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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)
EC Number:
278-207-2
EC Name:
Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)
Cas Number:
75431-69-5
Molecular formula:
C20H12N2O5S.1/3Al
IUPAC Name:
aluminum tris(7,14-dioxo-5,7,12,14-tetrahydroquino[2,3-b]acridine-2-sulfonate)
Constituent 2
Chemical structure
Reference substance name:
5,12-dihydroquino[2,3-b]acridine-7,14-dione
EC Number:
213-879-2
EC Name:
5,12-dihydroquino[2,3-b]acridine-7,14-dione
Cas Number:
1047-16-1
Molecular formula:
C20H12N2O2
IUPAC Name:
5,12-dihydroquino[2,3-b]acridine-7,14-dione
Constituent 3
Chemical structure
Reference substance name:
Dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)
EC Number:
243-319-2
EC Name:
Dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)
Cas Number:
19795-24-5
Molecular formula:
C20H12N2O8S2.2/3Al
IUPAC Name:
dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Municipal sewage treatment plant, Mannheim, Germany
- the inoculum was collected on 2016-11-14 from the aeration tank of the plant.
- Pretreatment/Concentration of sludge: s suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size of about 1 mm. To reduce the concent of inorganic carbon in the blank controls the activated slduge was aerated with carbon dioxide free air for about 72 hours at 22 ± 2°C. At the day of exposure the suspension was washed on time with drinking water. Therefore the aeration was stopped and the sludge was allowed to settle. After settling the supernatant was discarded and the remaining sludge suspension was filled up with drining water. The concentration of the sludge was adjusted to 6.0 g/L dry weight.






Duration of test (contact time):
28 d
Initial test substance concentrationopen allclose all
Initial conc.:
ca. 51 mg/L
Based on:
test mat.
Initial conc.:
20 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301 B / CO2 Evolution Test
- Test temperature: 22 +/- 2°C
- Dispersion treatment: Continuous magnetic stirring
- Aeration: carbon dioxide free air at a flow of approximately 800 mL per hour


TEST SYSTEM
- Culturing apparatus: 2000 mL incubation bottles filled up to a volume of 1500 mL
- Number of culture flasks/concentration: 1 for the reference item, 1 for inhibition control, 2 for the blank control, 2 for the test item
- Method used to create aerobic conditions: carbon dioxide free air at a flow of approximately 800 mL per hour
- Details of trap for CO2 and volatile organics if used:
CO2 adsorption vessels were connected to the air outlets of the incubation vessels via a series of 2 scrubbing bottles, each containing 100 mL of
a 0.05 mol/LNaOH solution.
- Course of the study:
The following incubation vessels were prepared:
- two for the test item concentration (TS1, TS2)
- one for the functional control (RS)
- two for the inoculum control (BC1, BC2)
- one for the inhibition control respectively (IH)

The test assays were prepared on the day of exposure. First, the required volumes of deionized water and the solutions of the mineral salts were dosed to all test vessels. The required amounts of the test substance aliquots for a concentration of 20 mg/L TOC were weighed into the vessels of the test substance assays and to the vessel of the inhibition control. Due to the poor water solubility of the test substance these test assays were treated for few minutes in an ultrasonic bath to ensure an even distribution of the test substance in the test medium. Finally enough reference substance (aniline) stock solution was added to reach 20 mg/L TOC in the reference substance assay and the inhibition control.
The pH-values in the test vessels were measured and adjusted to 7.4 +/- 0.2, if necessary. Aliquots of activated sludge suspension were added to all test vessels to adjust the concentration of activated sludge to 30 mg/L dry weight.
At being of exposure phase the test vessels were connected with an aeration unit and the bubble aeration with carbon dioxide free air was started after connecting with the CO2 adsorption scrubbin bottles to the air outlets of the incubation vessels.
At the end of exposure the pH values were measured in each test vessel. For stripping of carbon dioxide dissolved in the test medium each test vessel was acidified by adding 2 mL of concentrated hydrochloric acid. The concentration of DOC in the blank controls and the reference substance assays were determined. Due to the poor solubility of the test item DOC measurements could not be performed in the test substance assays.
Teh aeration was coninued for about 24 hours and the released carbon dioxide amounts in both traps of each test vessel were determined and added to the calculated amount of the previous day.

SAMPLING
- Sampling frequency:
TIC analysis was carried out on days 1, 4, 7, 11, 14, 18, 21, 25, 27, 28, 29
- Sampling method:
For each measurement the first stripping bottle was removed and a new bottle was connected to the last one.
Reference substance
Reference substance:
aniline

Results and discussion

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
9
Sampling time:
28 d
Details on results:
The test substance was not readily biodegradable in this carbon dioxide evolution test based on the quantitative determination of the formed carbon dioxide. in the test substance assays by comparison with the calculated maximal theoretical carbon dioxide production.
Because the test substance is a mixture of structural similar compounds, the criterion of the ten days window was not applied.
The degree of biodegradation after an exposure period of 28 days was > 10 %. The value represents the mean of two test assays.

BOD5 / COD results

Results with reference substance:
In the inhibition control containing both test and reference item a biodegradation of 29 % was determined within 14 days and it came to 44 % after 28 days. The biodegradation of the reference item aniline achieved a value of 66 % after 14 days and 94 % after 28 days.

Any other information on results incl. tables

total organic carbon of test substance: 395 mg/g

reference substance: aniline; stock solution: 401.5 mg/L, Theoretical Organic Carbon (ThOC): 311 mg/L

sample

BC1

BC2

RS

IC

TS1

TS2

Test substance concentration [mg/L]

-

-

-

50.3

50.9

50.7

TOC concentration of test substance [mg/L], nominal value

-

-

-

20

20

20

TOC concentration of reference substance [mg/L], nominal value

-

-

20

20

-

-

Stock solution reference substance [mL/test vessel]

-

-

96

96

-

-

Deionized water [mL/test vessel]

1473

1473

1377

1377

1473

1473

Mineral medium [mL/test vessel]

19.5

19.5

19.5

19.5

19.5

19.5

Inoculum (6 g/L dry matter) [mL/test vessel]

7.5

7.5

7.5

7.5

7.5

7.5

DIC values at start of exposure [mg/L]

0.7

0.5

-

-

-

-

 

0.8

0.5

-

-

-

-

pH values at start of exposure

7.3

7.3

7.4

7.4

7.3

7.3

pH values at start end exposure

7.4

7.4

7.3

7.2

7.3

7.3

Table 2: measurement of Total Inorganic Carbon

Day

BC NaOH

BC1

BC2

RS

IH

TS1

TS2

0

0.8

-

-

-

-

-

-

 

0.9

-

-

-

-

-

-

1

1.6

4.6

3.9

6.8

6.0

7.0

5.1

 

1.4

4.5

3.6

6.6

5.8

6.6

5.0

4

1.1

17.4

15.7

84.2

58.9

22.2

16.3

 

1.0

17.4

15.4

84.1

58.9

21.8

16.2

7

1.1

21.9

20.6

82.3

91.0

26.2

21.6

 

1.2

21.7

20.5

82.1

90.4

26.0

21.3

11

0.6

23.9

26.3

56.9

58.0

31.6

25.1

 

0.8

24.1

26.1

57.3

57.6

31.5

24.9

14

1.1

17.7

17.3

51.2

46.0

21.7

22.3

 

1.1

17.9

17.4

51.3

46.1

21.9

22.4

18

0.7

20.2

20.6

53.3

46.2

24.6

18.3

 

0.7

19.7

21.3

53.6

45.6

24.0

18.5

21

1.0

14.4

15.3

31.9

36.4

21.7

15.4

 

1.1

14.3

15.3

31.8

36.5

21.8

15.4

25

0.9

16.0

16.4

34.6

40.2

24.9

17.0

 

0.9

15.6

16.1

34.1

40.3

24.7

16.7

27

1.1

11.8

10.1

21.2

19.9

13.2

11.7

 

1.0

11.7

10.0

20.8

19.3

12.7

11.4

28

1.3

7.7

7.4

12.2

10.6

8.0

7.1

 

1.3

7.6

7.4

12.1

10.3

8.0

6.8

29

 

10.2

14.7

13.1

14.7

15.9

7.9

flask 1

 

10.4

14.8

13.2

14.9

15.9

7.8

29

 

5.4

5.1

5.6

4.9

5.8

7.6

flask 2

 

5.4

5.1

5.5

4.8

5.8

7.6

The NaOH blank value is determined on filling the absorption vessels. The measured values applies for the first and the second sampling respectively. The NaOH blank value was subtracted from each test vessel.

Table 3: degree of biodegradation [% CO2/ThCO2]

Test duration [days]

RS

IH

TS1

TS2

TS mean value

0

0

0

0

0

0

1

1

0

1

0

1

4

24

7

3

0

2

7

44

19

4

0

2

11

55

25

7

0

4

14

66

29

8

2

5

18

77

34

9

1

5

21

83

37

12

2

7

25

89

41

15

2

9

27

92

43

15

2

9

28

94

44

17

1

9

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the test conditions the test item is classified as not readily biodegradable within the 28 day period of the study.
Executive summary:

The ready biodegradability of the test item was determined with a non-adapted activated sludge over a test period of 28 days according to OECD 301B. The test item was tested at a concentration of 51 mg/L with 2 replicates corresponding to a carbon content (TOC) of 20 mg C/L in the test vessels.The test vessels were incubated at a temperature of 22 ± 2 °C.

The biodegradation of the test item was followed by TIC analysis of the quantity of CO2produced by the respiration of bacteria. The degradation was stopped on day 28 by acidification of the test solutions. The last measurement was made on day 29 after residual CO2had been purged from the test solutions over a period of 24 hours. The percentage CO2 production was calculated in relation to the theoretical CO2production (ThCO2) of the test item. The biodegradation was calculated for each TIC measurement.

 

To check the activity of the test system aniline was used as functional control. The percentage degradation of the functional control reached the pass level of 60 % within 14 days and a maximum biodegradation of 94 % after 18 days.

 

In the inhibition control containing both test and reference item a biodegradation of 29 % was determined within 14 days and it came to 44 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control.

 

The test substance was not readily biodegradable in this carbon dioxide evolution test based on the quantitative determination of the formed carbon dioxide. in the test substance assays by comparison with the calculated maximal theoretical carbon dioxide production.

Because the test substance is a mixture of structural similar compounds, the criterion of the ten days window was not applied.

The degree of biodegradation after an exposure period of 28 days was > 10 %. The value represents the mean of two test assays.