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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:
17 March 2017 - 14 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test method according to OECD 301F. GLP study.
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)
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: The coarse particles were removed by settling and the supernatant was discarded. The sludge was washed in the mineral medium. The concentrated sludge was suspended 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 A sample was withdrawn just before use for the determination of the dry weight of the suspended solids.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:
(77.8 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 3.5 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 #7, 8, 9 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 #10, 11, 12, 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 ≥ 99.7%, source: CHEMPUR, Piekary Śląskie, Polska.
Key result
Parameter:
% degradation (O2 consumption)
Value:
74.5
Sampling time:
28 d
Details on results:
- The test item attained 74.5% of biodegradation (>60% reached in a 10-day window within the 28-day period of the test)
- The reference item reached 83.6 % of biodegradation and the level for ready biodegradability by 5 days (>60% reached before day 14)
- In the toxicity test the biodegradation was equal to 55.3% in 14 days (>25% in 14 days). Therefore, the test item is not inhibitory.
- The oxygen uptake of the inoculum blank was equal to 38.9 mg/l in 28 days (<60 mg/l in 28 days)
- The pH values ( 7.43-8.78) of almost all flasks were inside the range (6-8.5, minimal overruns took place in 3 flasks without any influence in the test result)
Results with reference substance:
The reference item reached 83.6% of biodegradation and the level for ready biodegradability by 5 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

13.4

17.4

21.2

23.8

89.6

149.9

178.9

200.0

211.8

230.2

236.5

243.4

250.2

a2

12.9

18.7

22.1

25.3

32.1

136.3

165.2

186.6

207.8

232.3

240.1

249.1

257.5

a3

11.0

17.4

19.2

25.7

121.0

166.2

187.2

206.4

222.4

239.7

247.1

250.5

257.4

am. avg

12.4

17.9

20.8

24.9

80.9

150.8

177.1

197.7

214.0

234.1

241.2

247.6

255.0

Blank test O2uptake mg/l

b1

7.4

14.7

19.1

24.1

26.8

29.8

31.6

33.4

33.7

36.8

35.1

37.9

40.3

b2

10.8

16.1

18.4

19.1

22.3

23.7

24.9

25.9

27.2

28.8

31.2

32.1

34.5

b3

16.8

21.1

23.8

26.2

27.2

31.2

34.4

34.0

34.7

38.9

37.3

39.6

42.0

bm. avg

11.7

17.3

20.4

23.2

25.4

28.2

30.3

31.1

31.9

34.8

34.5

36.5

38.9

Reference item O2uptake. mg/l

w1

27.8

53.7

68.8

75.8

78.1

83.4

85.4

87.7

88.2

92.6

92.5

93.1

95.5

w2

29.3

56.5

70.7

77.5

83.0

88.4

90.9

94.3

96.8

99.1

101.2

104.3

106.0

w3

24.5

54.0

68.1

76.9

80.7

87.1

90.5

94.0

96.0

100.6

103.1

105.2

110.9

wm. avg

27.2

54.7

69.2

76.8

80.6

86.3

88.9

92.0

93.7

97.5

98.9

100.9

104.1

Toxicity control O2uptake. mg/l

tox1

22.4

59.6

74.8

82.2

101.6

211.1

233.2

248.5

259.3

273.9

281.3

290.4

300.0

tox2

21.7

56.7

71.7

82.1

145.1

221.5

249.3

266.6

282.1

300.3

308.5

315.4

326.5

tox3

20.8

56.3

70.7

78.7

117.2

196.8

219.3

235.4

249.7

264.5

273.5

279.6

288.9

toxm.avg

21.6

57.5

72.4

81.0

121.3

209.8

233.9

250.2

263.7

279.6

287.8

295.2

305.1

Corrected

test item O2uptake, mg/l

(a1-bm)

1.8

0.2

0.8

0.6

64.2

121.7

148.6

168.9

179.9

195.4

202.0

206.9

211.2

(a2-bm)

1.2

1.4

1.6

2.2

6.6

108.1

134.9

155.5

175.9

197.4

205.6

212.5

218.6

(a3-bm)

-0.6

0.2

-1.2

2.6

95.6

138.0

156.9

175.3

190.5

204.9

212.6

213.9

218.5

Reference item % degradation

ThOD = 0.78

mgO2/mg

C = 100 mg/l

R1(w1)

20.6

46.7

62.1

67.5

67.6

70.8

70.7

72.6

72.2

74.1

74.4

72.5

72.5

R2(w2)

22.6

50.2

64.4

69.7

73.9

77.1

77.7

81.0

83.2

82.4

85.4

86.9

86.0

R3(w3)

16.5

47.0

61.1

68.9

70.8

75.5

77.1

80.6

82.2

84.3

87.9

88.1

92.2

Rwavg

19.9

48.0

62.6

68.7

70.8

74.5

75.2

78.1

79.2

80.3

82.6

82.5

83.6

Test item% degradation

ThOD = 2.90

mgO2/mg

C = 100 mg/l

R1(a1)

0.6

0.1

0.3

0.2

22.1

42.0

51.2

58.2

62.0

67.4

69.6

71.3

72.8

R2(a2)

0.4

0.5

0.6

0.7

2.3

37.3

46.5

53.6

60.7

68.1

70.9

73.3

75.4

R3(a3)

0.0

0.1

0.0

0.9

33.0

47.6

54.1

60.4

65.7

70.6

73.3

73.8

75.3

Raavg

0.3

0.2

0.3

0.6

19.1

42.3

50.6

57.4

62.8

68.7

71.3

72.8

74.5

Toxicity test

% degradation

 

R1(tox1)

2.9

11.5

14.8

16.0

20.7

49.7

55.2

59.1

61.8

64.9

67.1

69.0

70.9

R2(tox2)

2.7

10.7

13.9

16.0

32.5

52.5

59.5

64.0

68.0

72.1

74.4

75.8

78.1

R3(tox3)

2.5

10.6

13.7

15.1

25.0

45.8

51.4

55.5

59.2

62.4

64.9

66.1

67.9

Rtoxavg

2.7

10.9

14.1

15.7

26.1

49.3

55.3

59.5

63.0

66.5

68.8

70.3

72.3

 

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

flask #

7

8

9

1

2

3

4

5

6

10

11

12

Test item

Inoculum blank

Reference item

Toxicity test

initial

7.43

7.45

7.46

7.38

7.44

7.44

7.42

7.43

7.45

7.47

7.47

7.48

final

7.84

7.64

7.85

7.43

7.44

7.44

8.71

8.78

8.69

7.85

7.66

8.13

 

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 74.5%. 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 55.3%. Thus, the test item is not inhibitory. At the 28th day of the test the measured aerobic biodegradation of the test item attained 74.5%. Thus, the test item can be deemed to be readily biodegradable.

Description of key information

Key study: Test method according to OECD 301F / EU C.4 (Manometric respirometry methods), GLP study. At the 28th day of the test the measured aerobic biodegradation of the test item attained 74.5%. Thus, the test item is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Key study. A 28-day ready aerobic biodegradability test in an aerobic aqueous medium with manometric respirometry method was performed on the test substance D-alpha fenchol, according to OECD 301F / EC C.4-D manometric respirometry methods (GLP study). 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 55.3%. Thus, the test substance is not inhibitory. At the 28th day of the test the measured aerobic biodegradation of the test item attained 74.5%. Thus, the test item is readily biodegradable.