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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:
Experimental start date: 06 November 2017 Experimental completion date: 04 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study conducted according to OECD TG 301F in compliance with GLP, without deviations that influence the quality of the results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: FRET 11-0539
CAS Number: 1631962-93-0
Physical state/Appearance: clear colorless liquid
Storage Conditions: room temperature in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
other: mixed population of sewage treatment micro-organisms
Details on inoculum:
Test System and Supporting Information
A mixed population of sewage treatment micro-organisms was obtained on 01 November 2017 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 Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at 21 ± 1 ºC prior to use.
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 Design and Study Conduct
Preliminary Solubility Work
Information provided by the Sponsor indicated that the water solubility of the test item was 3.4 mg/L. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation for this study.

Test Item Preparation
Following preliminary solubility work and the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper*. High shear mixing was also applied to break up the filter paper containing the test item. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.
A nominal amount of test item (1000 mg) was dissolved in 10 mL of acetone to give a
1000 mg/10 mL solvent stock solution. An aliquot (500 µL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition of inoculum. The volume was then adjusted to 500 mL with mineral medium to give a final concentration of 100 mg/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.
A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guidelines.
A filter paper* was added to each inoculum control vessel in order to maintain consistency between the test and control vessels. Acetone (500 µL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition of inoculum and adjusting to a final volume of 500 mL with mineral media for the inoculum control vessel.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Reference Item Preparation
A reference item, aniline (C6H5NH2), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 10 minutes. A filter paper* was added to each vessel in order to maintain consistency between the test and procedure control vessels. Acetone (500 µL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately
350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm,
5 minutes) prior to addition to each vessel. An aliquot (50 mL) of this stock solution was added to each test vessel with the inoculum (5 mL) and the volume adjusted to 500 mL with mineral media, to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.

Toxicity Control
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the test.
An aliquot (500 µL) of the test item solvent stock solution was dispensed onto a filter paper* and the solvent allowed to evaporate for approximately 15 minutes. The filter paper was dispersed in approximately 350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to the test vessel with an aliquot
(50 mL) of the 1000 mg/L aniline stock solution (see Section 3.5.2) and inoculum (5 mL). The volume was adjusted to 500 mL with mineral medium to give a final concentration of 100 mg test item/L plus 100 mg aniline/L.

Preparation of Test System
The 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 plus a filter paper.
b) Two replicate bottles containing inoculated mineral medium plus a filter paper and the reference item, aniline, at a concentration of 100 mg/L.
c) Three replicate bottles containing the test item on filter paper*in inoculated mineral medium at a concentration of 100 mg/L.
d) Two replicate bottles containing the test item on a filter paper* in inoculated mineral medium, and 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.
A filter paper with acetone evaporated to dryness was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item 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 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 was prepared correctly, a diluted, 100 mg/L stock solution (in reverse osmosis water) was also sampled for Dissolved Organic Carbon (DOC) analysis.
All remaining 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 (see Figure 1). 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 temperatures of between 21 and 22 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.
Reference substance:
aniline
Test performance:
Validation Criteria and Biodegradation
The mean BOD of the inoculated mineral medium (control) was 27.84 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 ranged from 7.6 to 7.7 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.
The toxicity control attained 30% biodegradation after 14 days and 32% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Key result
Parameter:
% degradation (O2 consumption)
Value:
1
Sampling time:
28 d
Details on results:
The test item attained 1% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
Results with reference substance:
Aniline (procedure control) attained 61% biodegradation after 14 days and 70% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

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

0.92

0.80

1.16

0.30

0.58

0.78

2

1.42

1.20

2.42

1.54

2.00

1.90

3

2.66

2.42

3.46

2.42

3.00

4.34

4

4.16

3.70

4.50

3.30

4.04

5.82

5

5.42

4.78

7.42

4.34

5.16

30.52

6

6.50

5.74

36.36

5.24

6.34

67.60

7

7.74

6.96

95.34

6.28

7.66

104.06

8

8.46

7.84

169.40

7.04

8.58

141.10

9

9.16

8.74

184.14

7.62

9.42

172.90

10

10.24

9.78

189.04

8.66

10.58

181.88

11

12.20

11.32

193.64

10.28

12.38

185.76

12

13.32

12.42

197.26

11.50

13.78

189.76

13

14.42

13.46

200.72

12.58

15.20

193.76

14

15.16

14.24

203.92

13.36

16.74

196.72

15

15.66

14.78

207.00

14.00

17.96

199.32

16

16.66

15.62

210.50

15.04

19.20

200.88

17

17.82

16.74

214.24

16.36

20.50

203.26

18

19.24

18.16

218.20

18.00

22.12

205.12

19

20.32

19.28

221.54

19.24

23.36

206.90

20

21.20

20.20

224.54

20.12

24.50

208.50

21

21.90

20.94

227.16

20.82

25.40

210.42

22

23.00

22.12

229.90

22.04

26.78

211.96

23

24.08

23.20

232.50

23.04

28.12

214.02

24

25.28

24.32

234.98

24.20

29.70

215.90

25

26.16

25.20

237.20

25.08

30.86

218.26

26

26.98

25.94

239.28

26.12

31.86

220.52

27

27.58

26.54

241.08

26.90

32.62

222.26

28

28.32

27.36

242.74

27.78

33.48

223.54

 

Percentage Biodegradation Values

Day

% Biodegradation

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

0

0

0

0

0

4

0

0

0

0

0

5

1

0

0

0

4

6

10

0

0

0

10

7

28

0

0

0

16

8

52

0

0

0

22

9

57

0

0

0

27

10

58

0

0

0

28

11

59

-1

0

0

29

12

60

0

0

0

29

13

60

0

0

0

30

14

61

0

1

1

30

15

62

0

1

1

30

16

63

0

1

1

31

17

64

0

1

1

31

18

65

0

1

1

31

19

65

0

1

1

31

20

66

0

1

1

31

21

67

0

1

1

31

22

67

0

1

1

31

23

68

0

2

1

32

24

68

0

2

1

32

25

68

0

2

1

32

26

69

0

2

1

32

27

69

0

2

1

32

28

70

0

2

1

32

 

pH Values of the Test Preparationson Days 0 and 28

Test Vessel

pH

Day 0

Day 28

Inoculum ControlR1

7.4

7.6

Inoculum Control R2

7.4

7.6

Procedure Control

7.4

7.9

Test Item R1

7.4

7.7

Test Item R2

7.4

7.6

Toxicity Control

7.4

7.6

 

 


R1– R2= Replicates 1 and 2

R     =      Replicate

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The substance showed 1% biodegradation in an OECD TG 301F test and is considered to be not readily biodegradable.
Executive summary:

The ready biodegradability of FRET 11 -0539 was investigated in a study conducted in accordance with OECD TG 301F (Manometric Respirometry Test) and GLP. The concentration tested was 100 mg/l test substance, with an activated sludge concentration of 1 % v/v. The test substance attained 1% biodegradation after 28 days. Aniline (procedure control) attained 61% biodegradation after 14 days and 70% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Description of key information

The ready biodegradability of FRET 11 -0539 was investigated in a study conducted in accordance with OECD TG 301F (Manometric Respirometry Test) and GLP. The concentration tested was 100 mg/l test substance, with an activated sludge concentration of 1 % v/v. The test substanceattained 1% biodegradation after 28 days.Aniline (procedure control) attained 61% biodegradation after 14 days and 70% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information