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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Apr - 08 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
adopted 17. Jul 1992
Deviations:
yes
Remarks:
Temperature range was 19.8 – 21.5 °C instead of 20.0 – 24.0 °C
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
Commission Regulation (EC) No. 440/2008, adopted 30 May 2008
Deviations:
yes
Remarks:
Temperature range was 19.8 – 21.5 °C instead of 20.0 – 24.0 °C
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Mainz, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of activated sludge: Activation basin of the biologic sewage treatment plant "Stadtentsorgung Neustadt" in Lachen-Speyerdorf, Germany mostly treating domestic sewage
- Date of collection: 05 Apr 2019
- Batch No: E20190405
- Pretreatment: The sludge was filtrated, washed with test medium (2x) and re-suspended in test medium. It was aerated until use. The dry matter was determined to contain 4.8 g of suspended solids/L.
- Concentration of sludge: 25.0 mg/L
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
20 mg/L
Based on:
TOC
Remarks:
corresponding to 30.1 mg test item/L
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium
- Test temperature: 19.8°C - 21.5°C (deviation: according to the study plan the temperature should have been 20.0 – 24.0 °C)
- pH: 7.0 - 7.7
- pH adjusted: no
- Suspended solids concentration: 4.8 g/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2000 mL SCHOTT-flasks, 100 mL scrubber flasks as absorbent vessels
- Flask volume: 1500 mL
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Flasks were aerated for 72 hours with purified, CO2-free, moistened air.
- Measuring equipment: Carbon analyser TOC multi N/C 2100S, Analytik Jena, data logger for temperature (ebro), pH-meter 3310 (wtw)
- Test performed in open system: yes
- Details of trap for CO2 and volatile organics if used: The scrubbing of carbon dioxide was achieved by bubbling the purified air through a flask containing 1.5 M NaOH. CO2 was trapped in 0.25 M NaOH. Two scrubbers containing 100 mL each were connected in series to the test vessels.
- Apparatus: The test vessels were aerated with purified (by activated charcoal), CO2-scrubbed, moistened air. Magnetic stirrers were used to prevent deposition of inoculum.

SAMPLING
- Sampling frequency: samples from each front scrubber flask on day 0, 2, 4, 7, 10, 15, 18, 22 and 29. On day 28, 5 mL HCl 2 M was added to each test flask in order to drive off dissolved CO2. On day 29, samples from both scrubber flasks were taken.
- Sampling method: sample volume: 1 mL, measurements in duplicates or triplicates, respectively (depending on the variation between the measured values), carbon analyser calibration: with freshly prepared reference solutions containing potassium hydrogen phthalate, sodium hydrogen carbonate and sodium carbonate

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2 (mineral medium and inoculum)
- Apparatus blanks: 2 (mineral medium only)
- Abiotic sterile control: 1 (test item, mineral medium and HgCl2)
- Toxicity control: 1 (test item, positive control, mineral medium and inoculum)
Reference substance
Reference substance:
aniline
Remarks:
24.5 mg/L, organic carbon content: 20.0 mg/L

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
79
Sampling time:
29 d
Details on results:
As degradation in the toxicity flask was 62.6 % after 14 days, the test item can be stated as “not toxic towards the inoculum in a concentration of 30.1 mg/L”.

BOD5 / COD results

Results with reference substance:
Degradation of aniline: 90.7% (mean)

Any other information on results incl. tables

Table 1: IC values in mg/L, front scrubber

Day

Apparatus blank 1

Apparatus blank 2

Blank Control 1

Blank Control 2

Positive Control 1

Positive Control 2

Test 1

Test 2

Abiotic Control

Toxicity Control

0

2.56

2.31

3.00

3.04

2.99

2.80

2.86

9.02

2.35

2.54

2

3.72

3.35

7.25

10.35

14.69

13.83

45.60

40.85

11.81

3.41

4

4.56

4.16

16.39

19.53

103.48

89.95

112.89

100.00

13.30

93.64

7

5.57

7.23

32.86

32.13

188.16

194.22

170.58

169.73

15.24

207.70

10

7.39

7.00

40.59

38.72

213.49

235.82

217.37

237.79

16.34

413.05

15

8.94

10.29

41.35

54.30

272.75

305.18

259.90

267.35

16.97

449.85

18

9.76

13.97

54.83

64.41

312.33

330.78

278.57

290.23

18.16

496.10

22

12.18

15.86

70.17

76.88

366.82

385.62

329.38

338.33

23.66

*594.56

29

17.30

24.02

83.98

89.88

377.97

387.57

345.31

353.15

27.46

*606.90

Table 2: IC values in mg/L, back scrubber

Day

Apparatus blank 1

Apparatus blank 2

Blank Control 1

Blank Control 2

Positive Control 1

Positive Control 2

Test 1

Test 2

Abiotic Control

Toxicity Control

0

2.93

2.76

2.66

2.76

2.41

4.21

2.48

2.57

2.90

2.78

29

3.67

3.09

3.36

3.00

3.15

3.67

3.05

3.10

2.86

3.85

Table 3: Net IC values in mg/L of front scrubber flasks

Day

Blank Control 1

Blank Control 2

Positive Control 1

Positive Control 2

Test 1

Test 2

Abiotic Control

Toxicity Control

0

3.0

3.0

3.0

2.8

2.9

9.0

2.4

2.5

2

6.1

9.2

13.6

12.7

44.5

39.7

10.7

2.3

4

14.5

17.6

101.6

88.0

111.0

98.1

11.4

91.7

7

28.9

28.2

184.2

190.3

166.6

165.8

11.3

203.7

10

35.8

34.0

208.7

231.1

212.6

233.0

11.6

408.3

15

34.2

47.1

265.6

298.0

252.7

260.2

9.8

442.7

18

45.4

55.0

302.9

321.3

269.1

280.8

8.7

486.7

22

58.6

65.3

355.2

374.0

317.8

326.8

12.1

583.0

29

65.8

71.7

359.7

369.3

327.1

334.9

9.2

588.7

Table 4: Net IC values in mg/L of back scrubber flasks

Day

Blank Control 1

Blank Control 2

Positive Control 1

Positive Control 2

Test 1

Test 2

Abiotic Control

Toxicity Control

0

2.7

2.8

2.4

4.2

2.5

2.6

2.9

2.8

29

2.8

2.5

2.6

3.1

2.5

2.6

2.3

3.3

Table 5: Degradation values in %

Day

Positive Control 1

Positive Control 2

Positive Control Mean

Test 1

Test 2

Test Mean

Abiotic Control

Toxicity Control

2

2.0

1.7

1.8

12.1

8.5

10.3

2.7

-0.8

4

27.9

23.6

25.7

30.8

24.6

27.7

2.9

12.4

7

50.3

52.3

51.3

44.3

42.0

43.1

2.9

28.3

10

55.6

62.8

59.2

56.4

60.9

58.6

2.9

59.5

15

71.2

81.5

76.3

66.6

67.0

66.8

2.3

63.4

18

79.1

84.9

82.0

68.0

69.7

68.9

2.0

68.1

22

90.8

96.7

93.8

78.6

79.5

79.0

3.0

80.4

29

89.2

92.2

90.7

78.5

79.0

78.8

2.1

79.4

Table 6: Validity criteria for OECD 301B.

Criterion from the guideline

Outcome

Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.

 Difference: 0.5%

 yes

Percentage degradation of the reference compound reached the pass level by day 14 (≥ 60%).

  59% on day 10

 yes

The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.

 62.6%

 non-toxic

The IC content of the test substance suspension in the mineral medium at the beginning of the test must be less than 5% of the TC.

 0%

 yes

The total CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/L medium.

 14.8 mg/L

 yes

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Interpretation of results:
readily biodegradable