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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-13 to 2018-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD and GLP guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Specific details on test material used for the study:
Batch number Op 13
EC No. 916-632-3
Chemical name Reaction mass of disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate and disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate) (3:1) (REACH)

Component 1:
EC No. 270-629-5
CAS No. 68460-07-1
Molecular weight 706.06 g/mol
Molecular formula C28H20N8O8S2Na2

Component 2:
EC No. 272-559-0
CAS No. 68877-33-8
Molecular weight 705.08 g/mol
Molecular formula C28H21N9O7S2Na2

Composition Comp. 1 and 2: 66.3 %
Water solubility not determined / estimated > 10 g/L
ThOD* 0.90 mg O2/mg
ThODNO3* 1.46 mg O2/mg
Appearance Dark grey, partly coarse powder (solid)
Expiry date 2027-11-20
Recommended storage Room temperature

* The ThOD and ThODNO3 were calculated at the test facility, based on the information provided by the sponsor.

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): Activated sludge from the municipal sewage treatment plant, 31137 Hildesheim, Germany
- Receipt: 2018-03-13
- Preparation of inoculum for exposure: The activated sludge was washed twice with chlorine free tap water. After the second washing the settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration with CO2 free air until test start (1 day). The amount of inoculum used to initiate inoculation was 4.5 mL/L (25.0 mg/L dw).
- Initial cell/biomass concentration: approx. 0.52 x 107 CFU/L in the final test solution
- Water filtered: no
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
56 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 301F
- Additional substrate: No
- Test temperature: Nominal: 20 - 24, ± 1 °C; Actual: 20.9 – 21.2 °C

pH-Value
Start End
Inoculum Control 7.72 1) 7.57
2) 7.55
Functional Control 7.72 7.71
Test Item 7.69 1) 7.57
2) 7.57
Toxicity Control 7.66 7.69

1), 2) = First, second replicate

- pH adjusted: no
- Dispersion treatment: Continuous stirring
- Continuous darkness: Dark, in an incubator


TEST SYSTEM
- Culturing apparatus: Incubator
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: Closed bottles with a rubber sleeve with soda lime
- Measuring equipment: The bottles were closed with OxiTOP measuring heads.
The oxygen consumption was determined in the incubation vessels by the OxiTop® measuring system at 360 measuring points (every 112 minutes) during the 28 d incubation period.



SAMPLING
- Sampling frequency: 360 measuring points (every 112 minutes) during the 28 d incubation period.
- Sampling method: Pressure measurements directly from the test vessels



CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium without test and reference item
- Abiotic sterile control: No
- Toxicity control: Test item and reference item in test concentrations



STATISTICAL METHODS:
Calculation of the theoretical oxygen demand (ThOD) of the test item and functional control.
According to OECD-guideline 301, the index of the atoms of the item CcHhClclNnNanaOoPpSs was used for the calculation of the ThOD.
The oxygen depletion of the test and reference item was calculated out of the difference of the test and inoculum control replicates.
The biodegradation of the test and reference item was calculated out of the BOD in relation to the ThOD.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
No preliminary study
Test performance:
Based on the calculated oxygen demand, the test concentration of 56 mg/L, corresponding to an oxygen demand of 50.4 mg O2/L (81.8 mg O2/L with nitrification) in the vessel, was selected.

The test solutions were prepared in measuring flasks and were placed in brown glass bottles as incubation vessels (inoculum control, test item and functional control) or were prepared directly in brown glass bottles (toxicity control):

• two for the inoculum control (C1, C2)
• one for the functional control (R1)
• two for the test item (P1, P2)
• one for the toxicity control (T1)

Inoculum control
The inoculated test medium, consisting of the required volumes of mineral medium stock solutions, ultrapure water and inoculum, was prepared in a measuring flask. 250 mL of this solution were filled in the brown glass bottles of the inoculum control, using a 250 mL measuring flask.

Functional control
The reference item was weighed out and transferred into a measuring flask, the required volumes of mineral medium stock solutions, ultrapure water and inoculum, were added. 250 mL of this solution were filled in the brown glass bottle of the functional control, using a 250 mL measuring flask.

Test Item
The test item was weighed out and transferred into a measuring flask with ultrapure water and stirred overnight. The required volumes of mineral medium stock solutions, ultrapure water and inoculum were added. 250 mL of this solution was filled in the brown glass bottle of the test item replicate, using a 250 mL measuring flask.

Toxicity control
The reference item was weighed out and directly transferred into the test vessel with 250 mL of the test item medium (see above), using a 250 mL measuring flask.

A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2. The bottles were closed with
OxiTop measuring heads and the measuring system was activated.
% Degradation
Key result
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
The biodegradation in % was calculated based on the ThOD of 0.90 mg O2/mg test item.

Colony forming units (CFU) of the inoculum were determined prior to test start by standard dilution plate count: 1.16 109 CFU/L. The CFU concentration of the inoculum corresponds to approx. 0.52 x 107 CFU/L in the final test solution.

Results of the Functional Control
The pass level for ready biodegradation (≥ 60% degradation) was reached within 3 days. The validity criterion that the degradation should be ≥ 60% after 14 d was fulfilled.
Stages of Biodegradation: The adaptation phase changed to degradation phase within 2 days (degradation > 10%), with a maximum of 95% on day 28 .

Results of the Toxicity Control
In the toxicity control the biodegradation achieved 39% after 6 days. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.
Until day 6, the course of the measurement was identical to the functional control and is therefore considered to be reliable. Until then, all the validity criteria of the toxicity control were fulfilled, therefore the test item was considered to be not toxic to the test system. Starting with day 7, there have been technical issues with the toxicity control replicate. From then on, the measurement was considered to be not reliable anymore and was not reported.

Course and Stages of Biodegradation of Functional Control and Test Item
The biodegradation of the test item is shown in comparison to the readily degradable functional control and the toxicity control. Both test item replicates did not reach the 10% level (beginning of biodegradation) within the 28-day-period of the study. The mean biodegradation on day 28 was 0%.

After a test period of 28 days, the test item is classified as not readily biodegradable within the 28 day period of the study.

BOD5 / COD results

Results with reference substance:
In the toxicity control the biodegradation achieved 39% after 6 days. The validity criterion that the test item should not inhibit the biodegradation of the reference item was fulfilled.
Until day 6, the course of the measurement was identical to the functional control and is therefore considered to be reliable. Until then, all the validity criteria of the toxicity control were fulfilled, therefore the test item was considered to be not toxic to the test system. Starting with day 7, there have been technical issues with the toxicity control replicate. From then on, the measurement was considered to be not reliable anymore and was not reported.

Any other information on results incl. tables

Biological Oxygen Demand (BOD) of Inoculum Control, Functional Control, Test Item and Toxicity Control

 

 

BOD
[mg O2/L]

Date

[d]

Inoculum
Control

Functional
Control

Test Item

Toxicity Control*
Test Item +
Reference Item

 

 

 

R1

P1

P2

T1

 

 

mean

gross

net

gross

net

gross

net

gross

net

2017-03-15

1

0.9

4.1

3.2

2.2

1.2

1.3

0.4

6.6

5.7

2017-03-16

2

4.5

32.2

27.7

5.8

1.3

5.1

0.6

35.1

30.6

2017-03-17

3

8.4

38.5

30.1

8.4

0.0

7.8

-0.6

40.5

32.0

2017-03-18

4

11.7

43.7

32.1

10.7

-0.9

10.8

-0.8

45.9

34.3

2017-03-19

5

13.8

49.6

35.8

13.0

-0.8

12.8

-1.0

51.8

38.0

2017-03-20

6

15.7

54.9

39.1

14.0

-1.7

13.7

-2.0

54.9

39.2

2017-03-21

7

15.8

57.7

41.9

14.3

-1.5

11.5

-4.3

 

 

2017-03-22

8

17.1

60.9

43.8

15.5

-1.6

12.3

-4.8

 

 

2017-03-23

9

18.1

62.8

44.6

15.7

-2.4

13.0

-5.1

 

 

2017-03-24

10

19.1

64.7

45.6

16.9

-2.2

13.9

-5.2

 

 

2017-03-25

11

19.8

65.3

45.5

16.9

-2.9

14.1

-5.7

 

 

2017-03-26

12

21.3

66.6

45.4

18.4

-2.9

15.1

-6.2

 

 

2018-03-27

13

21.8

67.6

45.8

19.1

-2.7

15.9

-5.9

 

 

2018-03-28

14

22.1

68.4

46.3

19.7

-2.4

16.6

-5.5

 

 

2018-03-29

15

23.0

69.0

46.0

20.5

-2.6

17.5

-5.5

 

 

2018-03-30

16

23.3

70.3

47.0

21.1

-2.2

18.3

-5.0

 

 

2018-03-31

17

24.7

70.5

45.8

21.9

-2.8

19.6

-5.1

 

 

2018-04-01

18

24.8

71.8

47.0

22.5

-2.3

19.9

-4.8

 

 

2018-04-02

19

25.7

71.9

46.2

23.7

-2.0

21.2

-4.5

 

 

2018-04-03

20

25.9

72.0

46.1

24.0

-1.9

21.5

-4.4

 

 

2018-04-04

21

25.5

71.9

46.4

24.0

-1.5

21.1

-4.4

 

 

2018-04-05

22

25.8

72.2

46.4

24.3

-1.5

21.4

-4.4

 

 

2018-04-06

23

26.1

73.1

47.0

25.0

-1.1

22.5

-3.6

 

 

2018-04-07

24

26.6

73.4

46.8

25.4

-1.2

22.8

-3.8

 

 

2018-04-08

25

26.9

74.0

47.1

25.8

-1.1

22.6

-4.2

 

 

2018-04-09

26

27.0

74.5

47.5

26.6

-0.4

23.5

-3.5

 

 

2018-04-10

27

26.8

74.1

47.3

26.8

0.0

23.4

-3.4

 

 

2018-04-11

28

27.0

74.4

47.4

26.7

-0.3

23.7

-3.3

 

 

*) for details please refer to section 7.4

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: under test conditions the test item is classified as not readily biodegradable
Conclusions:
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 non-adapted activated sludge in the Manometric Respirometry Test for a period of 28 days. The study was conducted from 2018 -03 -13 to 2018 -04 -11, according to OECD guideline 301 F at the test facility. The test item concentration selected as appropriate was 56 mg/L, corresponding to a ThOD of 50.4 mg O2/L (81.8 mg O2/L with nitrification) per test vessel. The oxygen was depleted by the respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation rate is therefore expressed as the percentage BOD (biological oxygen demand) and was calculated for each study day.

 

The mean oxygen depletion in the inoculum control was 27.0 mg O2/L on day 28.

 

In order to check the activity of the test system sodium benzoate was used as functional control. The pass level > 60% was reached within 3 days. The biodegradation reached a maximum of 95% degradation on day 28.

 

In the toxicity control containing both test and reference item 39% degradation occurred within 6 days. The degradation of the reference item was not inhibited by the test item.

 

The biodegradation of the test item is shown in comparison to the readily degradable functional control and the toxicity control. Both test item replicates did not reach the 10% level (beginning of biodegradation) within the 28 -day-period of the study. The mean biodegradation on day 28 was 0%.

 

The validity criteria of the guideline are fulfilled.

 

 

 

The test item is classified as

not readily biodegradable

within the 28 day period of the study.

 

 

 

Biodegradation [%] of the Test Item in Comparison to the Functional Control and Toxicity Control

 

Biodegradation [%]

 

Study Day [d]

 

Replicate

7

14

21

28

Test Item
56mg/L Test Item

1

0

0

0

0

2

0

0

0

0

Functional Control
30 mg/L Reference Item

 

84

92

93

95

Toxicity Control
56mg/L Test Item +
30 mg/L Reference Item

 

39*

-

-

-

                                     *) after 6 days