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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:
13 March 2018 - 10 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test method according to OECD 301F. GLP 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, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was aerated for 6 days, at the test temperature of about 22 °C, until application. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids. Before application the sludge was washed in a mineral medium.
- Concentration of sludge: 3.72 g suspended solids/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Remarks:
(50.8 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 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 4.1±0.42 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 ± 2ºC
- pH: 7.4 ± 0.2
- pH adjusted: no
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes
- Volume of test solution in flask, V: 0.164 L

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 repirometer.
- Details of trap for CO2 and volatile organics if used: potassium hydroxide solution.

SAMPLING
- Sampling frequency: O2 uptake data were read out every 112 min during the 28 day test (40 320 min that is 360 readings) and were recorded and stored in the measuring heads of the sample bottles.
- Sampling method: Readings from apparatus (closed WTW OxiTop OC 110 repirometer).

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 30mg/L SS.
- Toxicity control: flasks #16, 17, 18, containing test item, reference item at the same concentrations as in the individual solutions 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 ≥ 99.7%, source: CHEMPUR, Piekary Śląskie, Polska.
Key result
Parameter:
% degradation (O2 consumption)
Value:
61.7
Sampling time:
28 d
Details on results:
-The biodegradability attained 61.7 % within 28 days. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 25 days and was not reached in a 10-day window in which biodegradation attained 52.7%. Lag time was 3.5 day and degradation time was 12.5 day.
-The reference item reached 97.1% of biodegradation and the level for ready biodegradability by 4 days
-In the toxicity test the biodegradation was equal to 48.5% in 14 days
-The oxygen uptake of the inoculum blank was equal to 37.9 mg/L in 28 days
-The pH values of flasks were in the range of 7.41-8.67
Results with reference substance:
The reference item reached 97.1% of biodegradation and the level for ready biodegradability by 4 days (>60% reached before day 14)

Table 2. Sample oxygen uptake: biodegradability.

 

time, days

1

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2uptake, mg/L

a1

8.3

15.6

83.4

119.2

138.3

158.4

169.6

178.9

185.8

197.7

204.0

209.5

220.3

a2

8.6

16.1

103.7

127.4

144.7

162.6

171.7

178.1

183.4

190.9

195.3

198.2

204.1

a3

11.7

17.4

78.3

124.6

144.9

166.1

175.5

184.1

190.4

199.6

204.5

208.5

215.3

am. avg

9.5

16.4

88.5

123.7

142.6

162.4

172.3

180.4

186.5

196.1

201.3

205.4

213.2

Blank test O2uptake mg/L

b1

6.3

11.8

14.6

21.5

24.1

29.0

29.5

32.5

33.5

36.3

40.0

40.2

41.6

b2

5.6

10.4

13.7

16.7

18.2

23.9

24.2

26.9

27.9

29.3

34.3

35.9

37.1

b3

7.2

10.2

13.6

18.1

20.6

24.1

25.7

27.3

28.1

28.9

33.0

32.3

35.1

bm. avg

6.4

10.8

13.9

18.8

21.0

25.6

26.5

28.9

29.8

31.5

35.8

36.1

37.9

Reference item O2uptake. mg/L

w1

24.0

53.5

67.3

77.7

84.2

89.6

95.0

97.0

98.5

101.2

103.9

104.2

107.0

w2

23.3

55.3

68.6

78.3

85.7

92.1

95.8

98.6

100.1

105.1

107.4

108.8

111.9

w3

24.6

55.4

69.1

83.2

90.2

98.9

104.5

108.2

112.2

115.1

118.4

119.1

122.0

wm. avg

24.0

54.7

68.3

79.7

86.7

93.6

98.5

101.3

103.6

107.1

109.9

110.7

113.6

Toxicity control O2uptake. mg/L

tox1

19.1

57.2

71.2

113.2

144.0

188.7

212.9

231.0

244.9

261.6

272.0

279.3

291.3

tox2

20.1

57.2

73.6

113.1

135.1

166.1

186.8

207.7

227.2

257.2

273.7

284.8

301.8

tox3

23.5

60.5

84.2

115.9

141.1

180.3

206.1

231.6

246.2

263.5

270.9

278.5

291.6

toxm.avg

20.9

58.3

76.3

114.1

140.1

178.4

201.9

223.4

239.5

260.8

272.2

280.9

294.9

Corrected

test item O2uptake, mg/L

(a1-bm)

2.0

4.8

69.4

100.5

117.3

132.8

143.2

150.0

156.0

166.2

168.2

173.4

182.4

(a2-bm)

2.2

5.3

89.8

108.7

123.7

137.0

145.2

149.2

153.5

159.4

159.5

162.1

166.1

(a3-bm)

5.4

6.7

64.4

105.8

123.9

140.5

149.0

155.2

160.5

168.1

168.7

172.4

177.3

Reference item % degradation

ThOD = 0.78

mgO2/mg

C = 100 mg/L

R1(w1)

22.6

54.8

68.5

75.6

81.1

82.0

87.9

87.3

88.1

89.3

87.3

87.3

88.5

R2(w2)

21.8

57.0

70.1

76.3

83.1

85.2

88.9

89.4

90.0

94.3

91.8

93.1

94.8

R3(w3)

23.3

57.2

70.7

82.6

88.8

94.0

100.0

101.7

105.6

107.1

105.9

106.4

107.8

Rwavg

22.6

56.3

69.7

78.2

84.3

87.1

92.3

92.8

94.6

96.9

95.0

95.6

97.1

Test item% degradation

ThOD = 2.84

mgO2/mg

C = 100 mg/L

R1(a1)

0.7

1.7

24.5

35.4

41.3

46.8

50.4

52.8

54.9

58.5

59.2

61.0

64.2

R2(a2)

0.8

1.9

31.6

38.3

43.6

48.2

51.1

52.5

54.1

56.1

56.2

57.1

58.5

R3(a3)

1.9

2.3

22.7

37.3

43.6

49.5

52.5

54.6

56.5

59.2

59.4

60.7

62.4

Raavg

1.1

2.0

26.2

37.0

42.8

48.2

51.3

53.3

55.2

57.9

58.3

59.6

61.7

Toxicity test

% degradation

 

R1(tox1)

3.5

12.8

15.8

26.1

34.0

45.1

51.5

55.8

59.4

63.6

65.2

67.2

70.0

R2(tox2)

3.8

12.8

16.5

26.0

31.5

38.8

44.3

49.4

54.5

62.3

65.7

68.7

72.9

R3(tox3)

4.7

13.7

19.4

26.8

33.2

42.7

49.6

56.0

59.8

64.1

65.0

67.0

70.1

Rtoxavg

4.0

13.1

17.2

26.3

32.9

42.2

48.5

53.7

57.9

63.3

65.3

67.6

71.0

 

Table 3. The pH values of 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

7.58

7.59

7.61

7.52

7.57

7.59

7.59

7.63

7.57

7.70

7.57

7.69

final

7.43

7.45

7.47

7.42

7.43

7.41

8.47

8.67

8.65

8.15

8.26

8.27

 

Validity criteria fulfilled:
yes
Remarks:
See "overall remarks".
Interpretation of results:
not readily biodegradable
Conclusions:
Under the ready aerobic biodegradability test, the test item attained 61.7% biodegradation but not reached within the 10 day window. Thus, the test item can not be classed as readily biodegradable.

Executive summary:

A ready 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 48.5%. Thus, the test item is not inhibitory. The  biodegradability of the test item attained 61.7 % within 28 days. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 25 days and was not reached in a 10-day window in which biodegradation attained 52.7%. Thus, the test item can not be classed as readily biodegradable.

Description of key information

Key study: Test method according to OECD 301F / EU C.4 (Manometric respirometry methods), GLP study. The test item attained 61.7% biodegradation but not reached within the 10 day window. Thus, the test item can not be classed as readily biodegradable.

Key value for chemical safety assessment

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

Additional information

Key study. A ready 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 48.5%. Thus, the test item is not inhibitory.The  biodegradability of the test item attained 61.7 % within 28 days. The pass level for ready biodegradability is 60% of ThOD. This value was attained after 25 days and was not reached in a 10-day window in which biodegradation attained 52.7%. Thus, the test item can not be classed as readily biodegradable.