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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:
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
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
GLP compliance:
yes (incl. QA statement)
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 A sample was withdrawn just before use for the determination of the dry weight of the suspended solids.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 3mg/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 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:
79.8
Sampling time:
28 d
Details on results:
- The test item attained 79.8% of biodegradation (>60% reached in a 10-day window within the 28-day period of the test)
- The reference item reached 72.4 % 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 61.7% in 14 days (>25% in 14 days). Therefore, the test item is not inhibitory.
- The oxygen uptake of the inoculum blank was equal to 52.9 mg/l in 28 days (<60 mg/l in 28 days)
- The pH values ( 7.19-8.74) of almost all flasks were inside the range (6-8.5, minimal overruns took place in two flask without any influence in the test result)
Results with reference substance:
The reference item reached 72.4% 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

2.1

33.8

138.3

179.3

204.7

227.8

240.5

249.1

256.5

266.8

270.8

276.5

281.5

a2

2.1

34.6

139.5

181.7

204.2

225.9

236.3

250.0

257.2

266.7

273.5

278.7

284.0

a3

4.4

31.0

142.0

181.8

206.4

232.2

243.5

252.0

260.3

268.2

275.6

280.1

287.0

am. avg

2.9

33.1

139.9

180.9

205.1

228.6

240.1

250.4

258.0

267.2

273.3

278.5

284.2

Blank test O2uptake mg/l

b1

4.9

10.7

19.6

27.5

31.8

38.0

38.8

43.5

46.5

50.3

52.4

55.0

57.9

b2

4.0

12.0

18.8

22.7

26.2

32.3

34.0

37.5

40.7

46.2

48.9

52.1

56.3

b3

6.9

14.9

19.5

23.2

27.1

29.3

29.9

31.4

32.5

38.2

38.6

41.1

44.4

bm. avg

5.3

12.5

19.3

24.5

28.4

33.2

34.2

37.4

39.9

44.9

46.6

49.4

52.9

Reference item O2uptake. mg/l

w1

13.7

50.4

64.5

72.9

77.3

84.5

86.7

88.1

90.8

94.6

96.6

97.2

98.7

w2

15.0

52.8

67.3

76.5

82.1

89.8

93.1

95.7

98.7

103.0

105.8

109.0

111.4

w3

13.2

52.4

67.6

79.0

83.3

90.0

93.1

98.0

100.3

106.7

110.3

113.1

118.0

wm. avg

14.0

51.9

66.5

76.1

80.9

88.1

91.0

93.9

96.6

101.4

104.2

106.4

109.3

Toxicity control O2uptake. mg/l

tox1

8.6

57.1

99.7

164.2

214.1

244.0

257.1

270.1

281.5

295.3

302.5

308.5

318.8

tox2

5.3

55.2

108.4

170.5

224.6

254.7

269.8

281.7

294.8

309.5

319.2

327.6

338.4

tox3

5.7

53.6

113.0

172.7

216.9

243.5

256.5

270.6

280.9

293.7

300.2

304.5

312.4

toxm.avg

6.5

55.3

107.1

169.1

218.5

247.4

261.1

274.2

285.7

299.5

307.3

313.5

323.2

Corrected

test item O2uptake, mg/l

(a1-bm)

-3.1

21.3

119.0

154.8

176.4

194.6

206.2

211.7

216.5

221.9

224.2

227.1

228.7

(a2-bm)

-3.2

22.1

120.2

157.2

175.9

192.7

202.1

212.6

217.3

221.7

226.9

229.3

231.1

(a3-bm)

-0.8

18.5

122.7

157.3

178.0

199.0

209.3

214.6

220.4

223.3

229.0

230.7

234.1

Reference item % degradation

ThOD = 0.78

mgO2/mg

C = 100 mg/l

R1(w1)

10.8

48.5

57.9

62.0

62.8

65.8

67.2

64.9

65.3

63.6

64.0

61.2

58.7

R2(w2)

12.4

51.6

61.5

66.6

68.9

72.6

75.5

74.6

75.4

74.5

75.9

76.4

75.0

R3(w3)

10.2

51.1

62.0

69.8

70.4

72.8

75.5

77.6

77.4

79.2

81.6

81.7

83.5

Rwavg

11.2

50.4

60.5

66.2

67.4

70.4

72.8

72.4

72.7

72.4

73.9

73.1

72.4

Test item% degradation

ThOD = 2.90

mgO2/mg

C = 100 mg/l

R1(a1)

0.0

7.3

41.0

53.4

60.8

67.1

71.1

73.0

74.7

76.5

77.3

78.3

78.8

R2(a2)

0.0

7.6

41.4

54.2

60.6

66.4

69.7

73.3

74.9

76.5

78.2

79.1

79.7

R3(a3)

0.0

6.4

42.3

54.2

61.4

68.6

72.2

74.0

76.0

77.0

79.0

79.6

80.7

Raavg

0.0

7.1

41.6

53.9

61.0

67.4

71.0

73.4

75.2

76.7

78.2

79.0

79.8

Toxicity test

% degradation

 

R1(tox1)

0.9

12.1

21.9

37.9

50.5

57.3

60.6

63.2

65.6

68.0

69.5

70.4

72.3

R2(tox2)

0.0

11.6

24.2

39.7

53.3

60.2

64.0

66.4

69.3

71.9

74.1

75.6

77.6

R3(tox3)

0.1

11.2

25.5

40.3

51.2

57.1

60.4

63.4

65.5

67.6

68.9

69.3

70.5

Rtoxavg

0.3

11.6

23.8

39.3

51.7

58.2

61.7

64.3

66.8

69.2

70.8

71.8

73.5

 

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

7.61

7.61

7.59

7.54

7.65

7.61

7.64

7.62

7.51

7.57

7.59

final

7.33

7.96

7.33

7.20

7.19

7.20

8.48

8.58

8.28

8.28

8.22

8.74

 

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 79.8%. 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 61.7%. Thus, the test item is not inhibitory. At the 28th day of the test the measured aerobic biodegradation of the test item attained 79.8%. Thus, the test item can be deemed to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance isoborneol which shares the same functional groups with the substance L-borneol also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Parameter:
% degradation (O2 consumption)
Value:
79.8
Sampling time:
28 d
Remarks on result:
other: (% degradation of analogue substance isoborneol)
Details on results:
Based on the experimental results obtained with the analogue substance isoborneol (100 mg/L) which was found to degrade up to 79.8 % after 28 days incubation with activate sludge inoculum (30 mg/L SS), the read-across was applied and L-borneol was also considered to be readily biodegradable under test conditions.
Validity criteria fulfilled:
not applicable
Interpretation of results:
readily biodegradable
Conclusions:
Based on read-across approach from experimental data on analogue substance isoborneol, L-borneol is determined to be readily biodegradable.
Executive summary:

Based on experimental results on analogue substance isoborneol (100 mg/L) where 79.8% of biodegradation was reached after 28 days of incubation with activated sludge inoculum (30 mg/L SS) in aerobic conditions in a manometric respirometry test (OECD Guideline 301F), the read across approach was applied and L-borneol is determined to be readily biodegradable under aerobic aqueous conditions. Furthermore, it is assumed that L-borneol is not inhibitory to activated sludge in an estimated concentration of 100 mg/L based on the experimental results with the analogue substance where 61.7% of biodegradation was obtained in the toxicity test after 14 days of incubation.


Description of key information

Key study: Read-across approach. Test method EU method C.4 -D, OECD 301F. GLP study. Based on read-across approach from experimental data on analogue isoborneol, L-borneol is determined to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Key study: Read-across approach: In the readily biodegradability study conducted on analogue isoborneol according to OECD 301F (GLP study), 79.8% of biodegradation was obtained after 28 days of incubation with activated sludge inoculum in aerobic conditions. Based on these results, the read-across was applied and L-borneol is determined to be readily biodegradable under aerobic aqueous conditions.