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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:
18 June 2019 - 16 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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 for exposure: The sludge was aerated for 7 days, at the test temperature of about 22 ºC, 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 A sample was withdrawn just before use for the determination of the dry weight of the suspended solids.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.

Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Remarks:
(67.3 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 11 ml 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, with measured content of organic carbon below the measuring range of 2.0 mg/L, 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
- Aeration of dilution water: continuous stirring
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes
- Other: volume of test solution in flask, V: 0.164 L

TEST SYSTEM
- Number of culture flasks/concentration: triplicates were used, flasks #14, 15, 16 containing test item (100 mg/l) and inoculum (30mg/L SS).
- Method used to create aerobic conditions: constant stirring
- 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: 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, 7 containing reference item (sodium acetate 100 mg/l) and inoculum 30mg/L SS.
- Toxicity control: flasks #17, 18, 19, 20, 21 containing test item 100 mg/L, reference item 100 mg/L and inoculum 30mg/L SS.
- Other: Flasks used for correction in oxygen uptake due to interference by nitrification:
Flasks 0a1, 0a2: Test suspension containing test item (100 mg/L) and inoculum at start.
Flasks 0tox1, 0tox2: Test suspension containing test item and reference item at the same concentrations as in the individual solutions and inoculum at start.

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:
0
Sampling time:
28 d
Details on results:
- The test item attained 0.0% of biodegradation
- The reference item reached 83.9% of biodegradation and the level for ready biodegradability by 4 days (>60% reached before day 14)
- In the toxicity test the biodegradation was 0.0% in 14 days (<25% in 14 days). Therefore, the test item can be assumed to be inhibitory.
- The oxygen uptake of the inoculum blank was equal to 40.5 mg/l in 28 days (<60 mg/l in 28 days)
- The pH values (7.53-7.68) of all flasks were inside the range (6-8.5)
Results with reference substance:
The reference item reached 83.9% 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

0.0

0.0

0.3

2.6

2.8

5.6

7.9

9.0

11.3

13.6

15.2

17.2

19.4

a2

0.1

1.8

3.2

8.8

13.6

19.8

23.3

28.5

34.1

40.0

42.3

47.0

51.3

a3

0.0

0.0

0.3

3.0

2.8

5.0

5.6

5.6

7.9

8.4

8.4

10.4

12.0

am. avg

0

0

0.3

2.8

2.8

5.3

6.8

7.3

9.6

11.0

11.8

13.8

15.7

Blank test O2uptake. mg/L

b1

5.5

11.6

14.9

17.1

19.9

22.4

22.4

26.7

29.0

31.8

33.8

35.8

41.1

b2

6.0

11.4

14.1

15.5

17.8

21.5

22.5

26.8

29.7

31.5

33.6

35.1

38.8

b3

6.7

10.9

14.1

16.9

17.9

22.5

25.7

28.2

31.7

35.5

36.4

37.9

41.6

bmavg

6.0

11.3

14.3

16.5

18.5

22.1

23.5

27.2

30.1

33.0

34.6

36.2

40.5

Reference item O2uptake. mg/L

w1

13.9

54.1

67.0

76.2

80.2

87.8

93.2

97.6

101.1

103.2

106.6

107.7

111.7

w2

9.5

51.3

67.5

75.3

81.3

85.7

88.0

90.0

92.9

93.3

95.5

97.6

99.2

w3

14.7

56.0

69.0

78.9

82.8

90.2

93.2

95.9

98.8

99.6

101.6

103.8

107.2

w4

11.2

53.0

67.0

76.0

81.3

87.2

91.1

94.1

97.3

98.7

101.1

102.9

105.7

wm. avg

12.3

53.6

67.6

76.6

81.4

87.7

91.4

94.4

97.5

98.7

101.2

103.0

106.0

 

Toxicity

control O2uptake mg/L

a17tox1

0.1

0.3

0.3

1.7

2.8

5.1

5.6

7.6

9.3

11.4

12.3

14.6

19.3

a18tox2

0.1

0.0

2.0

2.8

5.7

9.1

11.3

14.1

16.9

19.7

22.5

25.3

28.0

a19tox3

0.0

0.0

0.0

0.0

2.9

5.1

7.0

11.1

14.0

20.8

36.8

68.9

81.7

toxm. avg

0.1

0.1

0.8

1.5

3.8

6.4

8.0

10.9

13.4

17.3

23.9

36.3

43.0

a20tox

0.00

0.00

0.00

0.03

1.97

4.05

7.17

-

-

-

-

-

-

a21tox

0.57

0.003

2.07

2.95

3.61

5.69

8.24

-

-

-

-

-

-

a20-21toxavg

0.29

0.002

1.04

1.49

2.79

4.87

7.71

-

-

-

-

-

-

Corrected

test item O2uptake, mg/L

a1- bm

-6.0

-11.3

-14.0

-13.9

-15.7

-16.5

-15.6

-18.2

-18.8

-19.4

-19.4

-19.1

-21.1

a2- bm

-6.0

-9.5

-11.1

-7.7

-4.9

-2.3

-0.3

1.3

4.0

7.0

7.6

10.7

10.8

a3- bm

-6.0

-11.3

-14.1

-13.5

-15.7

-17.1

-17.9

-21.6

-22.2

-24.6

-26.2

-25.9

-28.5

Reference item % degradation

ThOD = 0.78

mgO2/mg

C = 100 mg/L

R1(w1)

10.1

54.9

67.6

76.5

79.1

84.1

89.4

90.3

90.9

90.0

92.3

91.7

91.3

R2(w2)

4.4

51.3

68.1

75.4

80.5

81.5

82.6

80.5

80.5

77.4

78.1

78.6

75.2

R3(w3)

11.1

57.3

70.1

80.0

82.4

87.2

89.4

88.1

88.1

85.5

85.8

86.6

85.6

R4(w4)

6.6

53.5

67.5

76.3

80.4

83.5

86.6

85.7

86.1

84.2

85.2

85.5

83.6

Rtoxavg

8.1

54.3

68.3

77.0

80.6

84.1

87.0

86.1

86.4

84.3

85.3

85.6

83.9

The obtained test item oxygen uptake for a2, as very different from a1 and a3, was rejected.

The difference of extremes of a1 and a3 replicate values of the oxygen uptake of test item at the end of the test is ((19.4 – 12.0)/19.4)*100% =  38.1%  is greater than 20%.

Table 3. Correction for test item oxygen uptake for interference by nitrification.

           days

0

28

difference

1) Concentration of nitrate

(mg N-NO3/l)

Flask #0a1

Flask #0a2

Flask #14

Flask #16

 

-0.33

2.16

1.97

1.54

1.94

Avg: 2.07 ± 0.13

Avg: 1.74 ± 0.28

2) Oxygen equivalent (4.57× N-NO3) (mg/L)

 

-1.51

 

3) concentration of nitrite

 (mg N-NO2/l)

Flask #0a1

Flask #0a2

Flask #14

Flask #16

difference

0.521

0.481

0.39

0.47

-0.07

Avg: 0.501 ± 0.028

Avg: 0.43 ± 0.06

4) Oxygen equivalent (3.43×N-NO2) (mg/L)

 

-0.24

5) total oxygen equivalent 2) + 4)

 

-1.75

Table 4. Correction for toxicity test oxygen uptake for interference by nitrification.

           days

0

14

difference

1) Concentration of nitrate (mg N-NO3/l)

Flask #0tox1

Flask #0tox2

Flask #20

Flask #21

 

0.01

2.03

2.17

2.15

2.07

2.10 ± 0.01

2.11 ± 0.06

 

2) Oxygen equivalent (4.57× N-NO3) (mg/L)

 

0.04

 

3) concentration of nitrite (mg N-NO2/l)

Flask #0tox1

Flask #0tox2

Flask #20

Flask #21

difference

0.486

0.559

0.538

0.499

 

- 0.003

0.522 ± 0.052

0.519 ± 0.027

 

4) Oxygen equivalent (3.43×N-NO2) (mg/L)

 

-0.010

5) total oxygen equivalent 2) + 4)

 

0.03

Table 5. The pH values of test flasks (no adjustment of pH was conducted).

flask #

14

15

16

1

2

3

4

5

6

7

17

18

19

20

21

Test item

Inoculum blank

Reference item

Toxicity test

initial

7.55

7.63

7.64

7.65

7.62

7.53

7.51

7.51

7.61

7.63

7.63

7.65

7.55

7.46

7.61

final

7.53

7.65

7.68

7.36

7.38

7.32

8.35

8.94

8.70

8.67

8.12

7.84

8.26

7.81

7.89

Test item biodegradability:

After correction for oxygen uptake for nitrification and for the blank inoculum control, the following biodegradation calculation is suggested on the 28th day:

(15.7 -40.5 – (-1.75)) mg O2/L / (ThODNH4 x conc. Test Item) x 100% < 0.0 %

Toxicity test biodegradability:

Assuming, as above, after correction for the blank inoculum control and nitrification, the biodegradation in toxicity test on the 14th  test day is:

(7.71 -23.5 -0.03) mg O2/L / (ThODNH4 x conc. Test Item + ThOD x conc. Test Reference) x 100% < 0.0 %

 

Validity criteria fulfilled:
no
Remarks:
See "overall remarks".
Interpretation of results:
not readily biodegradable
Conclusions:
At the 28-day ready aerobic biodegradability testing the biodegradation of the test item equals 0.0 %. Thus, the test item cannot be deemed to be readily biodegradable.

Executive summary:

A 28-day 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 ± 1ºC) 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 the reference item, on the 14th test day the biodegradation (based on ThOD) was 0% (less than 25%), after correction for the blank inoculum control and nitrification, and then the test item can be assumed to be inhibitory. At the 28th day of the test the measured aerobic biodegradation of the test item equals 0.0% after correction for the blank inoculum control and nitrification. Thus, the test item cannot be deemed to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Internationally accepted method, EPI-Suite, EPA (USA)
Justification for type of information:
See attached the QMRF and QPRF for the QSAR model.

Guideline:
other: REACH Guidance on QSARs R.6
Principles of method if other than guideline:
Howard, P.H., Boethling, R.S., Stiteler, W.M., Meylan, W.M., Hueber, A.E., Beauman, J.A., Larosche, M.E. 1992. Predictive model for aerobic biodegradability developed from a file of evaluated biodegradation data. Environ. Toxicol. Chem. 11:593-603.
Specific details on test material used for the study:
SMILES: C12cc (O) ccc1nc3ccc (N (CC) CC) cc3n2 (c4ccccc4) OS (=O) (=O) On5 (c8ccccc8) c7cc (O) ccc7nc6ccc (N (CC) CC) cc56
Key result
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

BIOWIN1 (Linear Model): 0.4511

BIOWIN2 (Non-Linear Model): 0.0008

Expert Survey Biodegradation Results:

BIOWIN3 (Ultimate Survey Model): 1.1117 (recalcitrant)

BIOWIN4 (Primary Survey Model):2.2284 (months)

MITI Biodegradation Probability:

BIOWIN5 (MITI Linear Model): -1.2842

BIOWIN6 (MITI Non-Linear Model): 0.0000

Anaerobic Biodegradation Probability:

BIOWIN7 (Anaerobic Linear Model): -3.0296

Ready Biodegradability Prediction: NO

Validity criteria fulfilled:
not applicable
Interpretation of results:
not readily biodegradable
Conclusions:
The substance Violet LCC6D was predicted to be not ready biodegradable (EPI-Suite, BIOWIN v4.10)
Executive summary:

The substance Violet LCC6D was predicted to be not ready biodegradable (EPI-Suite, BIOWIN v4.10)

Description of key information

Weight of evidence approach:

1. Test method according to OECD 301F / EU C.4 (Manometric respirometry methods), GLP study. At the 28-day ready aerobic biodegradability testing the biodegradation of the test item equals 0.0 %. Thus, the test item is deemed not to be readily biodegradable.

2. Data from EPI-Suite, EPA (USA) / BIOWIN v4.10 calculation method. The substance Violet LCC6D was predicted to be not ready biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

Weight of evidence approach:

The test substance was investigated in a biodegradation test (OECD TG 301 F) and the measured aerobic biodegradation attained 0.0% at 28th day of the test.  Thus the test substance was deemed not to be readily biodegradable. However, in the toxicity test run in parallel containing both the test item and a reference chemical, 0.0% biodegradation occurred in 14 days (it should be higher than 25% as per OECD TG 301 criterion) so the test item can be assumed to be inhibitory. If this happens, the test series should be repeated, using a lower concentration of test substance (if this can be done without seriously impairing the accuracy of the DOC determination) and/or a higher concentration of inoculum, but not greater than 30 mg solids/L.

Taking into account that a high toxicity of the test item to activated sludge was found, a repetition of the test was deemed to be rather ineffective even though very low concentrations are considered to be tested. On the other hand, the test was conducted with a concentration of inoculum of 30 mg solids/L, therefore in order to comply with the guideline’s requirement it would not be possible to increase this concentration in a further testing.

For this reason, the information on the biodegradation provided by QSAR prediction from BIOWIN 4.10 was considered in combination with the experimental result as part of a weight of evidence approach. The test substance was predicted as not ready biodegradable by the BIOWIN models. The same result was obtained with both the experimental test and QSAR prediction, thus it is possible to assume the test substance as not ready biodegradable as a weight of evidence conclusive decision.