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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 30 November 2018 to 02 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
July 17, 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: National Standardization Technical Committee for the Management of Hazardous Chemicals. GB/T 21856-2008 Chemicals-Ready Biodegradability CO2 Evolution Test [S]. Beijing: China Standard Press. 2008
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Storage: Ambient temperature (10-30°C)
- Stability under storage conditions: Stable
- Water solubility: Insoluble
- Solubility in organic solvents: Soluble in dimethyl sulfoxide and acetone
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Inoculum: Activated sludge
- Source: The activated sludge derived from a sewage treatment plant to the east of Suzhou city (a conventional treatment plant for domestic waste) was selected as source of inoculum.
- Collection and pretreatment: The freshly sampled activated sludge was collected from the aeration basin and kept aerobic during transport. The activated sludge was filtered through fine filter sieve to remove the coarse particles, washed using the test medium and settled for about 10 minutes, after which the supernatant was discarded. The sludge was resuspended in test medium and the above operation was repeated 3 times. 10 mL activated sludge was sampled (4 replicates) and dried at 105°C for 2 hours to determine the concentration of suspended solid, which was determined to be 5.7 g SS/L. The activated sludge was diluted with test medium to give the concentration of 4.0g SS/L. The suspended sludge was kept aerated at 22+/-2°C until required for testing. Before test, the activated sludge suspended solid was determined to be 4.33 g SS/L. From this result, the added amount of activated sludge was calculated.
- Concentration of inoculum in test cultures: 30 mg SS/L
Duration of test (contact time):
28 d
Initial conc.:
> 15.1 - < 15.3 mg/L
Based on:
test mat.
Initial conc.:
72.1 other: %
Based on:
other: Carbon content
Initial conc.:
245.4 µg/L
Based on:
IC (inorganic carbon)
Remarks:
TS-1
Initial conc.:
206.8 µg/L
Based on:
IC (inorganic carbon)
Remarks:
TS-2
Initial conc.:
119.8 other: mg
Based on:
ThCO2
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Test temperature: 22+/-2°C
- Test gas: CO2-free air
- Aeration rate: 30-100 mL/min
- The test was performed in diffused light

TEST SYSTEM
- Culturing apparatus: 4 L brown flask. Approximately 2.9 L test medium were added in each test vessel, then 20.8 mL of activated sludge was added to give a concentration of 30 mg SS/L in the final 3 L of inoculated mixture. These inoculated mixtures were aerated with CO2-free air overnight to purge the carbon dioxide of system. Before adding the test item, the inorganic carbon concentration of the test suspension in flask TS-1 and TS-2 was measured to be 245.4 µg/L and 206.8 µg/L, respectively. See Table 5.2.1/1 in "Any other information on materials and methods incl. tables".
- Number of culture flasks/concentration: The test included four groups of test suspension, inoculum blank, procedure control and toxicity control.
Flasks IC-1 and IC-2: containing only inoculum (inoculum blank);
Flasks TS-1 and TS-2: containing test item and inoculum (test suspension);
Flask PC: containing reference item and inoculum (procedure control);
Flask TC: containing test item, reference item and inoculum (toxicity control).

TEST MEDIUM
The stock solution of medium was prepared using purified water and analytical grade reagents. 10mL of stock solution A was firstly mixed with purified water, and then 1 mL of stock solution B, C and D was added, mixed and made up to 1 L with purified water. 47 L of test medium was prepared according to the above method. The pH value was measured to be 7.86-7.92 and adjusted to 7.54 with HCl.

SAMPLING
- Sampling method: Three 250 mL gas-washing bottles, which containing 150 mL of 0.0125 mol/L Ba(OH)2 solution were connected in series as CO2 absorption bottles for each test flasks to absorb the CO2 produced in the test flask
- Sampling frequency: The analysis of CO2 was made on day 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 21, 26 and 29.
On the days of CO2 determination, the CO2 absorption bottle closest to the test flask was disconnected and the others were moved froward in turn, then a new one with freshly prepared Ba(OH)2 solution was connected to the terminal. 50 mL of the absorption solution was taken from previously disconnected absorption bottle to titrate with hydrochloric acid solution. According to the change of the concentration of Ba(OH)2 solution to calculate the amount of CO2.
On the 28th day, 1 mL of concentrated hydrochloric acid was added to each test flask. Test flasks were aerated overnight to drive off the CO2 present in the test suspensions. On the 29th day, all CO2 absorption bottles were disconnected and titrated for the last analysis of evolved CO2.
Reference substance:
benzoic acid, sodium salt
Remarks:
The carbon content of reference item is calculated to be 58.3% // the concentration of reference item in the procedure control and toxicity control was 17.7 mg/L
Key result
Parameter:
% degradation (CO2 evolution)
Value:
> 84
Sampling time:
28 d
Remarks on result:
other: 10d window not applicable
Details on results:
- Temperature: During the test, the temperature of test area was in the range of 21.1-22.9°C satisfying test requirement (22+/-2°C).
- Pass level: Due to the test item being a mixture, the 10-d window is not applicable. If the biodegradation percentage reaches 60% on day 28, it would be considered that the test item is readily biodegradable.
- Toxicity control: The biodegradation percentage of toxicity control on day 14 was 85.6%, greater than 25%, which indicated the test item had no toxicity or inhibition to the inoculum.
- Test substance: The mean biodegradation percentage of test item on day 28 was greater than 84.0% which reached the pass level of ready biodegradability (greater than 60%). See biodegradation curve in "Illustration".
Results with reference substance:
The biodegradation percentage of reference item on day 14 was 89.8%, which was greater than 60%.

Table 5.2.1/2: Cumulative CO2 production and biodegradation percentages

 

Inoculum blank

Procedure control

Test substance TS-1

Test substance TS-2

Toxicity control

Cumulative CO2 (mg)

Cumulative CO2 (mg)

Degradation (%)

Cumulative CO2 (mg)

Degradation (%)

Cumulative CO2 (mg)

Degradation (%)

Cumulative CO2 (mg)

Degradation (%)

Time (day)

Mean IC1 and IC-2

PC

TS-1

TS-2

TC

1

2

3

4

6

8

10

12

14

16

21

26

29-1

29-2

29-3

0.92

2.94

7.97

11.82

18.12

24.48

31.77

38.04

42.97

46.45

55.36

69.74

75.98

77.03

77.03

7.21

23.84

53.65

74.75

90.37

98.05

101.78

101.78

101.78

101.78

101.78

101.78

103.03

103.84

103.84

6.4

21.0

47.4

66.0

79.8

86.5

89.8

89.8

89.8

89.8

89.8

89.8

90.9

91.7

91.7

2.77

12.21

24.46

36.74

42.94

51.14

59.46

75.09

82.67

88.43

95.69

96.55

98.16

99.33

99.94

2.3

10.2

20.4

30.7

35.9

42.7

49.6

62.7

69.0

73.8

79.9

80.6

82.0

82.9

83.4

3.18

11.39

23.98

37.70

54.09

60.94

68.06

73.67

86.31

93.24

102.86

104.76

108.25

108.36

108.48

2.7

9.5

20.0

31.5

45.2

50.9

56.8

61.5

72.1

77.9

85.9

87.5

90.4

90.5

90.6

26.35

63.08

96.32

122.97

146.59

165.35

189.34

195.53

201.10

204.93

204.93

210.35

219.71

227.83

232.88

11.2

26.9

41.0

52.4

62.4

70.4

80.6

83.3

85.6

87.3

87.3

89.6

93.6

97.0

99.2

Note: 29 -1, 29 -2, 29 -3 were the first, second and third CO2 absorption bottles close to the test flask.

Table 5.2.1/3: Comparison of biodegradation of the test item

Time (day)

Biodegradation (%)

 

Mean (%)

Absolute difference (%)

TS-1

TS-2

1

2

3

4

6

8

10

12

14

16

21

26

29-1

29-2

29-3

2.3

10.2

20.4

30.7

35.9

42.7

49.6

62.7

69.0

73.8

79.9

80.6

82.0

82.9

83.4

2.7

9.5

20.0

31.5

45.2

50.9

56.8

61.5

72.1

77.9

85.9

87.5

90.4

90.5

90.6

2.5

9.9

20.2

31.1

40.5

46.8

53.2

62.1

70.6

75.8

82.9

84.1

86.2

86.7

87.0

0.3

0.7

0.4

0.8

9.3

8.2

7.2

1.2

3.0

4.0

6.0

6.9

8.4

7.5

7.1

Note: the difference of the degradation rate is the maximum value minus the minimum value of the parallel biodegradation rate of the test item

Validity criteria fulfilled:
yes
Remarks:
See "Overall remarks"
Interpretation of results:
readily biodegradable
Conclusions:
The mean biodegradation percentage of test item on day 28 was greater than 84.0%, which reached the pass level of ready biodegradability (greater than 60%). The 10-d window is not applicable for mixture, like the test substance. In conclusion, the test substance is readily biodegradable.
Executive summary:

The objective of the study was to evaluate the ready biodegradability of the test item using the CO2 evolution test. The activated sludge was selected as inoculum and sodium benzoate as reference item. The test was aerated by the passage of carbon dioxide-free air and agitated at 22 +/-2°C in the diffused light for the period of 28 days. The amount of carbon dioxide produced was measured during the test. The amount of carbon dioxide produced from the test item (corrected for that derived from the blank inoculum) was expressed as a percentage of ThCO2.

In the procedure control, the biodegradation percentage of reference item on day 14 was 89.8%, which was greater than 60%.

The biodegradation percentage of toxicity control on day 14 was 85.6%, greater than 25%, which indicated the test item had no toxic inhibition to the inoculum.

The mean biodegradation percentage of test item on day 28 was greater than 84.0%, which reached the pass level of ready biodegradability (greater than 60%). The 10 -d window is not applicable for mixture, like the test substance.

In conclusion, the test substance is readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 8 March 2019 to 24 October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992. Abbreviation CSCL301F
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Biodegradability Test of Chemical Substances by Microorganisms (corresponding to 301F). Japanese notification, Yakushokuhatsu 0331 No. 7, Heisei 23.03.29 Seikyoku No. 5, Kanpokihatsu No. 110331009, March 31, 2011.
Version / remarks:
The latest revision, March 29, 2018. Abbreviation OECD301F
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Solubility in water: Poorly soluble (QSAR predicted value < 2.5 µg/L)
- log Kow: QSAR predicted value > 7.84
- Stability: Stable at ambient temperature and normal conditions
- Solubility and stability in solvents: Soluble and stable in Dimethylsulfoxide and acetone
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Inoculum/activated sludge: Activated sludge from domestic sewage treatment plant was used in this study. The inoculum was kept under aerobic condition by continuous aeration until used.
- Source: sewage treatment plant mainly handling domestic wastewater in Kanagawa.
- Date of collection: March 4, 2019.
- Mixed liquor suspended solids (MLSS): 1800 mg/L at the start of BOD measurement.
- Concentration of sludge: 30 mg/L
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Initial conc.:
2.61 other: mgO2/mg
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
BOD measurement
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral medium. Aqueous solution (Grade A4 in Japanese Industrial Standards (JIS) K0557) containing 1% of solution A and 0.1% each of solution B, C and D.
Liquid A: In ultra-pure water, 8.50 g of KH2PO4, 21.75 g of K2HPO4, 33.40 g of Na2HPO4.2H2O and 0.50 g of NH4Cl was dissolved and filled up to 1L.
Liquid B: In ultra-pure water, 27.50 g of CaCl2 was dissolved and filled up to 1L.
Liquid C: In ultra-pure water, 22.50 g of MgSO4.7H2O was dissolved and filled up to 1L.
Liquid D: 400 mL of ultra-pure water was added to 0.10 g of FeCl3.6H2O.
- Test temperature: 22 +/- 1°C
- pH: at the start and the end of BOD measurement, pH values of the test solutions were measured (pH meter F-51, HORIBA, Ltd.)
- pH adjusted: no
- Other: continuous stirring with magnetic stirrer

TEST SYSTEM
- Culturing apparatus: Culture bottle, glass; test volume 300 mL
- Number of culture flasks/concentration: 2
- Details of trap for CO2 and volatile organics if used: Soda lime, for carbon dioxide absorption, Kanto Chemical Co., Inc.
- Test performed in closed vessels: yes
- BOD measurement: The BOD was measured continuously for 28 days. Apparatus: Closed system apparatus for measuring oxygen consumption, OM-3100A (ID code: T), Ohkura Electric Co. In the apparatus, the temperature, stirring, recording meter and air-tightness of the closed system were checked once a day except holidays during BOD measurement period. Observation was made on color of the contents in test bottles using the [Inoculum blank] as a comparative reference. After the end of BOD measurement, observations on color of the contents in test bottles, growth of the activated sludge and dissolution state of the test substance were also made.
- DOC measurement: The DOC in the test solutions of the [Sludge + test substance] and the [Inoculum blank] were measured under the following apparatus and conditions. In addition, samples for the DOC measurement in the recovery test and the blank test were measured under the same conditions to confirm whether the test substance dissolves in water, or undissolves. TOC analyzer: multi N/C 2100S (No. 1), Analytik Jena Japan Co., Ltd.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 2
- Abiotic sterile control: no
- Toxicity control: no
- Other: See samples description in the Table 5.2.1/1 in "Any other information on materials and methods incl. tables"
Reference substance:
benzoic acid, sodium salt
Remarks:
100 mg/L test substance / ThOD of 1.67 mgO2/mg
Key result
Parameter:
% degradation (O2 consumption)
Value:
89
Sampling time:
28 d
Remarks on result:
other: mean value (each value was 88% and 90%, respectively)
Remarks:
Within the 10-d window
Parameter:
% degradation (test mat. analysis)
Value:
> 99
Sampling time:
28 d
Details on results:
See tables in "Any other information on results incl. tables".
- Observation of the contents after the end of BOD measurement: The content of the [Sludge + test substance]-1 and 2 (bottle 1 and 2) and the [Sludge + reference substance] (bottle 3) was white. Growth of the sludge was observed in the [Sludge + test substance] and the [Sludge + reference substance]. The insoluble test substance was not observed in the [Sludge + test substance].
- pH measurement: At the end of BOD measurement, the pH values were 7.5 and 7.5 for the [Sludge + test substance], respectively.
- Degradability based on the BOD: The BOD in the [Sludge + test substance]-1 and 2 after 28 days (as corrected with the average value of the [Inoculum blank] were 2.29 and 2.35 mgO2/mg, respectively. The degradabilities based on the BOD after 28 days were calculated to be 88% and 90% for [Sludge + test substance]-1 and 2, respectively. The degradability based on the BOD after 14 days was calculated to be 88% for the [Sludge + reference substance]. For the [Sludge + test substance]-1 and 2, BOD degradabilities of 10-day window were 69% and 68% respectively. These were reached within the 28 day period of the test.
- Degradability based on the DOC: The DOC in the [Sludge + test substance]-1 and 2 after 28 days (as corrected with the average value of the [Inoculum blank] were 0.1 and 0.3 mgC, respectively. The degradability based on the DOC after 28 days was not calculated since the test substance was poorly soluble in water and the DOC in the [Sludge + test substance] of recovery test was less than 90% of the theoretical value (21.3 mgC).
- Degradability based on the residual test substance amount: The residual amounts of the test substance in the [Sludge + test substance]-1 and 2 after 28 days were <0.1 and <0.1 mg, respectively. The degradabilities based on the residual amount of the test substance after 28 days were calculated to be >99% and >99% for the [Sludge + test substance]-1 and 2, respectively.
- Confirmation of presence or absence of transformation products with reversed phase HPLC: The HPLC chromatograms of the [Sludge + test substance]-1 and 2 were compared to that of the standard solution of the test substance. The peak of test substance was disappeared and new peak derived from transformation products was not detected on the chromatograms.
Results with reference substance:
Percentage degradabilities of Sodium benzoate at day 14: 88%

Table 5.2.1/2: BOD measurement, pH

Bottle No.

Sample description

BOD (mgO2)

pH

Day 7

Day 14

Day 21

Day 28

Day 0

Day 28

1

[Sludge + test substance]-1

(Test suspension-1)

43.9

64.4

70.7

72.9

-

7.5

2

[Sludge + test substance]-2

(Test suspension-2)

42.1

63.5

72.0

74.5

-

7.5

3

[Sludge + reference substance]

(Procedure control)

44.2

48.1

48.7

48.7

-

8.1

4

[Inoculum blank]-1

(Control blank-1)

3.9

4.2

4.2

4.2

7.4

7.5

5

[Inoculum blank]-2

(Control blank-2)

3.8

3.9

3.9

3.9

7.5

7.5

Table 5.2.1/3: Measured values (day 28)

 

Sludge + test substance

Theoretical value

Bottle No. 1

Bottle No. 2

BOD (CSCL301F)*

mgO2/30.0mg

68.8

70.4

78.3

BOD (OECD301F)*

mgO2/mg

2.29

2.35

2.61

DOC*

mgC

0.1

0.3

21.3

Test substance (GPC)

mg

<0.1

<0.1

30.0

%

<1

<1

-

* Value of [Sludge + test substance] was corrected with average BOD or DOC value of [Inoculum blank].

Table 5.2.1/4: Degradability (%)

 

Sludge + test substance

Bottle No. 1

Bottle No. 2

Average

BOD (CSCL301F)

%

88

90

89

BOD (OECD301F)

%

88

90

89

DOC (CSCL301F and OECD301F)*

%

NA

NA

-

Test substance

%

>99

>99

>99

* Degradability was not calculated because the test substance was insoluble in water and DOC in the recovery test was less than 90% of theoretical value.

Table 5.2.1/5: 10-day window

Bottle No.

Sample description

10-day window

0-day

10-day

10-day (%)

Mean

1

[Sludge + test substance]-1

(Test suspension-1)

2019-03-12

2019-03-22

69

69

2

[Sludge + test substance]-2

(Test suspension-2)

2019-03-13

2019-03-23

68

4

[Inoculum blank]-1

(Control blank-1)

-

-

-

-

5

[Inoculum blank]-2

(Control blank-2)

-

-

-

-

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The average of the degradability results of the test substance based on the BOD was 89% (each value was 88% and 90%, respectively) and 10-day window were fulfilled. The degradability result of residual amount of the test substance were all > 99% and no transformation product was detected on reversed phase HPLC chromatograms. Therefore, it can be concluded that the test substance is readily biodegradable and no transformation product is generated under the conditions of this test.
Executive summary:

This study was conducted to evaluate the ready biodegradability of the test substance according to the test method relating to new chemical substances "Biodegradability Test of Chemical Substances by Microorganisms (corresponding to 301F)" (Japanese notification) (abbreviation CSCL301F) and OECD Guideline 301F (abbreviation OECD301F) with GLP compliance.

Activated sludge from domestic sewage treatment plant was used as inoculum in this study. Five bottles were used: bottles 1 and 2 for test suspension (sludge + test substance), bottle 3 for the procedure control (sludge + reference substance sodium benzoate) and bottles 4 and 5 for control blank (inoculum blank).

Different measurements were performed: The Biochemical oxygen demand (BOD) was measured for 28 days (measured with a closed system apparatus for measuring oxygen consumption); the Dissolved Oxygen Carbon (DOC) was measured with a TOC analyzer; the residual amount of the test substance was measured with a gel permeation chromatograph (GPC) and finally, the confirmation of presence or absence of transformation products was measured with a reversed phase high performance liquid chromatograph (RP-HPLC).

According to the results of this study, all validity criteria were fulfilled. The average of the degradability results of the test substance based on the BOD after 28 days was 89% (each value was 88% and 90%, respectively), within the 10-day window (69% and 68% at the 10-day window, respectively). The degradability based on the DOC after 28 days was not calculated since the test substance was poorly soluble in water and the DOC in the test suspension of recovery test was less than 90% of the theoretical value. The residual amounts of the test substance in the test suspensions after 28 days were <0.1 and <0.1 mg, respectively, therefore, the degradabilities based on the residual amount of the test substance after 28 days were calculated to be >99%. Finally, the HPLC chromatograms of the test suspensions were compared to that of the standard solution of the test substance. The peak of test substance was disappeared and new peak derived from transformation products was not detected on the chromatograms.

In conclusion, the test substance is readily biodegradable and no transformation product is generated under the conditions of this test.

Description of key information

OECD Guideline 301F, GLP, key study, validity 1:

89% biodegradation after 28 days within the 10-day window (mean value)

Readily biodegradable

No transformation product generated

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Two valid GLP studies are available, according to OECD Guideline 301F and OECD Guideline 301B respectively, to determine the readily biodegradation of the registered substance.

In the first study (LSI, 2019), assessed as the key study, the substance attained a mean of 89% biodegradation after 28 days within the 10 -day window and no transformation product was generated under the conditions of this test.

In the second study (CTI, 2019), assessed as a supporting study, the substance attained > 84% biodegradation after 28 days.

Therefore, based on these studies, the registered substance can be considered to be readily biodegradable.