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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:
The study was conducted between 01 July 2015 and 29 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):Not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Test SystemA mixed population of sewage treatment micro-organisms was obtained on 30 June 2015 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.Preparation of InoculumThe sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at temperatures of between 21 and 26 ºC prior to use. This was a deviation to the study plan which states that the inoculum is maintained on aeration prior to use at test temperature. However the temperature only deviated above the range of 22 ± 1 ºC for approximately 3 hours before the inoculum was used and the study started. Therefore it was considered to have no effect on the integrity of the study given that all validation criterion were met.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Experimental PreparationTest ItemThe test item was dispersed directly in mineral medium.An amount of test item (500 mg) was dispersed in mineral medium and the volume adjusted to 500 mL to give a 1000 mg/L stock dispersion. An aliquot (50 mL) of this stock dispersion was diluted with mineral medium (445 mL) and inoculum (5 mL) to give the final test concentration of 100 mg/L. The volumetric flasks containing the stock solution and the test concentration were inverted several times to ensure homogeneity.A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guideline. Preparation of Test SystemThe following test preparations were prepared and inoculated in 500 mL bottles:a) Three replicate bottles containing inoculated mineral medium to act as the inoculum control. b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L. c) Three replicate bottles containing inoculated mineral medium and the test item at a concentration of 100 mg/L. d) Two replicate bottles containing inoculated mineral medium the reference item, aniline, at a concentration of 100 mg/L and the test item at a concentration of 100 mg/L to act as toxicity control vessels.All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. The pH values were adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the addition of the inoculum and the volume in all the vessels being adjusted to 500 mL by the addition of mineral medium. In order to confirm that the aniline stock solution and test item stock dispersion were prepared correctly, a diluted, 100 mg/L stock (in reverse osmosis water) was also sampled for DOC analysis.All inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask. The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated computer.The test was conducted in diffuse light at a temperature of 22 ± 1 ºC.On Day 28, two inoculum control, one procedure control, two test item and one toxicity control vessel were sampled for pH analysis.The remaining vessels which were not sampled were discarded and are not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.EvaluationsOxygen Consumption MeasurementsThe daily Biological Oxygen Demand (BOD) values for the inoculum control, test item, procedure control and the toxicity control are given.Physico-chemical MeasurementsThe temperature of the water bath was recorded daily.pH MeasurementsIn order to confirm whether the pH of the test preparations changed, the pH was measured using a Hach HQ40d Flexi handheld meter on Days 0 and 28.Data EvaluationCalculation of Theoretical Oxygen DemandThe Theoretical Oxygen Demand (ThOD) for a compound CcHhClclNnPpSsOoNana was calculated by:ThOD (NO3) (mg O2 / mg) = (16 (2c + 1/2 (h-cl) + 5/2 n + 5/2 p + 3s + 1/2 na – o) / molecular weightPercentage BiodegradationThe percentage biodegradation in terms of oxygen consumption was calculated as follows:% degradation = ((BOD - B) / ThOD) x 1000Where:BOD = Biological Oxygen Demand of the test item or reference item (mgO2/L)B = Oxygen consumption in basal mineral medium to which inoculum is added (control) (mgO2/L)ThOD = Theoretical oxygen demand to completely oxidize the reference and/or test item (mgO2/L)
Reference substance:
aniline
Preliminary study:
No data
Test performance:
No data
Parameter:
% degradation (O2 consumption)
Value:
70
Sampling time:
28 d
Details on results:
BiodegradationThe test item attained 70% biodegradation after 28 days, calculated from the oxygen consumption values. Despite attaining in excess of 60% biodegradation the test item failed to satisfy the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation rate exceeding 10%, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F. However, the test item has exhibited the potential for rapid biodegradation.The toxicity control attained 59% biodegradation after 14 days and 66% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.
Results with reference substance:
Aniline (procedure control) attained 73% biodegradation after 14 days and 78% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validation Criteria

The mean BOD of the inoculated mineral medium (control) was 26.30 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

 

The pH of the inoculated test item vessels on Day 28 were 7.5 and hence satisfied the validation criterion given in the OECD Test Guidelines.

 

The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Biological Oxygen Demand (BOD) Values

Day

BOD (mg O2/L)

Inoculum Control

Procedure Control

Test Item

Toxicity Control

R1

R2

R1

R2

0

0.00

0.00

0.00

0.00

0.00

0.00

1

0.00

0.00

0.00

0.00

0.00

0.00

2

0.70

2.00

1.00

1.58

1.08

3.50

3

1.74

3.08

6.46

32.48

32.28

44.90

4

3.58

4.84

79.34

79.96

77.60

130.98

5

5.12

6.28

166.30

88.42

83.84

184.38

6

5.78

6.74

201.38

94.26

88.84

246.66

7

7.04

7.62

212.08

101.50

93.18

266.26

8

8.50

8.74

217.24

107.42

97.54

275.44

9

9.38

9.38

221.12

113.50

102.16

301.50

10

10.12

9.88

224.82

125.00

110.24

319.24

11

11.24

10.88

229.24

131.40

122.82

328.28

12

12.42

11.66

233.70

136.44

130.40

334.78

13

13.32

12.12

237.36

140.78

139.32

339.40

14

14.42

12.78

240.62

144.74

145.78

343.98

15

16.16

13.96

245.16

149.48

152.90

349.20

16

17.20

14.54

248.94

153.18

158.82

353.32

17

18.40

15.32

252.06

156.90

165.06

357.98

18

19.36

15.86

254.28

160.36

171.30

363.40

19

20.36

16.50

256.28

163.86

178.10

367.26

20

21.08

16.78

257.74

166.98

182.46

370.10

21

22.08

17.40

259.40

170.44

186.80

372.98

22

23.24

18.24

260.98

173.94

190.18

375.94

23

24.20

18.86

262.22

177.18

193.30

379.06

24

25.12

19.50

263.36

180.52

196.38

382.72

25

25.98

20.08

264.40

183.84

199.46

386.30

26

26.66

21.90

265.06

187.06

201.84

389.02

27

27.70

23.00

266.14

190.84

204.26

391.88

28

28.54

24.06

267.02

193.84

205.88

394.00

R1– R2= Replicates 1 and 2

Percentage Biodegradation Values

Day

Procedure Control

Test Item

Toxicity Control

R1

R2

Mean

0

0

0

0

0

0

1

0

0

0

0

0

2

0

0

0

0

0

3

1

12

12

12

8

4

24

30

29

30

23

5

52

33

31

32

32

6

63

35

33

34

43

7

66

38

34

36

46

8

68

40

36

38

48

9

69

42

37

40

52

10

70

46

40

43

55

11

71

48

45

47

57

12

72

50

47

49

58

13

73

51

51

51

58

14

73

52

53

53

59

15

74

54

55

55

60

16

75

55

57

56

60

17

76

56

59

58

61

18

77

57

61

59

62

19

77

58

64

61

62

20

77

59

65

62

63

21

78

60

67

64

63

22

78

61

68

65

64

23

78

62

69

66

64

24

78

63

70

67

64

25

78

64

71

68

65

26

78

65

71

68

65

27

78

66

72

69

66

28

78

67

72

70

66

R1– R2= Replicates 1 and 2

pH Values of the Test Preparations on Days 0 and 28

Test Vessel

pH

Day 0
Pre-Adjustment

Day 0
Post-Adjustment

Day 28

Inoculum ControlR1

7.8

7.4

7.8

Inoculum Control R2

7.8

7.4

7.7

Procedure Control

7.7

7.4

7.9

Test Item R1

7.8

7.4

7.5

Test Item R2

7.7

7.4

7.5

Toxicity Control

7.7

7.4

7.3

 R1– R2 = Replicates 1 and 2

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The test item attained 70% biodegradation after 28 days. Despite attaining in excess of 60% biodegradation the test item failed to satisfy the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation rate exceeding 10%, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F. However, the test item has exhibited the potential for rapid biodegradation.
Executive summary:

Introduction

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q)).

 

Methods….

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 21 and 23 ºC for 28 days. 

 

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values on Days 0 to 28. Control solutions with inoculum and the reference item, aniline, together with a toxicity control were used for validation purposes.

 

Results….

The test item attained 70% biodegradation after 28 days. Despite attaining in excess of 60% biodegradation the test item failed to satisfy the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation rate exceeding 10%, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.  However, the test item has exhibited the potential for rapid biodegradation.

Description of key information

The test item attained 70% biodegradation after 28 days. Despite attaining in excess of 60% biodegradation the test item failed to satisfy the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation rate exceeding 10%, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.  However, the test item has exhibited the potential for rapid biodegradation.

Key value for chemical safety assessment

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

Additional information