterion in a 28 day test. The test substance was added to the inoculated medium by means of a stock solution of test item in ultrapure water. Aliquots of this stock solution were added to inoculated medium to obtain test substance concentrations of 0.84 and 2.01 mg/L, corresponding to a theoretical oxygen demand (ThOD) of 2.50 and 5.97 mg O2/L, respectively. An inoculum was prepared from activated sludge taken from an oxidation ditch used to treat domestic sewage.

The test fulfilled the conditions of validity given by the Guidelines. Already after 14 days a maximum mean biodegradability of 92% was determined at a test concentration of 0.84 mg/l. At a test concentration of 2.01 mg/L a maximum biodegradability of 76% was determined after 21 days. Test item is regarded to be ready biodegradable according to the guidelines.

Description of key information

Readily biodegradable: 79 - 92% degradation after 28 days based on O2 consumption (OECD 301D); read-across

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Since no data for the target substance are available, the biodegradability is assessed on the basis of data from two structurally related menthols (stereo isomers). The investigations with both source substances (L-menthol, CAS 2216-51-5 and D-menthol, CAS 15356-60-2) were carried out under GLP conditions according to OECD guideline 301D (closed bottle test). Microorganisms from municipal sewage treatment plants were used as inoculum to determine the ready biodegradability. According to the closed bottle test design relatively low test item concentrations of 0.84 and 2.0 mg/L were used, and the degradation rate was determined based on the measurement of the oxygen consumption. In the key study with L-menthol (CAS 2216-51-5) a degradation of 79-92% was recorded after 28 d test duration. This result was confirmed by the supporting study with D-menthol (CAS 15356-60-2), where 76-92% degradation was determined after a test duration of 28 d. The pass level of 60% was reached in both closed bottle tests within 10 days window. Thus, both menthol isomers can be regarded as readily biodegradable according to OECD criteria. The target substance, which is an isomer mixture of the two read across substances, is considered as a readily biodegradable substance.

[Type of water: freshwater]