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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:
From June 29, 2017 to September 04, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
Activated sludge was obtained from Totnes Sewage Treatment Works, Totnes, Devon, UK on 28 June 2017. This works treats sewage of predominantly domestic origin. At the laboratory, the activated sludge was kept aerated at room temperature and the pH maintained at 7.0 ± 1.0. Seven days prior to the exposure start the activated sludge was centrifuged, washed and re-suspended in the mineral medium and the solids concentration determined. This sludge was then diluted in medium, added to test bottles and stirred until required for use. The seeded mineral medium was pre-conditioned for seven days to reduce the blank oxygen uptake readings in the test.
Duration of test (contact time):
ca. 60 d
Initial conc.:
ca. 100 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test apparatus
The measurement of oxygen uptake was conducted in the Oxitop™ respirometers (Wissenschaftlich-Technische Werkstätten, GmbH, Germany). Each individual unit consisted of a dark glass 500 mL bottle with an Oxitop™ bottle top containing a piezo resistive electronic pressure sensor. Bottles were situated on a magnetic stirrer in a constant temperature incubator. Carbon dioxide produced by microbial respiration was absorbed by potassium hydroxide solution (KOH) placed in a seal cup in the neck of each bottle, and the oxygen taken up was measured as a decrease in pressure. The Oxitop™ controller collected the pressure values from the measuring tops and calculated the BOD (as mg/L).
Reference substance:
benzoic acid, sodium salt
Remarks:
100 mg/L
Test performance:
Validity Criteria
The validity requirements of the OECD guideline state:
·  The difference between extremes of replicate biodegradation values should be less than 20% at the end of 10-day window, at the plateau or at the end of the test;
·  The positive control should achieve > 60% biodegradation by Day 14. 
·  The oxygen consumption of the inoculum blank should not exceed 60 mg/L in 28 d.
Mean oxygen uptake of the inoculum blank was below 60 mg/L, as required in the OECD guideline. The difference between replicate test substance extremes was < 20% after the 10 d window, at plateau, and on day 60. Sodium benzoate reached a mean 68% biodegradation by Day 14. The average of the mean oxygen consumed in the inoculum blanks was 11.3 mg/L after 28 d and 18.9 mg/L after 60 d. Therefore, this test has satisfied all the validity criteria.
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Test substance
Value:
ca. 3
Sampling time:
28 d
Remarks on result:
other: <60% Biodegradation confirmed that the test substance cannot be classified as readiily biodegradable.
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Reference substance
Value:
ca. 68
Sampling time:
14 d
Remarks on result:
other: >60% Biodegradability confirmed that the activated sludge contained viabale organism
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Toxicity control
Value:
ca. 22
Sampling time:
14 d
Remarks on result:
other: <25% Biodegradability confirmed that the test substance is assumed to be inhibitory at used concentration
Details on results:
pH and temperature measurements
At the end of the 60-d test period, the pH values ranged from 7.1 to 7.2 in the inoculum blank bottles, from 6.8 to 6.9 in the sodium benzoate bottles and from 7.5 to 7.7 in the test substance bottles and from 6.8 to 6.9 in the toxicity control bottles. Temperature measurements recorded in several of the test bottles on Days 0 and 60 indicated the temperature was within the range 22 ± 1°C. Continuous monitoring of the incubator temperature showed it to have remained within the range 22 ± 1°C throughout the study.
Results with reference substance:
Sodium benzoate did attain a mean level of biodegradation of 68% after 60 d, and the results showed good replication. More than 60% degradation was achieved within the 10 d window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms.

Results

Theoretical oxygen demand (ThOD)

The ThOD of sodium benzoate was calculated as 1.67 g O2/g of substance, using the formula given in the OECD guideline. Biodegradation of sodium benzoate has been calculated on the basis of calculated ThOD.

Chemical oxygen demand (COD)

The measured COD value of test substance was 2.52 g O2/g of substance, and its biodegradation was calculated on the basis of measured COD.

Biochemical oxygen demand (BOD)

The results for the reference substance did show that sodium benzoate attained a mean level of biodegradation (based on the BOD:ThOD ratio) of 68% after 60 d, and also showed a good replication. More than 60% degradation was achieved within the 10 d window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms.

Biodegradation results: Reference substance

 

Day

Sodium benzoate (blank corrected) BOD(mg/L)

 

BODa (gO2/g)

 

Biodegradationb (%)

Rep1

Rep2

Rep3

Rep1

Rep2

Rep3

Rep1

Rep2

Rep3

Mean

7

106

106

106

1.06

1.06

1.06

63

63

63

63

14

113

113

116

1.13

1.13

1.16

68

68

69

68

21

114

117

117

1.14

1.17

1.17

68

70

70

69

28

116

118

118

1.16

1.18

1.18

69

71

71

70

35

111

108

119

1.11

1.08

1.19

66

65

71

67

42

112

109

117

1.12

1.09

1.17

67

65

70

67

49

112

110

118

1.12

1.10

1.18

67

66

71

68

56

114

109

114

1.14

1.09

1.14

68

65

68

67

60

113

110

119

1.13

1.10

1.19

68

66

71

68

BOD: Biological Oxygen Demand

a BOD (g O2/g) =     Blank corrected biodegradation (mg/L)         

                             Sodium benzoate concentration (100 mg/L)

b Biodegradation (%) =Biological Oxygen Demand (g O2/g)× 100

                                                ThOD (1.67 g O2/g)

The following phases (approximate) were observed:

Phase

Time period

Lag phase

Days 0 to 1

Degradation phase

Days 1 to 7

10-day window

Days 1 to 11

The results for test substance did show that it attained a mean level of biodegradation (based on the BOD:COD ratio) of 3% after 28 d and 4% after 60 d, and also showed a good replication. Less than 60% degradation was achieved within 10 d window, so test substance could not be classified as readily biodegradable.

Biodegradation results: Test substance

 

Day

Test substance

(blank corrected)

BOD (mg/L)

BODa (gO2/g)

Biodegradationb (%)

Rep1

Rep2

Rep3

Rep1

Rep2

Rep3

Rep1

Rep2

Rep3

Mean

7

4.0

4.0

4.0

0.04

0.04

0.04

2

2

2

2

14

3.7

9.3

6.5

0.04

0.09

0.07

2

4

3

3

21

1.6

10.0

7.2

0.02

0.10

0.07

1

4

3

3

28

5.6

11.2

5.6

0.06

0.11

0.06

2

4

2

3

35

0.6

3.4

3.4

0.01

0.03

0.03

0

1

1

1

42

4.6

1.8

7.4

0.05

0.02

0.07

2

1

3

2

49

5.8

5.8

8.6

0.06

0.06

0.09

2

2

4

3

56

7.2

10.0

10.0

0.07

0.10

0.10

3

4

4

4

60

9.2

12.0

9.2

0.09

0.12

0.09

4

5

4

4

BOD:   Biological Oxygen Demand

a Biological oxygen demand (g O2/g) =Blank corrected biodegradation (mg/L)

                                               Substance concentration (100 mg/L)

b Biodegradation (%) =Biological Oxygen Demand (g O2/g) × 100

                                    COD (2.52 g O2/g)

The mean toxicity control degradation achieved on Day 14 was 22% (based on combined ThOD/COD), as this was <25% the test substance was assumed to be inhibitory at this concentration. 

 

Day

Toxicity control

(blank corrected)

BOD (mg/L)

BODa (gO2/g)

Biodegradationb (%)

Rep1

Rep2

Rep3

Rep1

Rep2

Rep3

Rep1

Rep2

Rep3

Mean

7

88.4

49.1

106

0.44

0.25

0.53

21

12

25

19

14

93.4

74.0

107

0.47

0.37

0.54

22

18

26

22

21

97.3

88.8

108

0.49

0.44

0.54

23

21

26

23

28

102

98.7

110

0.51

0.49

0.55

24

23

26

24

35

102

116

108

0.51

0.58

0.54

24

28

26

26

42

106

120

109

0.53

0.60

0.55

25

29

26

27

49

110

124

110

0.55

0.62

0.55

26

30

26

27

56

109

120

109

0.55

0.60

0.55

26

29

26

27

60

110

122

108

0.55

0.61

0.54

26

29

26

27

 BOD: Biological Oxygen Demand

a Biological oxygen demand (g O2/g) = Blank corrected biodegradation (mg/L) 

                                            Toxicity control concentration (200 mg/L)

b Biodegradation (%) =Biological Oxygen Demand (g O2/g) × 100

                                     Combined ThOD/COD (2.10 g O2/g)

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the study conditions, the test substance was not readily biodegradable.
Executive summary:

A study was conducted to determine to study the biodegradability of the test substance, 'di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS' (active: 101%) according to OECD Guideline 301F and EU method C.4-D by Manometric respirometry test, in compliance with GLP. Following the pre-conditioning, the test bottles (Prepared activated sludge inoculum in mineral medium) were set up for the test according to the experimental design. In the study, Inoculum blanks, positive control (Reference substance - sodium benzoate at 100 mg/L), test substance (100 mg/L), and toxicity controls (Test and reference substances, both at 100 mg/L), each these group bottles were prepared in triplicate, except inoculum blanks which were prepared in duplicate. Additional single replicate test substance and toxicity control bottles were prepared for determination of pH at Day 0, so undissolved test substance was not removed by the pH probe. Oxygen uptake was recorded automatically every 240 minutes during the 60 d experimental period. Oxygen uptake values were corrected for the inoculum blank and the biodegradation was calculated as a percentage of the chemical oxygen demand for the substance under test and as a percentage of the theoretical oxygen demand for the reference substance. The chemical oxygen demand (COD) of test substance was determined using spectrophotometer for evaluation. Test substance attained a mean level of biodegradation (based on the BOD:COD ratio) of 3% after 28 d and 4% after 60 d. The test substance results showed good replication. Sodium benzoate attained a mean level of biodegradation (based on the BOD:ThOD ratio) of 68% after 60 d, and also showed a good replication. More than 60% degradation was achieved within the 10 d window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. The mean toxicity control degradation achieved on Day 14 was 22% (based on combined ThOD/COD), as this was < 25% the test substance was assumed to be inhibitory at this concentration. The test was considered to have satisfied all the validity criteria. Under the study conditions, the test substance was not readily biodegradable (Scymaris, 2017).

Description of key information

Based on the study results, the test substance is considered to be not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A study was conducted to determine to study the biodegradability of the test substance, 'di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS' (active: 101%) according to OECD Guideline 301F and EU method C.4-D by Manometric respirometry test, in compliance with GLP. Following the pre-conditioning, the test bottles (Prepared activated sludge inoculum in mineral medium) were set up for the test according to the experimental design. In the study, Inoculum blanks, positive control (Reference substance - sodium benzoate at 100 mg/L), test substance (100 mg/L), and toxicity controls (Test and reference substances, both at 100 mg/L), each these group bottles were prepared in triplicate, except inoculum blanks which were prepared in duplicate. Additional single replicate test substance and toxicity control bottles were prepared for determination of pH at Day 0, so undissolved test substance was not removed by the pH probe. Oxygen uptake was recorded automatically every 240 minutes during the 60 d experimental period. Oxygen uptake values were corrected for the inoculum blank and the biodegradation was calculated as a percentage of the chemical oxygen demand for the substance under test and as a percentage of the theoretical oxygen demand for the reference substance. The chemical oxygen demand (COD) of test substance was determined using spectrophotometer for evaluation. Test substance attained a mean level of biodegradation (based on the BOD:COD ratio) of 3% after 28 d and 4% after 60 d. The test substance results showed good replication. Sodium benzoate attained a mean level of biodegradation (based on the BOD:ThOD ratio) of 68% after 60 d, and also showed a good replication. More than 60% degradation was achieved within the 10 d window as expected for a biodegradable substance, thus confirming that the activated sludge contained viable organisms. The mean toxicity control degradation achieved on Day 14 was 22% (based on combined ThOD/COD), as this was < 25% the test substance was assumed to be inhibitory at this concentration. The test was considered to have satisfied all the validity criteria. Under the study conditions, the test substance was not readily biodegradable (Scymaris, 2017).

[Type of water: freshwater]