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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:
From 2017-06-30 to 2017-09-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trihydrogen [29H,31H-phthalocyaninetrisulphonato(5-)-N29,N30,N31,N32]cuprate(3-), compound with N,N'-di(o-tolyl)guanidine (1:3)
EC Number:
277-086-3
EC Name:
Trihydrogen [29H,31H-phthalocyaninetrisulphonato(5-)-N29,N30,N31,N32]cuprate(3-), compound with N,N'-di(o-tolyl)guanidine (1:3)
Cas Number:
72928-60-0
Molecular formula:
C32H16-τN8Cu(SO3C15H18N3)τ
IUPAC Name:
Bis and tris and tetra [N-(2-methylanilino)-N’-(2-methylanilino) methaniminium][phthalocyaninesulfonato-κ4N29,N30,N31,N32]cuprate(II)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At test facility: 2017-04-19 to 2017-04-27: refrigerator (6 ± 2 °C) / since 2017-04-27: room temperature, protected from light, in the tightly closed original container; since 2017-11-02 additionally in evacuated desiccator



Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge:
Source: Municipal sewage treatment plant, 31137 Hildesheim, Germany
Reason for the selection of the test system: Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.


- 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 for 2 ½ hours. Thereafter the sludge was homogenized with a blender. After sedimentation the supernatant was decanted and maintained in an aerobic condition by aeration with CO2 free air until test start (7 days). 10 mL/L of this mixture were used to initiate inoculation.

- Initial cell/biomass concentration: Approx. 1.31 x 10e7 CFU/L
Duration of test (contact time):
ca. 28 d
Initial test substance concentration
Initial conc.:
ca. 18 mg/L
Based on:
test mat.
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
- Additional substrate: no

- Test temperature: 22 ± 2°C
- Aeration of dilution water: yes
- Continuous darkness: no, low light conditions (brown glass bottles)


TEST SYSTEM
- Culturing apparatus: A COMPLETER
- Number of culture flasks/concentration:
* 2 replicates for the inoculum control (test medium without test and or reference item)
* 1 repicate for the functional control, Sodium benzoate (at 20 mg/l)
* 2 replicates for the test iitem (at 18 mg/l)
* 1 replicate for the toxicity control (test item (at 18 mg/l) and reference item at 20 mg/l)

- Method used to create aerobic conditions: Flow meter , Typ DK 800 PV, Krohne Duisburg, used for the aeration of the vessels with CO2 free air.

- Measuring equipment: The produced carbon dioxide is absorbed into the barium hydroxide solution. Three gas wash bottles, containing 100mL of a 0.0125 mol/L barium hydroxide solution are placed closed to the test vessels. Determination of CO2 carried out by tittration with the barium hydroxide solution at 0.0125 mol/l. For each titration the first gas wash bottle was removed and a new bottle was connected to the last one. Back titration of the residual Ba(OH)2 with 0.05 N HCl was carried out three times a week during the first ten days and thereafter twice weekly.


SAMPLING
- Sampling frequency: D1, D4, D6, D8; D11, D14, DD18, D21, D25, and D28.
- Sampling method: The barium hydroxide flask close to the test mixture is replaced by another barium hydroxide solution, and the CO2 is measured by titrimetric analysis.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculum control, mineral medium + inoculum : 2 replicates
- Abiotic sterile control: (reference item= sodium benzoate): mineral medium + inoculum + sodium benzoate
- Toxicity control: mineral medium + inoculum + sodium benzoate + test item

Reference substance
Reference substance:
other: sodium benzoate

Results and discussion

Test performance:
The validity criteria were fulfilled according to the guideline:

* The total CO2 evolution in the inoculum control at the end of the test was 21.7 mg/L
(validity criterion: < 40 mg CO2 /L after 28 days).

* The degradation of the functional control reached the pass level of ≥ 60% within 6 days (degradation: 62% on day 6).

* The differences of extremes of replicate values of removal of the test item at the end of the test was less than 20% (2% difference on day 28).

* The degradation of the toxicity control reached the pass level of 25% within 6 days
(degradation: 35% on day 6).

% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
ca. 4
Sampling time:
28 d

Any other information on results incl. tables

Table 1: Biodegradation of the Test Item in Comparison to the Functional Control and Toxicity Control

 

Biodegradation [%]

Study Day [d]

6

14

21

28

Test Item, 1stReplicate

1

1

2

3

Test Item, 2ndReplicate

2

3

4

5

Functional Control

62

83

87

88

Toxicity Control
test item + reference item

35

47

49

51

Table 2: CO2-Production and Biodegradation after 28 Days

CO2-Production

Functional Control

Test Item

Toxicity Control test item +
reference item

1

2

Net      [mg/3 L]

113.1

3.5

6.2

124.7

           [mg/L]

37.7

1.2

2.1

41.6

Theor.  [mg/3 L]

127.8

116.6

244.4

           [mg/L]

42.6

29.2

81.5

Degradation [%]

88

3

5

51

Table 3: CO2-Production and Biodegradation in the Inoculum Control, the FunctionalControland the Toxicity Control

Day

Date

Inoculum
Control

Functional Control

Toxicity Control

 

 

[mg CO2/3 L]

[mg CO2/3 L]

Degr.

[mg CO2/3 L]

Degr.

 

 

mv

Gross

Net Sum

[%]

Gross

Net Sum

[%]

1

2017-08-25

2.5

4.1

1.6

1

9.2

6.7

3

4

2017-08-28

6.3

53.4

48.7

38

52.6

53.0

22

6

2017-08-30

5.4

35.9

79.2

62

36.9

84.5

35

8

2017-09-01

6.5

22.1

94.8

74

21.7

99.7

41

11

2017-09-04

6.2

14.0

102.6

80

14.9

108.4

44

14

2017-09-07

7.1

10.9

106.4

83

12.8

114.1

47

18

2017-09-11

6.6

9.6

109.4

86

10.0

117.5

48

21

2017-09-14

7.7

9.2

110.9

87

10.2

120.0

49

25

2017-09-18

6.7

7.6

111.8

87

9.1

122.4

50

28

2017-09-21

5.2

5.9

112.5

88

6.4

123.6

51

29*

2017-09-22

4.8

5.4

113.1

88

5.9

124.7

51

Degr. = degradation      mv = mean value      

*) results of last two gas wash bottles

Table 4: CO2-Production and Biodegradation in the Inoculum Control and Test Item Samples

Study Day

Date

Inoculum
Control

Test Item

Replicate 1

Replicate 2

 

 

[mg CO2/3 L]

[mg CO2/3 L]

Degr.

[mg CO2/3 L]

Degr.

 

 

mv

Gross

Net Sum

[%]

Gross

Net Sum

[%]

1

2017-08-25

2.5

3.3

0.8

1

3.4

0.9

1

4

2017-08-28

6.3

5.4

0.8

1

7.3

1.9

2

6

2017-08-30

5.4

6.1

1.5

1

5.9

2.4

2

8

2017-09-01

6.5

6.1

1.5

1

6.1

2.4

2

11

2017-09-04

6.2

5.9

1.5

1

6.5

2.7

2

14

2017-09-07

7.1

7.2

1.6

1

7.5

3.1

3

18

2017-09-11

6.6

6.9

1.9

2

7.2

3.7

3

21

2017-09-14

7.7

7.6

1.9

2

8.5

4.5

4

25

2017-09-18

6.7

7.3

2.5

2

7.5

5.3

5

28

2017-09-21

5.2

5.3

2.6

2

5.7

5.8

5

29*

2017-09-22

4.8

5.7

3.5

3

5.1

6.2

5

Degr. = degradation      

mv = mean value      

*) results of last two gas wash bottles

Table5: pH-Values on Day 28

Inoculum Control

Func-tional
Control

Test Item

Toxicity Control

No. 1

No. 2

No.1

No. 1

No. 2

No. 1

7.74

7.75

7.76

7.75

7.74

7.75

 


 

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 in the Modified Sturm Test. The study was conducted from 2017-08-23 to 2017-09-22, according to OECD 301 B at the test facility. The test item was tested at a concentration of 18 mg/L with 2 replicates corresponding to a carbon content (TOC) of 10.6 mg C/L in the test vessels. The test vessels were incubated at low light conditions and at a temperature of 22 ± 2 °C.

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

To check the activity of the test system sodium benzoate was used as functional control. The percentage degradation of the functional control reached the pass level of 60% within 6 days and a maximum biodegradation of 88% on day 28.

In the toxicity control containing both test and reference item a biodegradation of 47% was determined within 14 days and it came to 51% after 28 days. The biodegradation of the reference item was not inhibited by the test item in 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 4%.

 Under the test conditions the test item is classified as not readily biodegradable within the 28 day period of the study.