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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 May 24th to June 21st, 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
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 sample of activated sludge was taken from the aeration tank of Sewage Treatment Plant ”Czajka”, Warsaw.
- Storage conditions: aerated at 22ºC
- Storage length: 24h.
- Preparation of inoculum for exposure: The coarse particles were removed by settling and the supernatant was discarded. The sludge was washed in the mineral medium. The concentrated sludge was suspend ed in mineral medium to yield a concentration of 3-5 g suspended solids/l and it was aerated, at the test temperature of 22 °C, until application next day. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Remarks:
(54.9 mg/l of organic carbon)
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: 10ml of solution (A) in 800 ml water, plus 1 ml solutions (B), (C), (D) and 11ml water. The following stock solutions were used, prepared with analytical grade reagents: Solution (A) contains: 8.50 g monopotassium dihydrogen orthophosphate (KH2PO4), 21.75 g dipotassium monohydrogen orthophosphate (K2HPO4), 33.40 g disodium monohydrogen orthophosphate dihydrate (Na2HPO4·2H2O), 0.50 g ammonium chloride (NH4Cl), in 1 L double-distilled water. Solution (B) contains: 27.50 g calcium chloride, anhidrous (CaCl2) in 1L water. Solution (C) contains: 22.50 g magnesium sulphate heptahydrate (MgSO4·7H2O) in 1L water. Solution (D) contains: 0.25 g iron(III) chloride hexahydrate (FeCl3·6H2O) in 1L water.
- The water used is double-distilled, containing about 3 mg/L of organic carbon (< 10% of the organic carbon content introduced by the test item), checked by DOC analysis using spectrophotometer Hach DR 3900 and Hach-Lange reagents.
- Test temperature: 22 ± 2 ºC
- pH: 7.09 - 8.41
- pH adjusted: no
- Volume of test solution in flask, V: 0.164 L
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration: triplicates were used, flasks #13, 14, 15, containing test item (100 mg/l) and inoculum (30mg/L SS).
- Measuring equipment: O2 uptake was measured by a closed WTW OxiTop OC 110 respirometer.
- Details of trap for CO2 and volatile organics if used: potassium hydroxide solution.

SAMPLING
- Sampling frequency: The data were read out every 112 min during the 28 day test (40320 min that is 360 readings) and were recorded and stored in the measuring heads of the sample bottles.
- Sampling method: These collected data, using the controller, were infrared read out from the heads and stored in the controller.

CONTROL AND BLANK SYSTEM
- Inoculum blank: flasks #1, 2, 3, containing only inoculum 30mg/L SS.
- Procedure control: flasks #4, 5, 6, containing reference item (sodium acetate 100 mg/l) and inoculum 30 mg/L SS.
- Toxicity control: flasks #16, 17, 18, containing test item, reference item and inoculum 30mg/L SS.

STATISTICAL METHODS: The calculations and the graphs were performed using SigmaPlot 9.0 software of SYSTAT Software, Inc., USA purchased from GAMBIT CoiS Ltd, Poland.
Reference substance:
acetic acid, sodium salt
Remarks:
CAS No: 127-09-3, purity ≥ 98.5%, source: Eurochem.
Key result
Parameter:
% degradation (O2 consumption)
Value:
71
Sampling time:
28 d
Details on results:
- At the 28th day of the test the aerobic biodegradation of the testing, the test item attained 71.0% of biodegradation. The pH values of all flasks were inside the range 7.09-8.41.
- In the toxicity test, containing both the test item and reference item, more (42.7%) than 25% of biodegradation occurred in 14 days. Therefore, the test item is not inhibitory.
Results with reference substance:
The reference item reached 86.6% of biodegradation and the level for ready biodegradability by day 4.

Table 2. Sample oxygen uptake: biodegradability.

 

time, days

1

2

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2 uptake, mg/l

a1

0.5

7.0

31.3

45.6

86.2

100.2

114.5

118.3

124.1

127.3

141.4

143.2

149.7

155.8

a2

0.9

11.8

38.2

76.4

105.5

115.2

124.5

128.5

134.9

137.6

141.4

144.9

158.1

160.1

a3

3.5

9.2

26.4

57.4

77.3

92.9

105.7

112.2

117.7

121.4

128.9

133.0

141.2

146.0

am. avg

1.6

9.4

32.0

59.8

89.7

102.7

114.9

119.6

125.6

128.7

137.2

140.4

149.6

153.9

Blank test O2 uptake. mg/l

b1

0.0

0.9

7.2

12.7

17.4

19.8

22.7

24.7

28.4

29.8

30.7

32.6

36.6

44.4

b2

1.5

3.2

7.0

13.2

16.5

20.0

23.8

24.8

28.8

30.5

33.4

35.5

38.8

44.5

b3

3.3

7.5

10.9

13.3

13.9

16.9

17.8

20.7

22.6

24.6

30.0

33.1

39.2

45.0

bm. avg

1.6

3.9

8.4

13.1

15.9

18.9

21.4

23.4

26.6

28.3

31.4

33.7

38.2

44.6

Reference item O2 uptake. mg/l

w1

22.3

47.2

54.9

64.7

74.3

79.6

83.9

87.9

92.9

96.4

103.2

109.3

111.5

117.2

w2

26.1

44.4

55.1

66.9

74.6

78.9

86.0

89.6

92.8

95.7

101.3

105.6

110.4

115.4

w3

21.7

44.3

53.8

66.6

73.8

79.3

83.6

86.3

89.3

92.5

98.5

100.2

104.1

104.0

wm. avg

23.4

45.3

54.6

66.1

74.2

79.3

84.5

88.0

91.7

94.9

101.0

105.0

108.6

112.2

Toxicity control O2 uptake. mg/l

tox1

18.4

47.0

57.7

80.1

98.3

107.2

117.5

123.2

132.0

135.3

154.2

163.7

173.8

183.2

tox2

17.0

38.9

52.9

76.0

95.9

107.1

116.4

123.7

129.2

137.8

159.2

172.4

190.9

200.5

tox3

19.1

43.1

57.0

78.0

98.8

107.7

115.3

120.5

124.7

129.5

135.5

146.1

172.3

182.5

toxm. avg

18.1

43.0

55.9

78.0

97.6

107.3

116.4

122.5

128.7

134.2

149.6

160.7

179.0

188.7

Corrected

test item O2 uptake, mg/l

(a1-bm)

-1.1

3.2

23.0

32.6

70.3

81.3

93.1

94.9

97.5

99.0

110.0

109.5

111.5

111.1

(a2-bm)

-0.7

7.9

29.9

63.4

89.6

96.3

103.0

105.0

108.3

109.3

110.0

111.2

119.8

115.5

(a3-bm)

1.9

5.3

18.0

44.4

61.4

74.0

84.3

88.8

91.1

93.1

97.6

99.3

102.9

101.4

Reference item % degradation

ThOD = 0.78

mgO2/mg

C = 100 mg/l

R 1(w1)

26.5

55.6

59.6

66.2

74.9

77.9

80.1

82.7

85.1

87.3

92.2

96.9

93.9

93.0

R1 (w2)

31.4

51.9

60.0

69.0

75.2

76.9

82.8

84.9

84.9

86.4

89.7

92.1

92.5

90.7

R3 (w3)

25.7

51.8

58.3

68.7

74.2

77.4

79.7

80.7

80.4

82.3

86.0

85.2

84.5

76.1

Rwavg

27.9

53.1

59.3

68.0

74.8

77.4

80.8

82.8

83.4

85.4

89.3

91.4

90.3

86.6

Test item

% degradation

ThOD = 1.54

mgO2/mg

C = 100 mg/l

Rwavg

0.0

2.1

14.9

21.2

45.6

52.8

60.5

61.6

63.3

64.3

71.4

71.1

72.4

72.2

R 1(a1)

0.0

5.1

19.4

41.1

58.2

62.5

66.9

68.2

70.3

71.0

71.4

72.2

77.8

75.0

R1 (a2)

1.2

3.5

11.7

28.8

39.9

48.0

54.7

57.6

59.1

60.4

63.4

64.4

66.8

65.8

R3 (a3)

0.4

3.6

15.3

30.4

47.9

54.5

60.7

62.5

64.3

65.2

68.7

69.3

72.3

71.0

Toxicity control % degradation

 

 

Raavg

7.2

18.6

21.3

28.9

35.5

38.1

41.4

43.0

45.4

46.1

53.0

56.0

58.4

59.7

R1 (tox1)

6.6

15.1

19.2

27.1

34.5

38.0

40.9

43.2

44.2

47.2

55.1

59.8

65.8

67.2

R2 (tox2)

7.5

16.9

21.0

28.0

35.7

38.3

40.5

41.9

42.3

43.6

44.9

48.4

57.8

59.4

R3(tox3)

7.1

16.9

20.5

28.0

35.2

38.1

40.9

42.7

44.0

45.7

51.0

54.7

60.7

62.1


Table 3. The pH values of the test flasks (no adjustment of pH was conducted).

flask
#

13

14

15

1

2

3

4

5

6

16

17

18

Test item

Inoculum blank

Reference item

Toxicity test

initial

7.24

7.24

7.25

7.28

7.29

7.31

7.29

7.28

7.28

7.23

7.23

7.20

final

7.11

7.27

7.12

7.09

7.15

7.12

8.12

8.33

8.41

8.11

7.80

7.87



Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks'
Interpretation of results:
readily biodegradable
Conclusions:
At the 28th day of the test the measured aerobic biodegradation of the test item attained 71.0%, passing the 10-day window. Thus, the test item can be deemed to be readily biodegradable.
Executive summary:

A 28-day ready aerobic biodegradability test in an aerobic aqueous medium with manometric respirometry method was performed on the test item, according to OECD 301F / EC C.4 – D manometric respirometry methods, in accordance with GLP principles. 100 mg/l of test item was inoculated with activated sludge (30 mg/L SS) and incubated under aerobic conditions in a closed respirometer flask at constant temperature (22 ± 2ºC) for 28 days. A blank test, a procedure test with reference substance (sodium acetate) and a toxicity test were run in parallel. In the toxicity test, containing both the test item and a reference item, on the 14th test day the biodegradation (based on ThOD) attained 47.2%. Thus, the test item is not inhibitory. At the 28th day of the test the measured aerobic biodegradation of the test item attained 71.0%, passing the 10-day window. Thus, the test item is readily biodegradable.

Description of key information

Key study. Method according to OECD 301F / EU C.4 -D (manometric respirometry method), GLP study. At the 28th day of the test the measured aerobic biodegradation of the test item attained 71.0%, passing the 10-day window. Thus, the test item is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A 28-day ready aerobic biodegradability test in an aerobic aqueous medium with manometric respirometry method was performed on the test item, according to OECD 301F / EU C.4 – D manometric respirometry methods, in accordance with GLP principles. 100 mg/l of test item was inoculated with activated sludge (30 mg/L SS) and incubated under aerobic conditions in a closed respirometer flask at constant temperature (22 ± 2ºC) for 28 days. A blank test, a procedure test with reference substance (sodium acetate) and a toxicity test were run in parallel. In the toxicity test, containing both the test item and a reference item, on the 14th test day the biodegradation (based on ThOD) attained 47.2%. Thus, the test item is not inhibitory. At the 28th day of the test the measured aerobic biodegradation of the test item attained 71.0%, passing the 10-day window. Thus, the test item is readily biodegradable.