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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:
From 19 June 2019 to 21 August 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The test substance is adequately identified but some data on composition is missing. Therefore, validation applies with restrictions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 21/08/2018; Date of issue: 19/11/2018
Specific details on test material used for the study:
- Appearance: Pale yellow extremely viscous liquid
- Molecular Formula: C16H26O
- Molecular Weight: 234.4 g/mol
- Water Solubility: 8.07 mg/L at 25 ºC (iSafeRat ® v1.8)
- Vapour Pressure: 0.112 Pa at 25 ºC (iSafeRat ® v1.3)
- Log Kow: 5.0 at 20 °C (EEC A8, Shake-Flask Method, Phytosafe 2012)
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 mixed population of sewage treatment micro-organisms was obtained on 17 June 2019 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 for exposure: 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 temperatures of between 20 and 21 °C prior to use.
Duration of test (contact time):
60 d
Initial conc.:
100 mg/L
Based on:
test mat.
Initial conc.:
300 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
The BOD values for the inoculum control, test item, procedure control and the toxicity control were measured daily.
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral medium used in this study was that recommended in the OECD Guidelines. The deionized reverse osmosis water used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).
The pH of the mineral media was measured to be pH 7.7 and was adjusted to pH 7.4 using a diluted hydrochloric acid solution.

TEST SYSTEM
The following test preparations were prepared and inoculated in 500 mL amber glass bottles:
a) Five 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 to act as the procedure control.
c) Five replicate bottles containing inoculated mineral medium and the test item at a concentration of 100 mg/L.
d) Two replicate bottles containing the test item at a concentration of 100 mg/L in inoculated mineral medium plus the reference item, aniline, 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 to the mineral medium. The pH of the inoculum control and procedure control vessels were 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. The pH of the contents of the test item and toxicity control vessels were not measured prior to the addition of the inoculum due to the risk of test item being lost from the test system by adherence to the pH probe.
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 Total Organic Carbon (TOC) analysis.
Two inoculum control and two test item vessels were sacrificed for compound specific analysis on Day 0 and 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. 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 four times a day on the hard disk drive of a non-dedicated computer.
The test was conducted in diffuse light at temperatures of between 21 and 24 °C.
On Day 60, two inoculum controls, one procedure control, two test item and one toxicity control vessel that were considered to have given the most consistent BOD values with the smaller difference between the extremes in replicate BOD values over the study period were initially sampled for compound specific analysis and/or pH analysis.
At the end of the test, the remaining inoculum control and test item vessels which were not sampled were each separately split into three separate storage bottles and stored frozen for further analysis if required. The contents of the three frozen test item storage bottles containing test item plus inoculum R1 were analyzed at a later date due to a suspected erroneous analytical result from one of the duplicate test item vessels.
The remaining vessels which were not sampled were 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. However in this study, the extra inoculated test item vessel that was frozen and not initially selected for reporting was subsequently analyzed for compound specific analysis due to an erroneous analytical result from one of the duplicate test item vessels. This vessel was not initially selected for analysis and reporting as the percentage difference between the BOD values in the other test item vessel was greater than the two that had been selected.
Therefore as the extremes of the replicate BOD values from the three replicate test item vessels were less than 20%, it was considered that the BOD values from the three replicate test item plus inoculum vessels were selected for reporting.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes

TEST ITEM PREPARATION
A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) and subjected to ultrasonic disruption for 15 minutes prior to allowing to cool to temperatures of approximately 21 ºC. The inoculum (5 mL) was then added and the volume adjusted to a final volume of 500 mL with mineral medium to give the test concentration of 100 mg/L.
The inoculum control vessels were prepared in a similar manner without the addition of test item and ultrasonication. However prior to the addition of the inoculum the pH of the mineral media in each inoculum control vessel was measured using a Hach HQ40d Flexi handheld meter.
A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guidelines.

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 a nominal amount of freshly distilled aniline (500 mg), directly in mineral medium (500 mL) with the aid of ultrasonication for approximately 20 minutes. After allowing to cool to approximately 21 °C, an aliquot (50 mL) of this stock solution was diluted with mineral medium (350 mL) prior to measurement of the pH value using a Hach HQ40d Flexi handheld meter and then addition of inoculum (5 mL) and adjusting to a final volume of 500 mL with mineral medium, to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.
The pH of the reference item stock solution was 7.4. The pH value was measured using a Hach HQ40d Flexi handheld meter.

TOXICITY CONTROL PREPARATION
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effects of the test item on the sewage treatment micro-organisms used in the test.
A nominal amount of test item (50 mg) was dispersed in mineral medium (350 mL) and subjected to ultrasonication for 15 minutes prior to allowing to cool to temperatures of approximately 21 ºC and the addition of an aliquot (50 mL) of the 1000 mg/L aniline stock solution. The inoculum (5 mL) was then added prior to adjusting to a final volume of 500 mL with mineral medium to give the test concentration of 100 mg test item/L and 100 mg aniline/L.
Reference substance:
aniline
Remarks:
Purity: 99+%; Batch A0358342
Preliminary study:
Information provided by the Sponsor indicated that the water solubility of the test item was close to 8.0 mg/L at 25 ºC (iSafeRat ® v1.8). Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation. From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, 1995) it was concluded that the best testable dispersions were found to be obtained when using the ultrasonication, filter paper and the glass slide methods of preparation. As there was no difference in the observations of the test item dispersions it was considered appropriate to use ultrasonication to disperse the test item into the test system.
Key result
Parameter:
% degradation (O2 consumption)
Value:
1
Sampling time:
28 d
Remarks on result:
other: Not readily biodegradable
Key result
Parameter:
% degradation (O2 consumption)
Value:
4
Sampling time:
60 d
Details on results:
The test item attained 1% biodegradation after 28 days and 4% biodegradation after 60 days (mean of three replicate vessels) and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours showed measured concentrations of 107% of nominal were obtained for both replicate vessels (reported as test item plus inoculum R2 and R3 in Tables in "Any other information on results incl. tables"). After 60 days, chemical analysis of these replicate vessels showed measured test concentrations of 17 and 101% of nominal (calculated to be a 83 and 0% loss over the test duration assuming 100% recovery on Day 0). The low measured test item concentration in test item replicate R2 on Day 60 was an erroneous result which may have been due to an error in the work up of the sample prior to analysis.
Therefore, the additional test item vessel that was split into three bottles and frozen on Day 60 (reported as test item plus inoculum R1 in Tables in "Any other information on results incl. tables") was analyzed at a later date due to the erroneous analytical result from test item plus inoculum R2. The measured test concentration of this additional replicate vessel was observed on Day 60 to be 83% of nominal. This vessel was not initially selected for analysis as the percentage difference between the BOD values in this other test item vessel was greater than the two that had been selected initially. However the biodegradation values of 6, 2 and 3% from the three replicate test preparations containing inoculum on Day 60 were similar indicating that the measured concentration determined form analytical analysis on Day 60 for test item plus inoculum R2 was erroneous.
Results with reference substance:
Total Organic Carbon of the diluted aniline stock solution confirmed that it had been prepared correctly.
Aniline (procedure control) attained 73% biodegradation after 14 days in a 10-Day Window, 74% biodegradation after 28 days and 75% biodegradation after 60 days thereby confirming the suitability of the inoculum and test conditions (> 60%, in a 10 -day window, after 14 days).

Table 5.2.1/1: Biological Oxygen Demand Values

Day

BOD (mg O2/L)

Inoculum Control

Procedure Control

Test Item

Toxicity Control

R1

R2

R1

R2

R3

0

0.00

0.00

0.00

0.00

0.00

0.00

0.00

1

0.34

0.54

0.46

0.88

0.74

0.92

0.80

2

1.88

2.08

2.54

2.66

2.54

2.66

2.54

3

2.12

3.30

12.54

3.54

3.74

3.84

3.92

4

3.66

4.54

71.22

4.54

5.08

5.08

38.36

5

4.58

5.50

142.66

5.28

6.58

6.24

106.16

6

5.66

6.42

184.26

5.62

7.70

6.92

171.60

7

7.10

8.42

205.96

7.88

10.04

9.32

183.92

8

10.88

10.00

217.96

9.62

11.74

11.04

189.18

9

12.66

11.04

224.82

10.54

12.78

11.88

193.04

10

14.12

11.78

229.82

11.12

13.74

12.20

196.30

11

15.92

12.66

233.58

11.82

14.66

12.66

199.42

12

16.92

14.66

237.44

14.00

16.54

15.08

203.96

13

17.86

16.16

240.16

15.62

18.04

16.62

207.62

14

18.36

17.46

242.24

17.04

19.28

17.86

210.66

15

18.66

18.28

243.70

17.90

20.24

18.66

213.12

16

18.84

19.08

244.94

19.12

21.16

19.24

215.34

17

19.24

20.16

246.32

20.24

22.40

20.36

218.00

18

19.46

21.74

247.94

22.32

24.04

22.00

221.46

19

19.76

22.90

248.86

23.70

25.20

23.12

223.92

20

19.92

23.86

249.86

24.82

26.28

24.00

225.82

21

20.22

24.36

250.44

25.74

27.12

24.24

227.16

22

21.02

25.62

251.56

27.04

28.36

25.12

229.28

23

21.88

26.74

252.56

28.36

29.58

26.08

231.12

24

23.58

28.32

253.98

30.28

31.16

27.66

233.44

25

24.50

29.44

255.02

31.78

32.28

28.70

235.08

26

26.24

31.02

256.82

34.06

33.98

30.48

237.32

27

27.48

31.98

257.82

35.40

34.90

31.20

238.58

28

28.54

32.78

258.68

36.70

35.74

31.74

239.62

29

28.94

33.94

260.02

38.16

36.86

32.70

240.98

30

29.98

35.48

261.56

40.20

38.24

34.20

242.74

31

30.82

35.94

262.18

41.02

38.82

34.32

243.16

32

32.48

37.66

263.72

43.24

40.44

35.86

245.02

33

33.44

38.16

264.02

44.24

40.86

36.12

245.48

34

33.82

38.16

264.02

44.28

40.90

36.12

245.48

35

34.16

38.16

264.02

44.28

40.90

36.12

245.48

36

35.40

40.16

264.02

46.36

41.86

36.28

246.52

37

36.16

40.52

264.32

47.02

42.40

36.56

246.86

38

37.56

42.74

265.10

49.24

44.14

38.10

248.98

39

38.24

44.86

266.68

52.10

46.40

40.36

251.48

40

39.70

46.64

267.90

54.76

48.14

42.32

253.74

41

40.32

48.02

268.18

56.68

49.40

43.48

254.98

42

40.94

49.32

269.72

58.52

50.72

44.32

255.78

43

41.86

50.90

271.86

60.86

52.48

45.82

257.18

44

43.48

52.44

273.82

63.14

54.18

47.18

258.40

45

43.48

54.02

275.62

65.64

55.86

48.74

259.64

46

45.32

54.66

277.24

67.64

57.20

49.86

260.28

47

45.40

55.76

278.82

68.76

58.24

51.78

261.68

48

46.56

56.72

280.38

69.82

59.52

54.48

264.26

49

46.74

57.84

281.38

70.78

60.52

57.06

266.62

50

48.40

59.84

282.52

71.82

61.28

63.22

268.22

51

48.74

61.12

283.54

72.90

62.10

64.44

269.92

52

50.18

61.90

284.88

74.02

62.86

65.46

271.50

53

51.68

62.82

286.30

75.12

63.76

66.54

273.12

54

52.78

63.78

288.46

76.20

64.62

67.66

274.82

55

55.12

64.62

290.06

77.20

65.66

68.78

276.66

56

57.12

65.42

291.32

78.30

66.68

70.10

279.32

57

58.64

66.06

292.46

79.10

67.40

71.04

280.78

58

59.26

67.18

293.90

80.16

68.36

71.96

281.98

59

60.04

67.78

294.96

81.04

69.46

73.14

283.70

60

61.02

68.32

296.24

82.38

70.34

74.44

285.90


R= Replicate

Table 5.2.1/2: Percentage Biodegradation values

Day

Biodegradation (%)

Procedure Control

Test Item

Toxicity Control

R1

R2

R3

Mean

0

0

0

0

0

0

0

1

0

0

0

0

0

0

2

0

0

0

0

0

0

3

3

0

0

0

0

0

4

22

0

0

0

0

6

5

45

0

1

0

0

17

6

58

0

1

0

0

27

7

64

0

1

1

1

29

8

67

0

0

0

0

29

9

69

0

0

0

0

30

10

70

0

0

0

0

30

11

71

0

0

0

0

30

12

72

0

0

0

0

31

13

72

0

0

0

0

31

14

73

0

0

0

0

32

15

73

0

1

0

0

32

16

73

0

1

0

0

32

17

73

0

1

0

0

33

18

74

1

1

0

1

33

19

74

1

1

1

1

33

20

74

1

1

1

1

33

21

74

1

2

1

1

34

22

74

1

2

1

1

34

23

74

1

2

1

1

34

24

74

1

2

1

1

34

25

74

2

2

1

2

34

26

74

2

2

1

2

34

27

74

2

2

0

1

34

28

74

2

2

0

1

34

29

74

2

2

0

1

34

30

74

2

2

0

1

34

31

74

3

2

0

2

34

32

74

3

2

0

2

34

33

74

3

2

0

2

34

34

74

3

2

0

2

34

35

74

3

2

0

2

34

36

73

3

1

0

1

34

37

73

3

1

0

1

34

38

73

3

1

0

1

34

39

73

4

2

0

2

34

40

73

4

2

0

2

35

41

72

4

2

0

2

35

42

73

4

2

0

2

35

43

73

5

2

0

2

35

44

73

5

2

0

2

35

45

73

6

2

0

3

35

46

74

6

2

0

3

35

47

74

6

3

0

3

35

48

74

6

3

1

3

35

49

74

6

3

2

4

35

50

74

6

2

3

4

35

51

74

6

2

3

4

35

52

74

6

2

3

4

35

53

74

6

2

3

4

35

54

74

6

2

3

4

36

55

74

6

2

3

4

36

56

74

6

2

3

4

36

57

74

6

2

3

4

36

58

75

6

2

3

4

36

59

75

6

2

3

4

36

60

75

6

2

3

4

36


R= Replicate

Table 5.2.1/3: pH values of the test preparations on days 0 and 60

Test Vessel

pH

Day 0

Day 60

Inoculum ControlR1

7.4

7.8

Inoculum Control R2

7.4

8.0

Procedure Control

7.4

8.5

Test Item R1

-

*

Test Item R2

-

8.7

Test Item R3

-

8.7

Toxicity Control

-

8.4


 R  =  Replicate

-      =  The pH of the contents of the test item and toxicity control vessels were not measured prior to the addition of the inoculum due to the risk of test item being lost from the test system by adherence to the  pH probe

*         The pH of the contents of the test item vessel R1on Day 60 was not measured as this vessel was frozen prior to compound specific analysis.

Table 5.2.1/4: Analytical measurements

Time point (Day)

Nominal concentration of test item in test sample (mg/L)

Sample dilution factor (F)

Determined concentration of test item in test sample (mg/L)

Percentage of nominal concentration (%)

0

Control – R1

Control – R2

100 – R1

100 – R2

P. Rec 1 – 99.9

P. Rec 2 – 99.9

0.1

0.1

0.1

0.1

0.1

0.1

<LOQ

<LOQ

107

107

98.5

104

-

-

107

107

99

104

60

Control – R1

Control – R2

100 – R1

100 – R2

100 – R3

P. Rec 1 – 101

P. Rec 2 – 101

0.1

0.1

0.1

0.1

0.1

0.1

0.1

<LOQ

<LOQ

16.9

101

82.8

99.5

98.9

-

-

17

101

83

99

98

100 - R3 replicate analysed after storage frozen due to atypical result in 100 - R1. The P.Recs at 101 mg/L were 90 + 103%.

Note: The Day 60 samples are reported in the Ecotoxicology report as follows:

Sample 100 - R1 is reported as test item plus inoculum R2,

Sample 100 - R2 is reported as test item plus inoculum R3, and

Sample 100 - R3 is reported as test item plus inoculum R1.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item attained 1% biodegradation after 28 days, and 4% biodegradation after 60 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
Executive summary:

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 Guideline 301F, EU Method C.4 -D and EPA OPPTS 835.3110, with GLP statement.

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 24 ºC for 60 days. At the request of the Sponsor the study was extended from 28 to 60 days in order to assess if any further degradation would occur. The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values andcompound specific analyses on Days 0 and 60. Control solutions with inoculum and the reference item, aniline, and a toxicity control were used for validation purposes.

The test item attained 1% biodegradation after 28 days, and 4% biodegradation after 60 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Aniline (procedure control) attained 73% biodegradation after 14 days with greater than 60% degradation being attained in a 10 -Day window. After 28 days 74% biodegradation was attained with 75% biodegradation after 60 days.

Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours resulted in measured concentrations of 107% of nominal were obtained for both replicate vessels (reported as test item plus inoculum R2 and R3). After 60 days, chemical analysis of these replicate vessels resulted in measured test concentrations of 17 and 101% of nominal (calculated to be a 83% loss in R2 and 0% loss in R3 over the test duration assuming 100% recovery on Day 0). The low measured test item concentration in test item replicate R2 on Day 60 was thought ot be due to an error in the work up of the sample prior to analysis.

Therefore, the additional test item vessel (R1) that had been split into three bottles and frozen on Day 60 (reported as test item plus inoculum R1) was analysed at a later date due to the anomalous analytical result from test item plus inoculum R2. The measured test concentration of this additional replicate vessel was observed on Day 60 to be 83% of nominal.

The vessel was not initially selected for analysis as the percentage difference between the BOD values in this other test item vessel was greater than the two that had been selected initially. However, the biodegradation values of 6, 2 and 3% for the three replicate test samples from R1 containing inoculum on Day 60 were similar to each other indicating that the measured concentration determined from analytical analysis on Day 60 for test item plus inoculum R2 was almost certainly erroneous.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2 May 2012 to 2 July 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The test substance is adequately identified but some data on composition is missing. Therefore, validation applies with restrictions.
Qualifier:
according to guideline
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
yes
Remarks:
The mineral medium was erroneously prepared with 1% of phosphate buffer instead of 0.1%. The test item solution was prepared within 2 days before the start of the test.
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspected date: 11 January 2011 / Signed date: 11 March 2011
Specific details on test material used for the study:
- Appearance: Light yellow translucent liquid
- Molecular Formula: C16H26O
- ThOD = 3.01 g/g
- Sotrage: The test substance was stored at room temperature protected from direct sun light.
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): The inoculum was derived from surface waters collected in the near area of Pau, France and receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The freshly collected sample of surface water was previously pre-conditioned to the experimental conditions under aerobic conditions for 5 days at the test temperature in the mineral medium used for testing (400 mL of surface water were added with 200 mL of mineral medium). The resulting solution was continuously stirred for 5 days at 20+/- 1°C. The pre-conditioned inoculum was further used at a rate of 2 mL/L.
Duration of test (contact time):
28 d
Initial conc.:
2.59 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium according to Guideline. The mineral medium was strongly aerated for at least 20 minutes, and then allowed to stand for approximately 20 h at the test temperature before use.
- Test temperature: 20 +/- 1 °C
- Continuous darkness: yes
- Other: each BOD bottles was fitted with a glass stopper

TEST SYSTEM
- Culturing apparatus: 250 mL BOD bottles
- Number of culture flasks/concentration: 2
- Preparation of the bottles: The 250 mL BOD bottles were thoroughly cleaned before use using 5-10 mL of a wash solution (2.5 g iodine plus 12.5 g potassium iodide per litre of 1% w/v sulphuric acid). The bottles were shaken to coat the bottle walls and left to stand for 15 min. The wash solution was poured off and the bottles were thoroughly rinsed with tap water and finally demineralised water.
- Dissolved oxygen: Dissolved oxygen was assessed by the electrode method. The zero-time for dissolved oxygen was measured within 1h after the setting of the test system. Duplicate vessels of each the blank series, the test series and the reference series were sacrificed every 3-4 days for dissolved oxygen analysis. The toxic control series was measured out from one single vessel.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Blank group = mineral medium alone with inoculum (duplicate determinations)
- Abiotic sterile control: no
- Toxicity control: Toxic control = solution containing 2-5 mg/L of each the test substance and aniline in mineral medium, with inoculum (single determinations).
- Other: Reference item = 2-5 mg/L aniline solution in mineral medium with inoculum (duplicate determinations)

PREPARATION OF THE EXPERIMENTAL SOLUTIONS
10 L of water were set at the saturation level using excessive amount of the test item. The volume was placed at 20 +/- 2°C and stirred for the night with a magnetic stirrer. The solution was then allowed to remain undisturbed for 2 hours so that excess of the item separated. The test item concentration was checked in the resulting solution before use. The data showed that the test item concentration was 5.83 mg/L. The solution was then diluted 1/2 in water and the mineral medium was reconstituted. The concentration of the diluted solution was checked to be 2.59 mg/L.
Measured 300 mL volumes were placed in the BOD-bottles provided for the test group and for the toxic control group. The BOD bottles were kept open. On the day after, the test system was set as follows:
- Blank group: 300 mL of mineral medium + 0.6 mL of pre-conditioned inoculum
- Test group: The BOD bottles were added with 0.6 mL of pre-conditioned inoculum.
- Reference group: A stock solution was prepared using 0.1216 g Aniline for 20 mL of water. 300 mL of mineral medium were added with 100 µL of the stock solution and 0.6 mL of pre-conditioned inoculum.
- Toxic control group: The BOD bottles were added with 100 µL of the Aniline stock solution and 0.6 mL of pre-conditioned inoculum.
Reference substance:
aniline
Remarks:
Purity >99.0%; batch No. 034K0126
Test performance:
The initial value for dissolved oygen concentration was 8.37 mg/L for the control, 8.39 mg/L for the test item treatment and 8.53 mg/L for the reference item treatment. The initial value was thus taken as mean from the three measured values: 8.43 mg/L.
The consumption of oxygen in the controls was less than 1.5 mg/L over the 28d test period, as required.
For the test item treatment, the measured concentrations for dissolved oxygen were approximately the same as for the controls up to day 28 of the test period.
For the reference item treatment and for the toxic control, the difference with the controls was more tan 1 mg/L up from day 9 of the test period.
Key result
Parameter:
% degradation (O2 consumption)
Value:
14.24
Sampling time:
28 d
Remarks on result:
other: Not readily biodegradable
Details on results:
Oygen updake and related biodegradation:
See tables 5.2.1/1 and 5.2.1/2 in "Any other information on results incl. tables"
- Test substance: The calculated values for biodegradation were less than 20% for the entire test period.
- Toxic control: Based on total ThOD, the biodegradation was more than 25% up from day 6 of the test period.

Active ingredient content:
The measured initial concentrations represented 96-101% the nominal value (2.59 mg/L). The treatment application was considered as valid. At the end of the 28-day test period, the measured concentrations still represented more than 85% of the nominal value in the test item solutions and in the toxic control (see Table 5.2.1/3 in "Any other information on results incl. tables")

Results with reference substance:
Aniline: The beginning of the 10-day window was observed between day 6 and day 9 of the test period. The pass-level was reached on day 9: mean value for biodegradation was 72.7%. The biodegradation stabilized at approximately 80% afterwards and the measurements were not continued after day 23.

Table 5.2.1/1: Measured oxygen concentrations in the BOD bottles, mg/L

Days

Blank

Test substance

Toxic

Aniline

Rep. 1

Rep. 2

Rep. 1

Rep. 2

Rep. 1

Rep. 2

0

3

6

9

12

16

19

23

28

8.43

7.92

7.69

7.70

7.49

7.53

7.55

7.62

7.48

8.43

7.98

7.72

7.70

7.61

7.63

7.43

7.47

7.55

8.43

7.93

7.69

7.71

7.73

7.66

7.62

7.61

7.26

8.43

7.91

7.85

7.80

7.68

7.74

7.65

7.53

7.38

8.43

7.85

4.88

4.68

3.65

3.62

3.86

3.91

3.25

8.43

7.91

7.52

3.92

4.06

3.99

3.52

3.62

-

8.43

7.85

7.72

4.38

3.94

3.70

3.23

3.43

-

Table 5.2.1/2: Calculated biodegradation, %

Days

Test substance

Toxic

Aniline

Rep. 1

Rep. 2

Mean

Rep. 1

Rep. 2

Mean

0

3

6

9

12

16

19

23

28

0

6.41

9.49

9.24

8.98

9.88

10.39

10.52

15.01

0

6.67

7.44

8.08

9.62

8.85

10.00

11.54

13.47

0

6.54

8.47

8.66

9.30

9.36

10.20

11.03

14.24

0

4.57

27.99

29.57

37.69

37.92

36.03

35.64

40.84

0

0.82

3.79

77.35

71.42

73.47

81.24

80.32

-

0

2.05

-0.31

67.94

73.87

79.40

87.18

84.21

-

0

1.43

1.74

72.65

72.65

76.43

84.21

82.27

-

Table 5.2.1/3: Measured concentrations of the test substance

 

Replicate unit

Replicate determination

Test item (mg/L)

% recovery

Test inititation

1

1

2

3

4

2.62

2.60

2.56

2.49

101.2%

100.3%

98.8%

96.1%

Mean

2.57

99.1%

End of test

1

1

2

2.51

2.49

97.0%

96.1%

2

1

2

2.52

2.47

97.2%

95.2%

Mean

2.50

96.4%

Toxic control

1

2

2.31

2.26

89.0%

87.0%

Mean

2.28

88.0%

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance is not readily biodegradable (removal within 28 days <20%)
Executive summary:

The biodegradation of the test substance was assessed according to the closed-bottle test method, appropriate for poorly water soluble substances.


The solution of the test substance (2.59 mg/L) in mineral medium was inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely filled, closed bottles in the dark at constant temperature (20 +/- 1 °C).


Degradation was followed by analysis of dissolved oxygen over a 28-day period. The amount of oxygen taken up by the microbial population during biodegradation of the test substance, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of the theorical oxygen demand (ThOD).


Additional analysis assessments were performed at test initiation and at test completion in the test vessels for quantification of the test item, so as to judge the potential for primary degradation over the test period.


The calculated values for biodegradation of the test substance were less than 20% for the entire test period (mean of 14.24%). Analytical measurement at the end of the test verified that the initial concentration of parent still remained. Therefore, the test substance was considered as not readily biodegradable (removal within 28 days < 20%).

Description of key information

OECD Guideline 301F, EU Method C4-D, EPA OPPTS 835.3110, GLP, Key study, validity 2:

1% biodegradation after 28 days and 4% biodegradation after 60 days, with activated sludge

Not readily biodegradable

Key value for chemical safety assessment

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

Additional information

To assess the biodegradation potential of the registered substance, two experimental studies are available.


The most recent study (Covance, 2019), assessed as the key study, was performed on the registered substance to assess the ready biodegradability of the test item in an aerobic aqueous medium, according to OECD Guideline 301F, EU Method C.4 -D and EPA OPPTS 835.3110, performed under GLP (with statement included in the report). The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed amber culture vessels in diffuse light at temperatures of between 21 and 24 ºC for 60 days. The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values and compound specific analyses on Days 0 and 60. The test item attained 1% biodegradation after 28 days and 4% biodegradation after 60 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F. Chemical analysis of the 100 mg/L test preparations containing inoculum at 0 hours resulted in measured concentrations of 107% of the nominal concentration. After 60 days, chemical analysis resulted in measured concentrations of 101% and 83% of the nominal (calculated to be a 0% and 17% loss over the test duration assuming 100% recovery on Day 0).

The second study (Phytosafe, 2012) was also performed on the registered substance but according to the closed-bottle test method. The solution of the test substance (2.59 mg/L) in mineral medium was inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely filled, closed bottles in the dark at constant temperature (20 +/- 1 °C). Degradation was followed by analysis of dissolved oxygen over a 28-day period. Additional analysis assessments were performed at test initiation and at test completion in the test vessels for quantification of the test item, so as to judge upon the primary degradation over the test period. The calculated values for biodegradation of the test substance were less than 20% for the entire test period (mean of 14.24% biodegradation after 28 days). Analytical determinations at the end of the test proved that the initial concentration of the parent still remained in the flasks at the end of the test. Therefore, the test substance can be considered as not readily biodegradable. This result supports that of the key study.