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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:
weight of evidence
Study period:
March 3, 2016 - March 31, 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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: 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 22ºC, until application next day. A sample was withdrawn just before use for the determination of the dry weight of the suspended solids.
- Amount of inoculum suspended: 30 mg/L.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: (a) Monopotassium dihydrogen orthophosphate, KH2PO4 8.50 g; Dipotassium monohydrogen orthophosphate, K2HPO4 21.75 g; Disodium monohydrogen orthophosphate dihydrate Na2HPO4 · 2 H2O 33.40 g; Ammonium chloride, NH4Cl 0.50 g. dissolved in water and made up to 1 litre. The pH of the solution should be 7.4. (b) Calcium chloride, anhydrous, CaCl2 27.50 g dissolved in water and made up to 1 litre. (c) Magnesium sulphate heptahydrate, MgSO4 · 7 H2O 22.50 g dissolved in water and made up to 1 litre. (d) Iron(III) chloride hexahydrate, FeC13 · 6H2O 0.25 g dissolved in water and made up to 1 litre. It should be mixed 10 ml of solution (a) with 800 ml dilution water, added 1 ml of solutions (b) to (d) and made up to 11 with dilution water.
- Dilution water: double-distilled water which contained no more than 10% of the organic carbon content introduced by the test material (3 mg/l checked by DOC analysis using spectrophotometer Hach DR 3900 and Hach-Lange reagents)
- pH: 7.4 ± 0.2
- Test temperature: 22 ± 2°C
- Volume of test solution in flask: 0.164 l
- Initial test item concentration: 100 mg/l (49.8 mg/l of organic carbon)
- Calculated ThOD(NH4): 2.64 mgO2/mg of test item; Calculated ThOD(NO3): 3.26 mgO2/mg test item.
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: closed WTW OxiTop OC 110 respirometer flasks placed in a thermo-cabinet WTW TS 606 CZ-G/3-VAR
- 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. The 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. Dissolved Organic Carbon in water was determined using Hach-Lange reagents for TOC measurements in LCK 380: Photometer with Barcode-System. Nitrates were determined using LANGE Cuvette Test LCK339 and nitrites were determined using LANGE Cuvette Test LCK341.

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 ≥ 98.5%, source: Eurochem.
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
- The measured aerobic biodegradation of the test item after 28 days was 0.0%.
- The biodegradation in the toxicity test was 11.5% in 14 days so it could be assumed that the test item was inhibitory.
Results with reference substance:
The reference item reached 75.5% of biodegradation and the level for ready biodegradability by day 5.

Correction for oxygen uptake for interference by nitrification

days

0

28

difference

#13

#14

#15

#13

#14

#15

#13

#14

#15

Concentration of nitrate (mg N-NO3/l)

0.083

0.065

0.072

1.16

0.971

0.924

1.08

0.906

0.852

0.946 ± 0.119

Oxygen equivalent (4.57 x N-NO3) mg/l

 

4.32

Concentration of nitrite (mg N-NO2/l)

0.006

0.007

0.008

0.176

0.167

0.185

0.170

0.160

0.177

0.169 ± 0.009

Oxygen equivalent (3.43 x N-NO2) mg/l

 

0.580

Total oxygen equivalent

 

4.90

Toxicity test

days

0

28

14

difference

 

#16

#17

#18

#16

#17

#18

#16

#17

#18

Concentration of nitrate (mg N-NO3/l)

0.039

0.069

0.061

0.93

1.13

0.738

0.891

1.06

0.677

0.976 ± 0.120

0.677

Oxygen equivalent (4.57 x N-NO3) mg/l

 

4.46

3.09

Concentration of nitrite (mg N-NO2/l)

0.005

0.006

0.005

0.075

0.085

0.095

0.070

0.079

0.09

0.075 ± 0.006

0.09

Oxygen equivalent (3.43 x N-NO2) mg/l

 

0.257

0.309

Total oxygen equivalent

 

4.72

3.399

 

Sample oxygen uptake: biodegradability

 

time, days

3

5

7

9

12

14

16

18

21

23

25

28

Test item O2uptake, mg/l

a1

7.6

11.1

11.9

11.7

15.0

16.2

16.5

18.3

20.9

21.5

21.8

23.1

a2

5.2

8.0

7.9

9.1

10.0

12.0

11.7

12.4

12.4

13.9

14.2

15.1

a3

7.1

8.4

8.8

8.8

10.0

11.6

11.3

11.3

14.3

13.3

15.2

16.0

am.avg

6.6

9.2

9.6

9.9

11.7

13.3

13.2

14.0

15.9

16.2

17.1

18.1

Blank test O2uptake. mg/l

b1

17.1

20.3

25.6

28.4

32.8

34.5

37.4

38.2

40.2

42.9

43.4

45.0

b2

12.1

16.6

20.7

23.3

27.1

28.1

30.7

31.9

34.2

33.8

35.5

36.6

b3

13.3

16.1

19.7

22.1

24.6

26.9

28.9

30.8

32.6

34.3

36.7

37.6

bmavg

14.2

17.7

22.0

24.6

28.2

29.8

32.3

33.7

35.7

37.0

38.5

39.7

Reference item O2uptake. mg/l

w1

36.5

37.3

42.6

49.3

57.9

61.8

65.6

69.4

73.5

76.0

77.7

82.2

w2

51.2

63.7

73.8

80.2

87.8

90.6

92.9

96.1

99.0

99.3

101.4

101.6

w3

49.1

61.8

70.8

76.5

82.3

87.1

87.2

90.1

93.0

92.8

96.1

95.7

wm.avg

50.1

62.7

72.3

78.3

85.0

88.9

90.0

93.1

96.0

96.1

98.8

98.6

Toxicity control O2uptake. mg/l

a4tox1

61.3

63.8

67.2

69.3

70.3

71.7

72.7

75.4

77.6

78.5

79.5

79.7

a5tox2

62.1

64.8

66.7

68.5

73.0

73.2

75.0

76.0

78.1

78.6

78.9

79.2

a6tox3

60.1

63.6

66.8

66.6

69.7

70.5

0.0

0.0

0.0

0.0

0.0

0.0

toxm.avg

61.7

64.3

66.9

68.9

71.7

72.5

73.8

75.7

77.8

78.6

79.2

79.5

Corrected

test item O2uptake, mg/l

a1- bm

-6.5

-6.6

-10.0

-12.9

-13.2

-13.6

-15.8

-15.4

-14.7

-15.5

-16.7

-16.6

a2- bm

-9.0

-9.7

-14.1

-15.4

-18.1

-17.8

-20.7

-21.2

-23.2

-23.2

-24.3

-24.6

a3- bm

-7.1

-9.2

-13.2

-15.8

-18.2

-18.2

-21.1

-22.3

-21.3

-23.7

-23.3

-23.8

Reference item % degradation (BOD /

ThOD x C) x 100

ThOD = 0.78

mgO2 / mg

C = 100 mg / l

R1(w2)

47.4

59.0

66.4

71.3

76.5

77.9

77.6

80.0

81.1

79.9

80.6

79.3

R3(w3)

44.7

56.6

62.5

66.6

69.4

73.5

70.3

72.4

73.5

71.5

73.8

71.8

Rtoxavg

46.1

57.8

64.5

68.9

72.9

75.7

74.0

76.2

77.3

75.7

77.2

75.5

w1 was not taken into average value calculations, as it was distinctly lower than other reference item O2uptakes. 

a6tox3 result from flask #18 was not taken into average value calculation, as this flask was withdrawn from test after 14 day incubation to nitrate and nitrite analysis.

pH values of the test flasks (no adjustment of pH was conducted)

flask #

19

20

21

7

8

9

10

11

12

22

23

24

Test item

Control

Reference item

Toxicity test

initial

7.55

7.54

7.54

7.48

7.50

7.54

7.57

7.47

7.46

7.54

7.52

7.52

final

7.51

7.73

7.75

7.27

7.19

7.20

8.37

8.43

8.60

7.49

9.25

9.07

Validity criteria fulfilled:
yes
Remarks:
difference of replicates <20%,reference item pass level (60%) on day 5, blank oxygen uptake 39.7 mgO2/l in 28 days, pH inside the range 6-8.5,test item oxygen consumption < 60%. Biodegradation (b. on ThOD) 11.5% in toxicity test: test item inhibitory
Interpretation of results:
under test conditions no biodegradation observed
Remarks:
Test item might be inhibitory.
Conclusions:
The test substance is not readily biodegradable: at the 28th day of the test the measured aerobic biodegradation of the test item attained 0%.
Executive summary:

A 28-day ready aerobic biodegradability test in an aerobic aqueous medium was performed on the test item, according to OECD 301F / EC C.4 – D manometric respirometry methods. GLP study. 100 mg/l of test item was inoculated with 30 mg/l SS of activated sludge and incubated under aerobic conditions in a closed respirometer flask at constant temperature of 22ºC (± 2). A blank test, a control test with reference substance (sodium acetate) and a toxicity test were run in parallel. Correction for the oxygen uptake for interference by nitrifcation was made. Under the test conditions, the measured aerobic biodegradation of the test was 0%. According to OECD criteria, the pass level for ready biodegradability for respirometric methods are 60% of ThOD, thus the test item cannot be deemed to be readily biodegradable. Furthermore, the biodegradation in the toxicity test was equal to 11.5% (<25%) after 14 days, so the test item might be inhibitory.

Endpoint:
biodegradation in water: screening test, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
Japanese MITI study.
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
not specified
GLP compliance:
not specified
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Concentration of sludge: 30 ppm
Duration of test (contact time):
4 wk
Initial conc.:
100 other: ppm
Based on:
not specified
Parameter followed for biodegradation estimation:
O2 consumption
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Key result
Parameter:
% degradation (test mat. analysis)
Value:
99
Sampling time:
28 d
Details on results:
The test substance formed N,N'-dicyclohexylurea.
Validity criteria fulfilled:
not specified
Remarks:
It was a Japanese MITI study.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The biodegradation measured as BOD was of 0%.
The degradation of the substance measured by GC was of 99%. The test substance formed N,N'-dicylohexylurea.
Executive summary:

A screening test of ready biodegradability of the test substance was performed according to the Japanese MITI test, one of the methods adopted in the OECD TG 301C. The substance, present at 100 ppm, reached 0% of its theorical BOD in 4 weeks, using an activated sludge inoculum at 30 ppm. The degradation of the substance measured by GC was of 99%. The test substance formed N,N'-dicylohexylurea.

Description of key information

Weight of evidence. Based on the available information, the test item is not readily biodegradable:

- Method according to OECD TG 301F / EU Method C.4 - D. GLP study. The test item was considered not to be readily biodegradable.

- Method according to OECD TG 301C (MITI, Japan). The test item was found not to be biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Weight of evidence:

- A Ready biodegradability test according to OECD Guideline No. 301F/EC C.4 -D manometric respiratory methods was conducted under GLP. 30 mg/l SS of activated sludge were exposed to 100 mg/ml of the test item during 28 days under aerobic conditions at 22ºC. The measured aerobic biodegradation of the test was 0%. after 28 days of incubation. According to OECD criteria the test item cannot be deemed to be readily biodegradable. Furthermore, the biodegradation in the toxicity test was equal to 11.5% (<25%) after 14 days, so the test item can be assumed to be inhibitory under the test conditions.

- A screening test of ready biodegradability of the test substance was performed according to the Japanese MITI test, one of the methods adopted in the OECD Guideline No. 301C. The substance, present at 100 ppm, reached 0% of its theoretical BOD in 4 weeks, using an activated sludge inoculum at 30 ppm. The degradation of the substance measured by GC was of 99%. The test substance formed N,N'-dicyclohexylurea.