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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:
2016-06-23 to 2017-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: International Standard ISO 9439
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Version / remarks:
2008-05-30
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)
EC Number:
915-758-6
Molecular formula:
C32H18CrN6O8.Na / C32H22CrN10O8.Na / C32H20CrN8O8.Na
IUPAC Name:
Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)
Test material form:
solid
Details on test material:
Appearance:
- physical state: solid
- color: dark brown


Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of activated sludge: Municipal activated sludge from the wastewater treatment plant of Mannheim, Germany. The inoculum was collected on 27 June 2016 from the aeration tank of the plant. A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm.
- Preparation of inoculum for exposure: To reduce the content of inorganic carbon in the blank controls the activated sludge was aerated with carbon dioxide free air for about 24 hours at 22 ± 2 °C.
- Pretreatment: At the day of exposure the suspension was washed one 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 drinking water.
- Concentration of sludge: 6.0 g/L dry weight
Duration of test (contact time):
28 d
Initial test substance concentration
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: The used mineral medium complies with the test guideline OECD 301B. It was prepared as follows:
Solution A:
KH2PO4: 8.50 g
K2HPO4: 21.75 g
Na2HPO4 x 2 H2O: 33.40 g
NH4Cl: 0.50 g
The compounds were dissolved with deionized water to 1000 mL; the pH value was adjusted to 7.4.

Solution B:
CaCl2 x 2 H2O: 36.40 g
The compound was dissolved with deionized water to 1000 mL.

Solution C:
MgSO4 x 7 H2O: 22.50 g
The compound was dissolved with deionized water to 1000 mL.

Solution D:
FeCl3 x 6 H2O: 0.25 g
The compound was dissolved with deionized water to 1000 mL.

15 mL solution A, 1.5 mL solution B, 1.5 mL solution C and 1.5 mL solution D was used for the preparation of the test assays.

- Test temperature: 22 ± 2 °C
- pH: 7.2 - 7.4
- pH adjusted: yes, 7.4 ± 0.2

TEST SYSTEM
- Culturing apparatus: 2 L incubation bottles filled up to a volume of 1.5 L.
- Number of culture flasks/concentration: 2 (blank control, test substance), 1 (inhibition control, reference substance)
- Method used to create aerobic conditions: At begin of the exposure phase the test vessels were connected with an aeration unit and the bubble aeration with carbon dioxide free air was started after connecting the several test vessels with the absorption units. The test assays were stirred using magnetic stirrers.
- Measuring equipment: two serial scrubbing bottles (total volume 250 mL) filled with 100 mL 0.05 mol sodium hydroxide solution for the adsorption of carbon dioxide from biodegradation processes.
- Test performed in open system: Yes
- Details of trap for CO2 and volatile organics if used: Usually twice a week the Total Inorganic Carbon (TIC) values of the adsorption solutions of the first trap were determined and used for the calculation of the produced carbon dioxide. After each sampling the second trap was moved forward and the new trap with fresh sodium hydroxide solution was placed into the second position. Each trap was analyzed separately.

SAMPLING
- Sampling frequency: twice a week
- Sampling method: sodium hydroxide solution for the adsorption of carbon dioxide

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Abiotic sterile control: no
- Toxicity control: yes
- Other: reference substance
Reference substance
Reference substance:
aniline

Results and discussion

% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
< 10
Sampling time:
28 d
Details on results:
Degree of biodegradation in the inhibition control after 14 days: 28 % CO2/ThCO2

BOD5 / COD results

Results with reference substance:
Degree of biodegradation of the reference substance after 14 days: 77 % CO2/ThCO2

Any other information on results incl. tables

Table 1: Measured data of Total Inorganic Carbon (TIC) [mg/L]

Day

 

BC NaOH

BC1

BC2

RS

IH

TS1

TS2

0

a

1.2

-

-

-

-

-

-

b

1.2

3

a

1.3

11.3

17.4

25.7

15.2

15.4

17.9

b

1.3

11.3

17.4

25.7

15.2

15.4

17.8

6

a

1.4

16.2

22.4

145

143

16.8

20.3

b

1.4

16.2

22.4

145

144

16.7

20.3

8

a

1.3

16.2

9.8

54.2

37.0

14.1

16.2

b

1.2

15.6

9.4

53.8

37.0

13.9

16.0

10

a

1.1

15.0

23.3

38.6

23.1

13.2

17.7

b

1.0

14.9

23.4

38.9

23.0

13.0

17.4

14

a

1.6

25.5

32.9

60.5

46.4

20.0

28.3

b

1.9

25.2

32.9

60.0

45.4

19.5

27.6

17

a

2.5

20.3

18.1

52.0

26.6

17.1

22.6

b

2.1

20.2

18.1

51.8

26.0

17.1

22.6

20

a

1.1

19.1

7.5

39.7

20.2

16.4

20.8

b

1.0

19.1

7.4

39.8

20.3

16.4

21.0

23

a

0.9

17.6

13.0

36.5

20.2

17.9

22.1

b

0.8

17.7

13.2

36.8

20.1

17.9

22.4

27

a

0.8

19.7

14.8

33.7

23.4

17.7

23.4

b

0.8

19.6

14.8

33.5

23.2

17.6

23.2

28

a

1.1

8.4

7.8

9.2

11.8

8.7

11.2

b

1.3

8.4

7.7

9.3

12.0

8.9

11.1

flask 1

29

a

 

22.9

19.8

6.8

18.1

16.7

16.8

b

23.0

19.7

6.7

18.1

16.8

17.1

flask 2

29

a

3.5

3.7

2.8

3.8

3.6

3.6

b

3.7

3.7

3.0

3.6

3.4

3.3

 

 

Table 2: Produced carbon dioxide amount in the test vessels

[mg CO2/test vessel]

mg CO2 added up after subtraction of the mean value of the blank controls

Duration of exposure [days]

BC mv

RS

IH

TS1

TS2

RS

IH

TS1

TS2

3

4.8

9.0

5.1

5.2

6.1

4.2

0.3

0.4

1.3

6

6.6

52.8

52.2

5.7

7.0

50.4

45.9

-0.5

1.7

8

4.2

19.3

13.1

4.7

5.4

65.5

54.8

0.0

2.9

10

6.5

13.7

7.9

4.3

5.9

72.7

56.2

-2.2

2.3

14

10.2

21.7

16.4

6.8

9.8

84.2

62.4

-5.6

1.9

17

6.7

18.7

9.3

5.9

7.9

96.2

65.0

-6.4

3.1

20

4.2

13.9

6.8

5.4

7.0

105.9

67.6

-5.2

5.9

23

4.8

12.6

6.6

5.7

7.3

113.7

69.4

-4.3

8.4

27

5.9

11.9

8.2

6.1

8.2

119.7

71.7

-4.1

10.7

28

2.7

3.1

4.1

2.9

3.8

114.5

72.0

-5.6

10.2

29

8.4

2.8

7.3

6.7

6.8

 

 

mg CO2 in the test assay = (TICmv*44*100)/(12*1000)

 

TICmv= measured TIC mean value after subtraction of the NaOH blank value

12 = atomic weight of carbon

44 = molecular mass of carbon dioxide

100/1000 = conversion into 100 mL absorption solution

mg CO2 added up = CO2mv - CO2mv BC + CO2pd

CO2mv = CO2 amount in the test assay at sampling day

CO2mv BC = CO2 amount in the blank control at sampling day

CO2pd = CO2 amount in the test assay at previous sampling day

 

 

Table 3: Theoretical Carbon dioxide [mg/test assay]:

RS : 109.6

IH : 220.7

TS1 : 109.6

TS2 : 110.9

 

Table 4: Degree of Biodegradation [% CO2/ThCO2]

Test duration [days]

RS

IH

TS1

TS2

TS mv

0

0

0

0

0

0

3

4

0

0

1

1

6

46

21

0

2

1

8

60

25

0

3

2

10

66

25

-2

2

0

14

77

28

-5

2

-2

17

88

29

-6

3

-2

20

97

31

-5

5

0

23

104

31

-4

8

2

27

109

32

-4

10

3

28

104

33

-5

9

2

 

[% CO2/ThCO2] = (CO2Ta)/(ThCO2Ta* 100)

 

CO2Ta = CO2 amount at sampling day

ThCO2Ta = Theoretical CO2 amount in the test assay

 

Table 5: Measured values [mg/L], determination of DOC after centrifugation

 

[mg/L DOC]

BC1

BC2

BC mv

RS

IH

TS1

TS2

TS mv

At begin of exposure

a

0.2

0.2

0.2

19.2

-

-

-

 

b

0.2

0.2

19.3

-

-

-

At the end of exposure

a

0.9

0.8

0.9

1.1

-

-

-

b

0.9

0.8

1.1

-

-

-

Degradation of DOC [%]

99

-

-

-

-

 

 

Degradation of DOC [%] = [1 -(CTAt - CBCt)/(CTA0 - CBC0)] * 100

 

CTAt= average DOC conc. of the reference substance assays; current values

CBCt= average DOC conc. of the blank control assays; current values

CTA0= average DOC conc. of the reference substance assays at day 0

CBC0= average DOC conc. of the blank control assays at day 0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test substance is not readily biodegradable according to OECD criteria. It is poorly biodegradable.