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Biodegradation in water: screening tests

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Reference
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
Justification for type of information:
Data is from experimental report.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Qualifier:
equivalent or similar to guideline
Guideline:
other: Annex to EEC- Directive 92/69/EEC from 31 July 1992 C02 Evolution Test (Method CA-C)
Qualifier:
equivalent or similar to guideline
Guideline:
other: International Standard ISO 9439:1999 Water Quality - Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds - Method by analysis of released carbon dioxide .
Principles of method if other than guideline:
The carbon dioxide evolution test (former Sturm test) is a static method to evaluate the ultimate aerobic biodegradability of a test substance in water . Mixtures of the test substance, a defined inorganic medium and a non pre-adapted inoculurn (e .g. activated sludge or effluent of a municipal or laboratory waste water treatment plant) are incubated and aerated at room temperature up to 28 days. The biologically produced carbon dioxide is trapped in a 0,25 mol. potassium hydroxide solution . The conductivity shift of the absorption solution is used for calculating of the C02 production .
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test substance: Sudan Orange 220
- Chemical name: 1 -Phenylazo-2-naphtholl
- Batch number: 02-0103
- Substance number: 03/0063-1
- Date of production: 22 November 2001
- Molecular formula: C16H12N2O
- Molecular weight: 248 g/mol
- Aggregate state: solid, powder
- Water-solubility: <100 mg/I
- Colour: orange-red, clear
- Homogeneity: homogeneous
- Expiry date: unlimited useable
- Special conditions for storage: storage at room temperature
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Other: The identity of the test substance was confirmed by IR-spectroscopy.
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): Activated sludge from laboratory wastewater plant treating municipal sewage .
- Concentration of activated sludge in the test assay : 30 mg/l (dry substance).
- Pretreatment: Activated sludge of a concentration of 30 mg/I dry substance from a laboratory waste water treatment plant jed with municipal sewage was pre-aerated for about two days before the start of the test.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Remarks:
ThCO2
Details on study design:
- Test temperature: 22 ± 2 OC
Reference substance:
aniline
Preliminary study:
TC [mg/g] : 776
The analysis was performed in accordance to ISO/EN 17025-criteria .
Key result
Parameter:
% degradation (CO2 evolution)
Remarks:
ThCO2
Value:
< 10
Sampling time:
28 d
Remarks on result:
other: Other details not known
Details on results:
The test substance is in this test poorly biodegradable (not readily biodegradable according to OECD criteria).
Results with reference substance:
Biodegradation degree of the reference substance after 14 days (% CO2/ThCO2): 60 - 70%

Biodegradation degree in the inhibition control after 14 days (% C02/ThCO2): 40-50%.

As the following criteria is met, the validity criteria is fulfilled:

Deviation of the degradation degree of the test subst . in the plateau phase <20% - yes

Degradation degree of the reference substance >60% after 14 days - yes

Degradation degree in the inhibition control >25 %after 14 days - yes

DIC in the blank control by test begin at 20 mg/l TOC test concentration <1mg/l - yes

C02-oroduction in the blank control <70mg/l - yes

Table: C02 - Evolution Test

Test substance: Sudan Orange 220

Summary of the test results

ThC02[mg/test vessel]:

RS: 109,9

IH: 219,3

TS1: 109,9

TS2: 110,2

PC: -

 

Degradation degree Dt%(CO2/ThCO2)

Test duration (days)

RS

IH

PC

TS1

TS2

TSmv

0

0

0

-

0

0

0

1

2

1

 

2

3

3

2

2

2

 

2

4

3

3

7

7

 

3

5

4

4

7

16

 

3

6

5

5

19

24

 

4

7

6

6

29

30

 

4

8

6

7

36

34

 

4

7

6

8

40

37

 

4

7

6

9

47

39

 

4

7

6

10

52

40

 

4

6

5

11

56

42

 

5

6

6

12

61

43

 

4

5

5

13

63

45

 

5

6

6

14

67

46

 

4

5

5

15

69

47

 

5

5

5

16

72

47

 

4

5

5

17

76

47

 

4

4

4

18

78

48

 

4

4

4

19

81

48

 

4

4

4

20

81

48

 

4

4

4

21

83

48

 

4

4

4

22

85

48

 

3

4

4

23

86

48

 

3

4

4

24

87

48

 

4

4

4

25

88

49

 

4

4

4

26

89

49

 

4

4

4

27

90

49

 

3

4

4

28

92

48

 

3

4

4

 

DOC -elimination degree (%)

Measured values [mg/1]

 

BC1

BC2

BCmv

RS

IH

PC

TS1

TS2

 

DOC at test begin

0,3

0,0

 

0,1

20,7

-

-

-

-

0,1

0,0

21,1

-

-

-

-

DOC at test end

1,0

0,7

 

1,1

1,3

-

-

-

-

TSmv

1,1

1,4

1,3

-

-

-

-

DOC elimination (%)

 

99

-

-

-

-

-

 

 

Remark: The test substance is poorly soluble in water, therefore no DOC elimination was determined.

ACCOUNTOF THETEST RESULTS

C02-Evolution Test                Test details

Determination of C02 bymeasuring of the conductivity

Test substance:Sudan Orange 220

TC: 776mg/g

Reference substance:aniline

Stock solution:409,4 mg/I

TOCvalue:312mg/I

Direct addition of the test substance

 

Sample

Blank control

BC1 – BC2

Reference substance RS

Inhibition control

IH

Abiotic control

PC

Test substance

TS1 – TS2

Test substance concentration

[mg/1]

-

-

25,6

-

25,7 – 25,8

TOCconcentration of the test

substance [mg/1]; nominal value

-

-

20

-

20

TOCconcentration of the reference

substance [mg/1]; nominal value

-

20

20

-

-

Deionized water

[mi/vessel]

1000

1000

1000

-

1000

Inorganic medium

[ml/vessel]

19,5

19,5

19,5

-

19,5

Inoculum(6g/Idry matter)

[ml/vessel]

7,5

7,5

7,5

-

7,5

Stock solution mercury chloride

[ml/vessel]

-

-

-

-

-

Direct addition of the test substance

[mg/vessel]; nominal value

-

-

38,7

-

38,7

Weighed sample of the test

substance [mg/vessel]

-

-

38,4

-

38,6 – 38,7

Stock solution reference

substance [ml/vessel]

-

96

96

-

-

Remaining water

[ml/vessel]

473

377

377

-

473

Test volume total [ml]

1500

1500

1500

-

1500

pH-value at pre-incubation before and

after correction

7,7

7,7

7,7

7,7

-

7,7

7,7

7,4

7,4

7,4

7,4

 

7,4

7,4

pH-value at test end

7,3

7,3

7,2

7,2

-

7,3

7,3

 

Inoculum: Activated sludge of a concentration of 30mg/I dry substance from a laboratory waste wate treatment plant, fed with municipal sewage was pre-aerated for about two days before the start of the test.

 

Remarks: The pH-values of the test assays with test substance were measured at the begin of the test in an additional test vessel TS 3 with mineral medium and test substance at the required test concentration. A correction of the pH value was not necessary. The test mixture was discarded after the measurement of the pH. Weighed sample test vessel TS3: 38,7 mg, pH-value vessel TS3 at pre-incubation before/and after correction : 7,7 / 7,4

C02 -Evolution-Test

Summary of the conductivity values

Test substance:Sudan Orange 220

Reference substance:aniline

 

Conductivity: measured valuesbydaily reading [mS/cm]          SC02:summarized C02-production

[mg/test vessel]

 

 

Date

Day

BC1

BC2

RS

IH

PC

TS1

TS2

RS

SCO2

IH

SCO2

PC

SCO2

TS1

SCO2

TS2

SCO2

16.09.03

0

51,8

51,8

52,1

52,0

 

51,4

51,6

0,0 

0,0

 

0,0 

0,0

17.09.03

1

51,7

51,6

51,9

51,8

 

51,2

51,3

2,6

2,6

 

2,6

3,4

18.09.03

2

51,4

51,3

51,6

51,3

 

50,9

50,8

2,6

4,1

 

2,6

4,9

19.09.03

3

51,0

51,9

50,6

49,3

 

50,4

50,3

7,2

16,3

 

3,4

5,7

20.09.03

4

50,7

50,6

48,5

46,4

 

50,1

49,9

20,9

36,1

 

3,4

6,4

21.09.03

5

50,4

50,2

46,8

43,8

 

49,7

49,4

31,5

53,6

 

4,1

7,9

22.09.03

6

50,0

49,9

45,4

41,8

 

49,3

48,9

39,1

65,8

 

4,1

8,7

23.09.03

7

49,6

49,5

44,3

40,3

 

48,9

48,6

44,5

74,2

 

4,1

7,9

24.09.03

8

49,2

49,2

43,1

39,1

 

48,5

48,3

51,3

81,0

 

4,9

7,9

25.09.03

9

48,9

48,8

42,0

38,1

 

48,1

48,0

56,6

85,6

 

4,9

7,2

26.09.03

10

48,6

48,5

41,0

37,4

 

47,8

47,8

62,0

88,6

 

4,9

6,4

27.09.03

11

48,3

48,3

40,2

36,7

 

47,5

47,6

66,5

92,4

 

5,7

6,4

28.09.03

12

48,0

47,9

39,4

36,0

 

47,2

47,3

69,6

94,7

 

4,9

5,7

29.09.03

13

47,7

47,7

38,7

35,4

 

46,9

47,0

73,4

97,7

 

5,7

6,4

30.09.03

14

47,4

47,5

38,1

34,8

 

46,7

46,8

75,7

100,0

 

4,9

5,7

01.1 0.03

15

47,2

47,2

37,4

34,3

 

46,4

46,6

79,5

102,3

 

5,7

5,7

02.1 0.03

16

46,9

46,9

36,6

33,8

 

46,2

46,3

83,3

103,8

 

4,9

5,7

03.1 0.03

17

46,6

46,7

36,0

33,5

 

46,0

46,1

85,6

103,8

 

4,1

4,9

04.1 0.03

18

46,4

46,5

35,4

33,2

 

45,8

45,9

88,6

104,6

 

4,1

4,9

05.1 0.03

19

46,2

46,3

35,1

33,0

 

45,6

45,7

89,41

104,6

 

4,1

4,9

06.1 0.03

20

46,0

46,1

34,6

32,7

 

45,4

45,5

91,7

105,4

 

4,1

4,9

07.1 0.03

21

45,8

45,9

34,3

32,5

 

45,2

45,3

92,4

105,4

 

4,1

4,9

08.1 0.03

22

45,6

45,7

33,9

32,2

 

45,1

45,2

93,9

106,1

 

3,4

4, 1

09.1 0.03

23

45,4

45,5

33,6

32,0

 

44,9

45,0

94,7

106,1

 

3,4

4, 1

10.1 0.03

24

45,3

45,4

33,4

31,9

 

44,7

44,8

95,5

106,1

 

4,1

4,9

11.1 0.03

25

45,81

45,3

33,1

31,7

 

44,6

44,7

97,0

106,9

 

4,1

4,9

12.10 .03

26

44,9

45,1

32,8

31,5

 

44,4

44,5

97,7

106,9

 

4,1

4,9

13.10 .03

27

44,7

44,9

32,5

31,3

 

44,3

44,4

98,5

106,9

 

3,4

4, 1

14.10 .03

28

44,6

44,7

32,2

31,2

 

44,1

44,2

1008

106,1

 

3,4

4, 9

15.10 .03

29

44,0

44,2

31,5

30,7

 

43,6

43, 6

 

 

 

 

 

 

Remark The measured values of day 29 are influenced by C02 stripping and therefore added to the values of day 28.Correlation for calculation from the conductivity change to the produced C02 amount in the test vessels: y=7,6097x+2,6191 .

 

Legend BC=blank control; RS=reference substance; IH=inhibition control; PC =abiotic control; TS =test substance

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The percentage degradation of the test substance 1-phenylazo-2-naphthol (Sudan Orange 220) was determined to be <10% in 28 days by CO2/ThCO2 parameter. Thus, the test substance is considered to be not readily biodegradable according to OECD criteria.
Executive summary:

The carbon dioxide evolution test (former Sturm test) is a static method to evaluate the ultimate aerobic biodegradability of a test substance in water . Activated sludge was obtained from laboratory wastewater plant treating municipal sewage. Activated sludge of a concentration of 30 mg/I dry substance was pre-aerated for about two days before the start of the test.Mixtures of the test substance, a defined inorganic medium and a non pre-adapted inoculurn (e .g. activated sludge or effluent of a municipal or laboratory waste water treatment plant) are incubated and aerated at room temperature up to 28 days. The biologically produced carbon dioxide is trapped in a 0,25 mol. potassium hydroxide solution. The production of carbon dioxide (C02) is a clear indication of biodegradation . The measured amount of carbon dioxide at the end of the test is compared with the calculated maximal theoretical production (ThC02) and indicated as biodegradation degree in percent. The conductivity shift of the absorption solution is used for calculating of the C02 production. Biodegradation degree of the reference substance after 14 days (% CO2/ThCO2): 60 - 70%. The percentage degradation of the test substance was determined to be <10% in 28 days by CO2/ThCO2 parameter. Thus, the test substance 1-phenylazo-2-naphthol (Sudan Orange 220) is considered to be not readily biodegradable according to OECD criteria.

Description of key information

The carbon dioxide evolution test (former Sturm test) is a static method to evaluate the ultimate aerobic biodegradability of a test substance Sudan Orange 220 (CAS no. 842-07-9) in water (Sustainability Support Services (Europe) AB has letter of access, Experimental report no. 03/0063/36/1, 2003). Activated sludge was obtained from laboratory wastewater plant treating municipal sewage. Activated sludge of a concentration of 30 mg/I dry substance was pre-aerated for about two days before the start of the test. Mixtures of the test substance, a defined inorganic medium and a non pre-adapted inoculurn (e .g. activated sludge or effluent of a municipal or laboratory waste water treatment plant) are incubated and aerated at room temperature up to 28 days. The biologically produced carbon dioxide is trapped in a 0,25 mol. potassium hydroxide solution. The production of carbon dioxide (C02) is a clear indication of biodegradation. The measured amount of carbon dioxide at the end of the test is compared with the calculated maximal theoretical production (ThC02) and indicated as biodegradation degree in percent. The conductivity shift of the absorption solution is used for calculating of the C02 production. Biodegradation degree of the reference substance after 14 days (% CO2/ThCO2): 60 - 70%. The percentage degradation of the test substance was determined to be <10% in 28 days by CO2/ThCO2 parameter. Thus, the test substance 1-phenylazo-2-naphthol (Sudan Orange 220) is considered to be not readily biodegradable according to OECD criteria.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Experimental study and publication data for the test compound 1-phenylazo-2-naphthol (C.I. Solvent Yellow 14) (CAS No. 842-07-9) were reviewed for the biodegradation end point which are summarized as below:

 

The carbon dioxide evolution test (former Sturm test) is a static method to evaluate the ultimate aerobic biodegradability of a test substance Sudan Orange 220 (CAS no. 842-07-9) in water (Sustainability Support Services (Europe) AB has letter of access, Experimental report no. 03/0063/36/1, 2003). Activated sludge was obtained from laboratory wastewater plant treating municipal sewage. Activated sludge of a concentration of 30 mg/I dry substance was pre-aerated for about two days before the start of the test. Mixtures of the test substance, a defined inorganic medium and a non pre-adapted inoculurn (e .g. activated sludge or effluent of a municipal or laboratory waste water treatment plant) are incubated and aerated at room temperature up to 28 days. The biologically produced carbon dioxide is trapped in a 0,25 mol. potassium hydroxide solution. The production of carbon dioxide (C02) is a clear indication of biodegradation. The measured amount of carbon dioxide at the end of the test is compared with the calculated maximal theoretical production (ThC02) and indicated as biodegradation degree in percent. The conductivity shift of the absorption solution is used for calculating of the C02 production. Biodegradation degree of the reference substance after 14 days (% CO2/ThCO2): 60 - 70%. The percentage degradation of the test substance was determined to be <10% in 28 days by CO2/ThCO2 parameter. Thus, the test substance 1-phenylazo-2-naphthol (Sudan Orange 220) is considered to be not readily biodegradable according to OECD criteria.

 

Biodegradation study of Sudan I was performed (Haiyan Xu et. a; 2010) (CAS no. 842-07-9). The test is carried at a temperature of 37ᵒC with a duration period of 2 days. The anaerobic bacterial species used in the study were obtained from the American Type Culture Collection (ATCC). All strains were preserved at -80ᵒC in 10 to 15% glycerol stocks and revived as needed. The bacterial strains were grown anaerobically at 37ᵒC by using BHI broth or MRS supplemented with various Sudan dye. A loopful for each strain was cultured in static conditions at 37ᵒC for 24 h in an Erlenmeyer flask containing 10 ml medium for use as a seed culture. The bacterial seed cultures of 1.5 ml were inoculated into flasks containing 100 ml BHI broth. Dye stock solutions were added to the medium at final concentrations of 10µg/ml, the cultures were incubated at 37ᵒC in an anaerobic chamber for 2 days without agitation. Metabolites of the reduction of the test compound Sudan I were isolated and identified by HPLC andLiquid chromatography/ electrospray ionization mass spectrometric (LC/ESI-MS).Reduction of the dyes was determined by measuring the disappearance of the absorbance at 500 nm immediately after extraction with ethyl acetate as well. The results are presented in percentage (%) obtained by the means from triplicate incubations. Among the tested bacterial strains, B.ovatus, B. uniformis, B. distasonis, B. fragilis, B. thetaiotaomicron, B. caccae, B. infantis, C. perfringens, C. butyricum, C. difficle, C. indolis, C. clostridioforme, E. aerofaciens, E. faecalis, E. faecium, F. russi, F. nucleatum, L. paracasei, L. rhamnosus, R. obeum and R. gnavus were able to completely degrade (100%) the test substance Sudan I.

The bacteria which are unable to degrade the Sudan I are Bifidobacterium longum, B. Adolescentis, B. catenulatum, B. angulatum, Clostridium ramosum, Clostridium leptum, Eubacterium tenue, Escherichia coli, Lactobacillus bifidus, Lactobacillus reuteri, Lactobacillus ruminis, and Peptostreptococcus magnus, respectively. The metabolite produced from Sudan I by E. faecalis was identified as aniline, based on an identical retention time of 4.05 min and ions atm/z94 [MH+] and 135 [MH++acetonitrile].1 -amino-2 -naphthol from Sudan I dye could not be detected in the extracted samples. No metabolites of Sudan I dye produced by E. coli was detected by LC/ESI-MS, indicating that the dyes were not degraded by the bacterium. Thus, based on percentage degradation of test substance, 1-phenylazo-2-naphthol (Sudan I) is considered to be readily biodegradable in nature.

 

Although publication study result indicates that the chemical is readily biodegradable in anaerobic conditions, but based on the experimental result for aerobic biodegradation (key study), it is concluded that the test substance 1-phenylazo-2-naphthol (C.I. Solvent Yellow 14) is considered to be not readily biodegradable in nature.

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