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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 21 2016 to July 26 2016
Reliability:
1 (reliable without restriction)
Justification for type of information:
Study was conducted in accordance with internationally recognised guidelines in a GLP laboratory.
Qualifier:
according to
Guideline:
OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))
Deviations:
no
Qualifier:
according to
Guideline:
other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals Degradation and Accumulation, 302 C Inherent Biodegradability: Modified MITI Test (II) [M]. Second Edition. Beijing: China Environmental Science Press. 2013: 74-81.
Deviations:
no
Qualifier:
according to
Guideline:
other: The national hazardous chemical management standardization technical committee. GB/T 21818-2008 Chemical Inherent Biodegradation: Modified MITI Test (II) [S] . Beijing: China Standards Press.2008.
Deviations:
no
Qualifier:
according to
Guideline:
other: State Environmental Protection Administration of China. HJ/T 153-2004 The Guidelines for the test
Version / remarks:
of chemical [S]. Beijing: China Environmental Science Press. 2004. [5] State Environmental Protection Administration of China. GB 11914-89 Water quality- Determination of the chemical oxygen demand-Dichromate method [S]. 1989.
GLP compliance:
yes
Specific details on test material used for the study:
Name: NovaSpec Base Oil
CAS: 1472010-43-7
Molecular formula:
Molecular weight:
Batch No.: TS13096
Purity: 100% (UVCB substance)
Description: Colourless liquid
Melting point: -39 °C at 101.3 kPa
Boiling point: 216 °Cat 101.3 kPa
Volatility: Non-volatile
Vapour Pressure: 1.22 kPa at 37.8 °C
Solubility in water:Using the US EPA On-Line EPI Suite™ WSKOW vl .42 model, the water solubility range is predicted to be 2.64E-27 to 0.004421mg/L. This indicates that the substance is insoluble, with water solubilities much lower than 0.1 mg/L.
Solubility in solvent: Water: none
Density: 0.82Odor: None
Storage conditions: Controlled Room Temperature, 15-25 deg C, below 70% humidity, away from bright lights.Stability: Stable in container after opening/in lightExpiration date: December 31, 2016
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
Fresh samples from the ten sites, mainly in areas where varieties of chemicals were used and discharged were collected. Sites samples included sewage treatment works, industrial waste-water treatment works, rivers, lakes, seas. 1 L samples of sludge, surface soil, water, etc. were collected and mixed thoroughly together. After removing of floating matter and allowing to stand, the supernatant was adjusted to pH=7.0±1 .0 with phosphoric acid. An appropriate volume of the filtered supernatant was filled in a vessel and the liquid was aerated for about 23 .5h. 30min after ceasing the aeration of the solution obtained above, one third of the whole volume of the supernatant was discarded and an equal volume of a solution containing 0.1 % each of glucose, peptone and monopotassium phosphate was added to the remaining portion of the supernatant and aeration re-commenced. This procedure was repeated once per day. The unit was maintained in a good operation and kept at 25°C±2°C with the pH=7.0±1.0; the final obtained sludge should settle well and be aerate continuously.The dry weight of the inoculum was detennined as 3.5g/L. 229mL of this inoculum was centrifuged (1000g, 5min) and the supernatant was decanted, then the sludge was made up to 200mL with test medium to obtain a concentration equivalent to 4.0g suspended solid per liter, the sludge was then kept under aerobic at the test temperature until required.
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test groups
:Bottle 1 (abiotic control): Containing test substance and deionized water
Bottles 2, 3, 4, A (test suspension): Containing test substance, inoculum and test medium
Bottle 5 (procedure control): Containing reference substance, inoculum and test medium
Bottles 6 (inoculum blank 1): Containing inoculum and test medium (inoculum concentration is 100mg/L)
Bottle 7 (inoculum blank 2): Containing inoculum and test medium (inoculum concentration is 30mg/L)
Bottle 8 (toxicity control): Containing test substance, reference substance, inoculum and test medium (inoculum concentration is 100mg/L)
Note: Bottle A for determination of pH value of the test suspension at the start of the test.

Preparation of the test substance suspension
15.0mg of the test substance and 400mL of deionized water were added into bottle 1, with the aid of high shear mixing for 15min, then the mixing propeller was washed with 100mL of deionized water.15.0mg of the test substance were added into bottles 2, 3, 4, A and 8, respectively, and 400mL, 400mL, 400mL, 400mL and 350mL of test medium were further added into, respectively, with the aid of high shear mixing for 15min, then the mixing propellers were washed with 87.5mL of test medium.The purity of the test substance is 100%.

Preparation of the reference substance stock solution
200mg of the sodium benzoate was weighted into a beaker and dissolved with some test medium, then this solution was transferred to 200mL volumetric flask and further diluted with test medium to 200mL, and the reference substance stock solution with the concentration of 1000mg/L was obtained.

Test conditions
All of the test bottles were stirred and incubated on C.E.S Respirometer Systems at 25°C±2°C in the dark and sealed condition over 28 days.Reference substance
Name: Sodium benzoate
Batch No.: 20131101-2
CAS No.: 532-32-1
Molecular Weight: 144.10
Purity: 99.5%
Characteristics: White particle or crystalline powder
Storage conditions: Room temperature
Source: Guangzhou Chemical Reagent Factory
Expiration date: October 22, 2019
Solvent Name: n-hexane
Batch number: 152841
Source: Fischer
Reference substance:
benzoic acid, sodium salt
Preliminary study:
No details specified in the report.
Test performance:
The temperature was in the range of 23.5°C-27.0°C during the test. At the end of the test, the pH values of each test bottle were in the range of 7.18-7.90.
Key result
Parameter:
% degradation (test mat. analysis)
Value:
11
Sampling time:
28 d
Remarks on result:
other: Mean
Details on results:
Percentage biodegradation of BOD
Procedure control (14d): 90.7%
Procedure control (28d): 94.3%
Toxicity control (14d): 56.3%
Toxicity control (28d): 60.9%
Test suspension (28d) 9.1% 12.5% 11.5% (3 replicates), 11.0% (mean)
Residual content of the test substance at the end of the test
Abiotic control: 14.8mg (1.6% abiotic degradation)
Test suspension 1: 12.7mg (13 .8% primary biodegradation)
Test suspension 2: 13. 7mg (7 .3% primary biodegradation)
Test suspension 3: 13.0mg (12.2% primary biodegradation)
Recovery test
Prepared triplicate test solutions of the abiotic control and the test suspension with the test substance concentration of 30mg/L, respectively, then extracted test substance with n-hexane.The percent recovery of the abiotic control 1 1, 2, 1 were 99.9%, 99.4% and 105%, with a mean of 102%. The percent recovery of the test suspensions 1, 2, 3 were 104%, 104% and 97.1%, with a mean of 102%. It revealed that the recovery methods for the test substance used in the study were satisfactory. The results and the chromatogram of analysis methodology validation were shown in (( NovaSpec Base Oil analysis methodology validation test report)) (report number for A16015-T14037-1R).
Results with reference substance:
Sodium benzoate was used to be reference substance. The percentage biodegradation of the reference substance was calculated by oxygen consumption. It was 86.6% on day 7 and was 90.7% on day 14.

Composition of the test systems

Testvessels

Abiotic control

Test suspension

Procedure control

Inoculum blank

Toxicity control

pH value determination

Bottle 1

Bottle 2

Bottle 3

Bottle 4

Bottle 5

Bottle 6

Bottle 7

Bottle 8

Bottle a

Test substance (mg)

15.0

15.0

15.0

15.0

-

-

-

15.0

15.0

Reference substance stock solution (mL)

-

-

-

-

50.0

-

-

50.0

-

Deionized water (mL)

400

-

-

-

-

-

-

-

-

Test medium (mL)

-

400

400

400

446.2

487.5

496.2

350

400

Deionized water or test medium adding after high shear mixing (mL)

100

87.5

87.5

87.5

-

-

-

87.5

87.5

Inoculum concentration (g/L)

-

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

Inoculum (mL)

-

12.5

12.5

12.5

3.80

12.5

3.80

12.5

3.8

Total volume (mL)

500

500

500

500

500

500

500

500

500

Test substance concentration (mg/L)

30

30

30

30

-

-

-

30

30

Reference substance concentration (mg/L)

-

-

-

-

100

-

-

100

-

Inoculum concentration (mg/L)

-

100

100

100

30

100

30

100

100

 


 

Percentage biodegradation calculated by BOD (%)

Time (d)

Test suspension

Procedure control

Toxicity control

Bottle 2

Bottle 3

Bottle 4

Mean

Bottle 5

Bottle 8

1

0.3

1.1

0.7

0.7

19.2

28.1

2

0.3

1.4

0.6

0.8

47.4

43.6

3

-0.2

0.8

-0.1

0.3

64.2

50.7

4

-0.7

0.3

-0.3

0.1

77.2

56.0

5

-1.2

0

-0.5

-0.6

83.0

57.1

6

-2.0

-0.8

-1.2

-1.4

85.4

57.2

7

-2.6

-1.4

-1.9

-2.0

86.6

57.2

8

-2.6

-1.8

-2.4

-2.3

87.5

57.2

9

-3.0

-2.6

-3.4

-3.0

88.1

56.9

10

-3.7

-3.5

-4.6

-3.9

88.7

56.7

11

-5.2

-5.0

-6.4

-5.6

89.1

56.6

12

-6.0

-6.2

-8.0

-6.8

89.5

56.4

13

-5.9

-6.7

-9.2

-7.3

90.0

56.3

14

-5.4

-6.2

-10.0

-7.2

90.7

56.3

15

-4.6

-5.7

-10.9

-7.1

90.6

56.2

16

-3.8

-5.6

-11.7

-7.0

91.

56.1

17

-2.8

-5.1

-12.1

-6.7

91.6

56.0

18

-1.6

-4.1

-11.6

-5.8

92.0

56.0

19

-0.3

-.6

-99.8

-4.2

92.4

55.9

20

1.1

-1.0

-7.3

0.4

92.6

55.9

21

2.6

0.8

-4.5

1.1

92.9

56.0

22

3.6

2.8

-2.0

2.1

93.2

56.2

23

4.4

5.1

0.8

3.5

93.4

56.5

24

5.4

7.5

4.0

5.6

93.6

56.9

25

6.4

9.6

7.0

7.7

93.8

57.7

26

7.6

10.7

8.9

9.1

94.1

56.4

27

8.6

11.7

10.4

10.2

94.4

60.3

28

9.1

12.5

11.5

11.0

94.3

60.9

 

pH values of the test solutions

Test bottle no.

1

2

3

4

5

6

7

8

ph value day 0

-

7.44 (bottle A)

-

-

-

-

ph value day 28

7.90

7.64

7.60

7.60

7.81

7.18

7.57

7.76

 

Oxygen uptake of each test bottle during the test (mg O2)

Time (d)

Abiotic control

Test suspension

Procedure control

Inoculum blank

Toxicity control

Bottle 1

Bottle 2

Bottle 3

Bottle 4

Bottle 5

Bottle 6

Bottle 7

Bottle 8

1

2.56

4.62

4.89

4.75

17.74

4.5

1.75

38.3

2

2.92

6.25

6.66

6.35

41.94

6.14

2.4

58.58

3

3.35

7.87

8.25

7.9

56.85

7.96

3.21

68.91

4

3.81

9.48

9.87

9.64

68.1

9.75

3.64

77.03

5

4.08

10.72

11.16

10.97

71.41

11.16

4.07

79.82

6

4.37

11.87

12.33

12.16

75.84

12.62

4.5

81.43

7

4.48

12.83

13.27

13.06

77.41

13.77

5.09

82.53

8

4.85

14.1

14.41

14.16

78.68

15.06

5.6

83.8

9

4.98

15.22

15.37

15.06

79.63

16.31

6.1

84.74

10

5.21

16.45

16.54

16.12

80.63

17.81

6.56

86.01

11

5.33

17.62

17.7

17.18

81.4

19.55

7.02

87.55

12

5.58

19.05

18.97

18.31

82.13

21.26

7.41

89.07

13

5.91

20.66

20.35

19.45

82.93

22.82

7.75

90.51

14

6.48

22.22

21.93

20.53

83.9

24.2

8.14

91.9

15

6.48

23.3

22.91

20.99

84.17

25.01

8.52

92.61

16

6.48

24.82

24.18

21.91

84.97

26.22

8.66

93.71

17

6.56

26.18

25.36

22.78

85.55

27.22

9.04

94.61

18

6.77

27.74

26.84

24.07

86.28

28.34

9.47

95.65

19

7.08

29.28

28.41

25.76

87.03

29.38

9.9

96.63

20

7.27

30.61

29.82

27.51

87.57

30.2

10.29

97.42

21

7.54

31.97

31.34

29.36

88.21

31.03

10.62

98.34

22

7.79

33.13

32.82

31.05

88.82

31.8

10.97

99.38

23

8.06

33.99

34.24

32.67

89.4

32.36

11.41

100.25

24

8.25

34.92

35.71

34.42

90.01

32.95

11.85

101.42

25

8.48

35.92

37.07

36.13

90.61

33.55

12.25

102.98

26

8.75

37.17

38.3

37.63

91.19

34.36

12.62

105.83

27

8.91

38.23

39.38

38.9

91.71

35.07

12.93

107.62

28

9.1

39.01

40.23

39.86

92.13

35.65

13.42

108.83

 

Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
Under the conditions of the study, the percentage biodegradation of the test substance on day 28 which was calculated by BOD was 11 .0%, the primary biodegradation which was calculated by the residual test substance was 11 .1 %.
Executive summary:

The aim of the study was to determine the Biochemical Oxygen Demand (BOD) of the test substance in order to evaluate its inherent degradability. This study was conducted in a GLP accredited laboratory to the following test guidelines:

-Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals Degradation and Accumulation,302C Inherent Biodegradability: Modified MITI Test (II)[M]. Second Edition.Beijing: China Environmental Science Press. 2013:74-81.  

-The national hazardous chemical management standardization technical committee. GB/T 21818 -2008 Chemical Inherent Biodegradation:Modified MITI Test(II) [S].Beijing: China Standards Press.2008.  

-OECD Guidelines for Testing of Chemicals,302C Inherent Biodegradability:Modified MITI Test (II).Paris: OECD.Adopted:12 th May,1981.  

-State Environmental Protection Administration of China.HJ/T 153 -2004 The Guidelines for the test of chemical [S].Beijing: China Environmental Science Press. 2004.

-State Environmental Protection Administration of China.GB 11914 -89 Water quality- Determination of the chemical oxygen demand-Dichromate method [S]. 1989.  

Under the conditions of the study,the percentage biodegradation of the test substance on day 28 which was calculated by BOD was 11.0%,the primary inherent biodegradation which was calculated by the residual test substance was 11.1%.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 July 2013 - 28 August 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Recent study conducted to recognised method with due consideration for dispersant methods. Non-GLP but in accordance with ISO 17025 ; hence K2 assigned.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" for details of deviations.
Principles of method if other than guideline:
A method modification was provided by the customer to add the sample to a silica gel substrate and test the substrate adhered sample material for biodegradation. Silica gel adhesion is thought to provide a better dispersal of the sample and no additional organic or nutrient material to the sample test chamber.
GLP compliance:
no
Remarks:
Conducted in accordance with ISO 17025
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material:Not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Test conditions:- inoculum: Surface water from Skokie, IL water district.- proportion and nature of industrial waste water in sewage: unknown, discharge from waste treatment facility within 1 mile.- test duration and temperature: 28 days or as indicated, 22C +/- 2C- bacterial inoculum ~1E5 cfu/ml
Duration of test (contact time):
28 d
Initial conc.:
7.5 mg/L
Based on:
other: TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
As per OECD 301B. Six test samples was submitted for OECD 301B biodegradation testing, of which one is the substance subject to the registration. For this course of sample testing a method modification was provided by the customer to add the sample to a silica gel substrate and test the substrate adhered sample material for biodegradation. Silica gel adhesion is thought to provide a better dispersal of the sample and no additional organic or nutrient material to the sample test chamber.
Reference substance:
acetic acid, sodium salt
Preliminary study:
Not discussed
Parameter:
% degradation (CO2 evolution)
Value:
86.7
Sampling time:
35 d
Remarks on result:
other: 60% biodegradation was achieved at 8.9 days; thus passing the 10 day window.
Details on results:
The substance is considered to be readily biodegradable.
Results with reference substance:
The reference sample achieved 74.6% biodegradation, also passing the 10-day window; confirming the suitability and viability of the test.
Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance achieved the requirements for Ready Biodegradability by the OECD 301B standard, and can be considered to be readily biodegradable under the conditions of the test.
Executive summary:

The test substance achieved the requirements for Ready Biodegradability by the OECD 301B standard, achieving 86.7% bidegradation. 60% biodegradation was achieved at 8.9 days; thus passing the 10 day window. The test material can be considered to be readily biodegradable under the conditions of the test. This test is assigned a Klimish rating of two, reliable with restrictions on the basis that is is not GLP. However, it is conducted to a recognised current OECD Guideline, in accordance with ISO 17025. The results are considered to be appropriate and reliable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
24 May 2012 to 20 July 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Recent study conducted to recognised method with due consideration for dispersant methods. Non-GLP but in accordance with ISO 17025 ; hence K2 assigned.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
no
Remarks:
Conducted in accordance with ISO 17025
Specific details on test material used for the study:
Not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Not adapted activated sludge from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant. 30 mg/l dry matter in the final mixture
Duration of test (contact time):
28 d
Initial conc.:
23.7 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
The activated sludge was used after sampling from the treatment plant without adaptation. However, the sludge was pre-conditioned for 3 days (aerated but not fed) to reduce the amount of CO2 produced by the blank controls.Prior to the test the sludge was washed twice with tap water and once with mineral medium. After centrifugation the sludge, at a tenfold concentration of the final concentration to be achieved for the test, was suspended in test medium as described in Table 1.A stock solution of the test substance was prepared in hexane. Aliquots of this stock solution were transferred to the empty test vessels to give a final test concentration of about 20 mg TOC/l. The test vessels were shaken gently and the hexane was evaporated. The reference material was applied by direct addition to give a final test concentration of about 20 mg/l with respect to the total organic carbon (TOC).For each test series the following number of test flasks was set up:T: Test suspension containing inoculum, test medium and test substance (three replicates)B: Inoculum blank containing inoculum and test medium (three replicates)R: Procedure control containing inoculum, test medium and sodium benzoate as ready biodegradable reference compound (three replicates)The test vessels were stirred and aerated with synthetic CO2-free air for a maximum test period of 56 days. The air leaving the individual vessels was passed through gas-absorption bottles filled with KOH.The pH-value was checked at the beginning and at the end of the test. At the beginning it was adjusted to pH 7.4 (± 0.2) with NaOH or HCl, where necessary.The biodegradation of the test material was followed by CO2 measurements at frequent intervals to allow the assessment of the 10-d window. The trapped CO2 was determined as inorganic carbon (IC).Validity criteriaThe test is considered valid if the difference of extremes of replicate values at the end of the 10-d window or at the end of the test, as appropriate, is less than 20% and if the percentage degradation of the reference substance has reached the pass level of 60% by day 14.Due to the potential variability of the test system such as low test material concentration and open vessels with respect to aeration three replicates of the test, reference and control units, respectively, were prepared. For the evaluation of biodegradation only two of the three replicates were selected and, consequently, mentioned in the study report. The selection was based on the criteria that the difference of the selected replicate values at the end of the 10-d window or at the end of the test, as appropriate, is less than 20%.According to the OECD guideline the CO2 evolution in the inoculum blank at the end of the test should not normally exceed 40 mg/l (corresponding to an inorganic carbon concentration of 10.9 mg/l).
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not conducted
Test performance:
The positive control, sodium benzoate, reached 86% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.
Parameter:
% degradation (CO2 evolution)
Value:
78
Sampling time:
28 d
Details on results:
The biodegradability - based on CO2 evolution - of TS2235 was calculated to be 78% and 88% of the theoretical value (ThCO2) after an incubation time of 28 and 56 days, respectively (Table 2, below). The biodegradation of TS2235 reached 52% at the end of the 10-d window. Significant biodegradation of the test substance was observed after a lag phase of about 1 day.TS2235 did not reach the pass level of 60% for ready biodegradability in the CO2 Evolution Test within the 10-d window and, therefore, cannot be termed as readily biodegradable. However, the test substance reached the pass level of 60% after 28 days.

Table 2  CO2 produced by the test units, the inoculum blank and the corresponding degradation data.

  

 

Inoculum

Test unit no. 1

Test unit no. 2

 

blank *

containing test material

containing test material

 

Total CO2 release in test sample

Total CO2 release in test sample

Degradation

Total CO2 release in test sample

Degradation

Mean Degradation of no. 1+2 (%)

Time (days)

(mg IC/l)

(mg IC/l)

(%) **

(mg IC/l)

(%) **

 

0

0

0

0

0

0

0

1

0.3

0.7

2

-0.2

-2

0

5

2

2.8

3.7

2.3

1.5

2.6

7

2.7

3.7

4.9

5.2

11.8

8.3

11

4.8

9.7

23.9

9.7

23.5

23.7

14

6.2

14.9

41.9

12.5

30.2

36

18

6.5

18.8

59.4

16.3

47

53.2

21

6.1

19.9

66.5

17.8

56.3

61.4

25

6.9

22.9

77

21.1

68.6

72.8

28

7

23.8

80.8

22.6

74.9

77.9

32

7

24

81.5

24.2

82.8

82.1

35

7

24.4

83.8

25.3

88

85.9

39

6.9

24.7

85.7

26.6

94.6

90.2

42

7.8

24.6

81.2

27.1

93

87.1

46

7.6

24.7

82.4

27.1

93.7

88

49

7.3

24.2

81.8

27.2

95.9

88.8

53

7

23.5

79.2

26.8

95.3

87.2

56

6.2

22.8

79.8

26.2

96

87.9

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The biodegradability - based on CO2 evolution - of TS2235 was calculated to be 78% and 88% of the theoretical value (ThCO2) after an incubation time of 28 and 56 days, respectively The biodegradation of TS2235 reached 52% at the end of the 10-d window. Significant biodegradation of the test substance was observed after a lag phase of about 1 day.The positive control, sodium benzoate, reached 86% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.TS2235 did not reach the pass level of 60% for ready biodegradability in the CO2 Evolution Test within the 10-d window and, therefore, cannot be termed as readily biodegradable. However, the test substance reached the pass level of 60% after 28 days.
Executive summary:

The biodegradability - based on CO2 evolution - of TS2235 was calculated to be 78% and 88% of the theoretical value (ThCO2) after an incubation time of 28 and 56 days, respectively. The biodegradation of TS2235 reached 52% at the end of the 10-d window. Significant biodegradation of the test substance was observed after a lag phase of about 1 day.

The positive control, sodium benzoate, reached 86% biodegradation after 14 days, thus confirming suitability of inoculum and test conditions.

TS2235 did not reach the pass level of 60% for ready biodegradability in the CO2 Evolution Test within the 10-d window and, therefore, cannot be termed as readily biodegradable. However, the test substance reached the pass level of 60% after 28 days.

This test is assigned a Klimish rating of two, reliable with restrictions on the basis that is is not GLP. However, it is conducted to a recognised current OECD Guideline, in accordance with ISO 17025. The results are considered to be appropriate and reliable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
November 5, 2014 to December 3, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study performed in accordance with OECD and Japanese testing guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Qualifier:
according to
Guideline:
other: "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances"
Principles of method if other than guideline:
"Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances" (March 31, 2011, No. 0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No. 5, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan; April 2, 2012 partial revision, No. 0402-1, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 28, 2012, No. 2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 120402001, Environmental Policy Bureau, Ministry of the Environment, Japan)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Physicochemical properties
Vapor pressure: 1.22 Pa (37.8°C)
Water solubility: <0.1 mg/L (calculated value)
Melting point: -39ºC
Boiling point: 216ºC
Density: 0.822 g/cm3 (20°C)
Oxygen conditions:
anaerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
On-site sludge sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lakes, and inland sea; return sludge from sewage plants). Activated sludge, which was prepared and controlled in this laboratory, was used in this study (sampling period: September, 2014, initiation date of use: October 22, 2014).The activated sludge, which was cultivated for 1 hours after the synthetic sewage was added, was used. The synthetic sewage was prepared according to the following method; glucose, peptone, and potassium dihydrogenphosphate were dissolved in purified water, and the pH of the solution was adjusted to 7.0±1.0.
Duration of test (contact time):
28 d
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
TOC removal
Details on study design:
Preparations for testDecision of additive amount of activated sludge: Additive amount of activated sludge into the test vessel was 2.68 mL on the basis of the concentration of suspended solid in the activated sludge which was determined by the following methods;Result: 3360 mg/LPreparation of basal culture medium: The basal culture medium (3 L) was prepared at the same proportion as the following method; purified water (The Japanese Pharmacopoeia, Takasugi Pharmaceutical) was added to each 3 mL aliquot of solutions A, B, C and D, in order to prepare 1 L of solution. The pH of this solution was then adjusted to 7.0.Validity of activated sludge: Aniline was used as a reference item in order to confirm that the sludge was sufficiently active.Preparation of test solutionsThe following test solutions were prepared and incubated under the conditions described below (Instruments and conditions of incubation). Addition of test item or aniline1) Test solution (water + test item) (n=1, Vessel No. 1)In one test vessel, 30 mg of the test sample was accurately weighed with an electronic analytical balance and added to 300 mL of purified water, so that the concentration of the test item reached 100 mg/L.2) Test solution (sludge + test item) (n=3, Vessel Nos. 2, 3 and 4)In each test vessel, 30 mg of the test sample was accurately weighed with an electronic analytical balance and added to the basal culture medium [the volume subtracting the volume (2.68 mL) of activated sludge from 300 mL], so that the concentration of the test item reached 100 mg/L.3) Test solution (sludge + aniline) (n=1, Vessel No. 6)In one test vessel, 29.5 μL (30 mg) of aniline was taken out by microsyringe and added to the basal culture medium [the volume subtracting the volume (2.68 mL) of activated sludge from 300 mL], so that the concentration of aniline reached 100 mg/L.4) Test solution (control blank) (n=1, Vessel No. 5)In one test vessel, nothing was added to the basal culture medium [the volume subtracting the volume (2.68 mL) of activated sludge from 300 mL].Inoculation of activated sludge: The activated sludge was added to each test vessel described in 2), 3) and 4), so that the concentration of the suspended solid reached 30 mg/L.Instruments and conditions of incubationInstruments for incubationClosed system oxygen consumption measuring apparatus: Temperature controlled bath with measuring unit: OM3100A (Ohkura Electric); Data sample0:r OM7000A (Ohkura Electric)Vessel: Glass vesselAbsorbent for carbon dioxide: Soda lime No.1 (for absorption of carbon dioxide, Wako Pure Chemical Industries)Conditions of incubationIncubation temperature: 25±1ºCIncubation duration: 28 days (under dark conditions)Stirring method: Each test solution was stirred by a stirrer.Room: Apparatus room 1AObservation and measurementObservation of test solution: During the incubation period, the appearance of the test solution was observed once a day.Measurement of biochemical oxygen demand (BOD): During the incubation period, BOD of the test solutions was measured continuously by a closed system oxygen consumption measuring apparatus. The incubation temperature was measured and recorded once a day.Analysis of test solutionAfter the end of the incubation, the test item in the test solutions were determined. In order to confirm presence or absence of water-soluble converted products, the amount of dissolved organic carbon (DOC) was determined. Converted products, which were expected from results of the preliminary test prior to this study and authentic samples of which were not available, were analyzed qualitatively.Pre-treatment of test solutions for analysisThe test solution (water + test item), the test solutions (sludge + test item), and the test solution (control blank) were pretreated to prepare samples for total organic carbon (TOC) analysis of DOC and high-performance liquid chromatography (HPLC) analysis of the test item and liquid chromatography-mass spectrometry (LC-MS) analysis of converted products
Reference substance:
aniline
Remarks:
Lot number KPQ4413
Parameter:
% degradation (TOC removal)
Value:
26 - 27
Sampling time:
28 d
Parameter:
% degradation (test mat. analysis)
Value:
24 - 28
Sampling time:
28 d
Remarks on result:
other: by HPLC
Details on results:
Test solution (water + test item)From the results of determination of the test item, the percentage residue of the test item was 97% and no change of the peak shape of the test item was observed on the chromatograms of HPLC analysis. No peak of converted product was detected in the qualitative analysis of converted product by LC-MS. Moreover, no water-soluble converted product was produced because DOC was not detected. From the results mentioned above, it was considered that the test item was not converted in the test solution.Test solutions (sludge + test item)The percentages biodegradation by BOD were 26-27% and growth of the sludge was observed.These results showed that some of the test item was biodegraded.The peak area of the test item was decreased and the peak shape of it was changed on the HPLC chromatograms of determination of the test item. Moreover, four converted products were detected in the qualitative analysis by LC-MS. These converted products were thought to be intermediates which have a hydroxyl group and a carboxyl group from the results of the structural analysis. The retention times of the converted products were shorter than that of the test item in the qualitative analysis with a reversed-phase column. The poralities of the converted products were considered to be higher than that of the test item. The amounts of the converted products were unknown because they could not be separated with the test item under the quantitative analytical condition of the test item.From the results mentioned above, it was concluded that some of the test item was biodegraded, and the converted products and the rest of the test item were not biodegraded under the test conditions of this study. In addition, any water-soluble converted products were thought not to be produced because DOC was almost not detected.

Validity of test conditions

The validity criteria of the tests and the values in this test are shown in the following table. This test was valid because all the values in the test met the criteria. In the calculation of TOD of aniline, ammonia was adopted as the form of nitrogen.

 

Value in this test

Value of criterion

Difference between extremes of replicate values of percentage biodegradation

Percentage biodegradation by BOD

1%

<20%

Percentage biodegradation of test item

4%

Percentage biodegradation an aniline by BOD

After 7 days

77%

>40%

After 14 days

90%

>65%

BOD value of control blank

After 28 days

21.7 mg*1

≤60 mg/L (<18 mg)

*1 Measurement value of BOD (6.5 mg)/[test volume (300 mL)/1000] = 21.7 mg/L

 

Factors that affected reliability of test.

No adverse effects on the reliability of this test were noted.

 

Appearance of test solutions

Appearance of test media in incubation vessels were as follows:

 

Test solution

Appearance (visual)

pH

At the start of incubation

Water + test item

The test item was not dissolved.

The test solution was colorless.

-

Sludge + test item

The test item was not dissolved.

The test solution was colorless.

-

Control blank

Insoluble compound except the sludge was not observed.

The test solution was colorless.

-

At the end of incubation

Water + test item

Insoluble compound was observed.

The test solution was colorless.

Vessel No. 1 6.5

Sludge + test item

The presence or absence of insoluble compound could not be confirmed.

Growth of the sludge was observed.

The test solution was colorless.

Vessel No. 2 7.1

Vessel No. 3 7.1

Vessel No. 4 7.1

Control blank

Insoluble compound except the sludge was not observed.

The test solution was colorless.

Vessel No. 5 7.3

 

Analytical result of test solutions

Analytical results of the test solutions after 28 days were as follows:

 

Water + test item

Sludge + test item

Theoretical amount

Vessel No. 1

Vessel No. 2

Vessel No. 3

Vessel No. 4

BOD*2

mg

0.4

27.2

28.5

26.8

104*3

Detected amount and percentage detection of DOC

mgC

0

0.6

0.8

0.8

25.5*3

%

0

2

3

3

-

Residual amount and percentage residue of test item*4(by HPLC)

mg

29.1

22.0

21.0

21.2

30.0

%

97

73

70

71

-

Detection of converted product (by LC-MS)

-

Not detected

Detected (four components)

-

*2 The value of the test solution (control blank) was subtracted from the values of the test solutions (sludge + test item).

*3 Calculated from the molecular formula C25H52

*4 The residual amount and percentage residue of the test item included converted products in the test solutions (sludge + test item) because converted products could not be separated with the test item under the analytical condition.

*5 Converted products were detected in the test solutions (sludge + test item). However, they could not be determined because authentic samples of them were not available.

 

Percentage biodegradation

Percentage biodegradation after 28 days were as follows:

 

Sludge + test item

Vessel No. 2

Vessel No. 3

Vessel No. 4

Average

Percentage biodegradation by BOD

%

26

27

26

26

Percentage biodegradation by DOC

-

The percentage biodegradation by DOC was not calculated because the test item is not dissolved in water at the test concentration (100 mg/L) and more.

Percentage biodegradation of test item (by HPLC)

%

24

28

27

26

 

Validity criteria fulfilled:
yes
Interpretation of results:
other: Under the test conditions of this study, some of the test item was biodegraded and four converted products were produced. The converted products and the rest of the test item were not biodegraded.
Conclusions:
Under the test conditions of this study, some of the test item was biodegraded and four converted products were produced. The converted products and the rest of the test item were not biodegraded.
Executive summary:

Summary

Test item: NovaSpec base oil

Objective: This study was aimed at evaluating the biodegradability of NovaSpec base oil by microorganisms.

 

Test method

a) "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances" (March 31, 2011, No. 0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No. 5, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan; April 2, 2012 partial revision, No. 0402-1, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 28, 2012, No. 2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 120402001, Environmental Policy Bureau, Ministry of the Environment, Japan)

b) OECD Guidelines for Testing of Chemicals, No. 301C, July 17, 1992, "Ready Biodegradability: Modified MITI Test (I)"

 

Conditions of incubation

Concentration of test item: 100 mg/L

Concentration of activated sludge: 30 mg/L (as concentration of suspended solid)

Volume of test solution: 300 mL

Incubation temperature 25±1°C

Incubation duration: 28 days (under dark conditions)

 

Measurement and analysis for calculation of percentage biodegradation

a) Measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus

b) Determination of test item by high-performance liquid chromatography (HPLC)

 

Results

 

Sludge + test item

Vessel No. 2

Vessel No. 3

Vessel No. 4

Average

Percentage biodegradation by BOD

%

26

27

26

26

Percentage biodegradation by TOC

-

The percentage biodegradation by DOC was not calculated because the test item is not dissolved in water at the test concentration (100 mg/L) and more.

Percentage biodegradation of test item (by HPLC)

%

24

28

27

26

 

Conclusion

Under the test conditions of this study, some of the test item was biodegraded and four converted products were produced. The converted products and the rest of the test item were not biodegraded under the conditions of the test.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
October 14, 2014 to November 12, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD and Chinese test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Qualifier:
according to
Guideline:
other: [1] Chemical Registration Center of MEP.
Qualifier:
according to
Guideline:
other: [2] Dangerous Chemicals Administration.
Qualifier:
according to
Guideline:
other: [4] State Environmental Protection Administration of China.
Qualifier:
according to
Guideline:
other: [6] China National standardizing committee.
Principles of method if other than guideline:
[1] Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals Degradation and Accumulation, 301F Ready Biodegradability: Manometric Respirometry Test. Second Edition. Beijing: China Environmental Science Press. 2013: 56-61.[2] Dangerous Chemicals Administration. GB/T 21801-2008 Chemicals—Ready Biodegradability: Manometric Respirometry Test. Beijing: Standards Press of China, 2008.[4] State Environmental Protection Administration of China. HJ/T 153-2004 The Guidelines for the test of chemical. Beijing: China Environmental Science Press. 2004. [6] China National standardizing committee. GB/T 11914-1989 Chemicals-Degradation screening test - Chemical oxygen demand [S]. China Standards Press, 1989.
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Physicochemical properties
Melting point: -39 °C at 101.3 kPa
Boiling point: 216 °C at 101.3 kPa
Volatility: Non-volatile
Vapour Pressure: 1.22 kPa at 37.8 °C
Solubility in Water: Using the US EPA On-Line EPI SuiteTM WSKOW v1.42 model, the water solubility range is predicted to be 2.64E-27 to 0.004421mg/L. This indicates that the substance is insoluble, with water solubilities much lower than 0.1mg/L.
Solubility in Solvent Water: none
Density: 0.82
Odor: None
Oxygen conditions:
anaerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Name: Activated sludgeSource: Aeration tank of Liede Sewage Treatment Plant of Guangzhou
Batch No.: IAs20141008-1
Selection reason: The inoculum is recommended by "The Guidelines for the Testing of Chemicals".Treatment: The sludge was removed any coarse particles and impurities on the surface, then was washed with the test medium (1100g, 10min, repeated for 6 times), the supernatant liquid phase was decanted and the solids re-suspended in the test medium. The sludge suspension was determined as 4.5g/L. Before the test, 88.9mL of the above inoculum was taken to the triangular flask and suspended in 11.1mL of test medium to obtain a concentration equivalent to 4.0g suspended solids per liter. Kept it aeration until required. The inoculum was added to the test vessels to give a final concentration of 28mg suspended solids per liter.
Duration of test (contact time):
28 d
Initial conc.:
28 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test medium: 8L of the test medium was prepared.Equipment: The test equipment include: test bottles, triangular flasks, beakers, graduated cylinder, pipette, volumetric flasks, etc.Test methodsSelection of the test method: As the test substance is poorly soluble in water, so the ready biodegradability: Manometric Respirometry test was adopted.Preparation of the test substance stock solution: 800mg of the test substance was weighted into a beaker and dissolved with some n-hexane, then transferred to 100mL volumetric flask and diluted with n-hexan to 100mL, and the test substance stock solution 1 with the concentration of 8000mg/L was obtained.55.0mg of the test substance was weighted into a beaker and dissolved with some n-hexane, then transferred to 10mL volumetric flask and diluted with n-hexan to 10mL, and the test substance stock solution 2 with the concentration of 5500mg/L was obtained.The purity of the test substance is 100%.Addition of the reference substance: 12.0mg and 8.2mg of the sodium benzoate were added directly into bottles 5 and 6, respectively.Test groupsTest groups include:Bottles 1, 2 & A (test suspension): Containing test substance, inoculum and test mediumBottles 3, 4 & B (inoculum blank): Containing inoculum and test mediumBottle 5 (procedure control): Containing reference substance, inoculum and test mediumBottle 6 (toxicity control): Containing test substance, reference substance, inoculum and test mediumBottle 7 (cosolvent control): Containing only the inoculum and test medium after the cosolvent volatilizing completelyNote: Bottles A & B for determination of pH values of the test suspension and the inoculum blank at the start of the test.Preparation of the test systemAddition of the test substance: 1.0mL of the test substance stock solution 1 were added to bottles 1, 2 and A respectively; 1.0mL of the test substance stock solution 2 was added to bottle 6.The above bottles were placed into fume hood to make n-hexane volatilized completely.Addition of the cosolvent: 1.0mL of the n-hexane was added to bottle 7 which then placed into fume hood to make n-hexane volatilized completely.Addition of the reference substance: 12.0mg and 8.2mg of the reference substance were added into bottles 5 and 6 respectively.Preparation of the inoculated mixture: 35mL of the inoculum with the concentration of 4.0g/L was added into 5L of the test medium, mixing and then the inoculated mixture with the concentration of 28mg/L was obtained.Dispense the inoculated mixture: The above inoculated mixture was dispensed into each bottle to made a final volume of 365mL of bottles 1, 2, A and 5, 432mL of bottles 3, 4, B and 7, and 250mL of bottle 6.Test substance concentration: 22mg/LReference substance concentration: 33mg/LInoculum concentration: 28mg/LTest conditions: All of the test bottles were stirred and incubated on OxiTop® Control BOD Test System at 22°C±1°C in the dark and closed condition over 28 days.
Reference substance:
benzoic acid, sodium salt
Remarks:
Batch No. 20080401-2
Preliminary study:
No details reported in the study report.
Test performance:
The oxygen uptake of mean in the inoculum blank was 18.3mg/L and the oxygen uptake of the cosolvent control was 15.4 mg/L, which were less than 60mg/L in 28 days.At the end of the test, the pH values of each test bottle were 7.52 - 7.71, which was within the range of 6.0 - 8.5.The difference of extremes of replicate values of the removal of the test substance at the plateau, at the end of the test or at the end of the 10-d window were less than 20%.The percentage biodegradation of the procedure control and the toxicity control were 81.9% and 61.3%, which had reached the pass levels of 60% and 25% of ThOD within 14 days.
Parameter:
% degradation (O2 consumption)
Value:
49
Sampling time:
28 d
Remarks on result:
other: Average %
Details on results:
Test conditions
Temperature: 21.8°C - 22.2°C
Light: Dark 8.2
Test results
Percentage biodegradation
Procedure control (14d): 81.9%Procedure control (28d): 79.9%Toxicity control (14d): 61.3%Toxicity control (28d): 75.0%Test suspension (28d): 50.0% 48.0% (two replicates); 49.0% (average)
On day 14 of the test, the percentage biodegradation of the procedure control and the toxicity control were 81.9% and 61.3%, which showed that the inoculum activity could meet the requirement of the test, and the test substance was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study.
Determination of the recovery
The recovery test was set to verify whether the test substance loss during the volatilization of the cosolvent. Three replicates were set in the recovery tests and the theoretical concentration of them were 32mg/L. The recovery concentrations of the three replicates were 31.80mg, 32.23mg and 31.51mg, respectively and the percent recovery were 99.37%, 100.7% and 98.46%, with the mean of 99.5%. It revealed that the test substance had no obvious loss during the volatilization of the cosolvent. Oxygen uptake of the inoculum blank and the cosolvent controlAt the end of the test, the oxygen uptake of the inoculum blanks were 23.9mg/L and 12.6mg/L with the mean of 18.3mg/L, and the oxygen uptake of the cosolvent control was 15.4mg/L.pH values of each test groupsAt the end of the test, the pH values of the test suspensions were 7.53 and 7.52, and those of the inoculum blanks were 7.54 and 7.56. The pH values of the cosolvent control was 7.54.The pH values of the procedure control and the toxicity control were 7.71 and 7.66, respectively.

Composition of test systems in the test vessels

Test vessels

Test suspension

Bottle A

Inoculum blank

Bottle B

Procedure control

Toxicity control

Cosolvent control

Bottle 1

Bottle 2

Bottle 3

Bottle 4

Bottle 5

Bottle 6

Bottle 7

Test substance stock solution 1 (mL)

1.0

1.0

1.0

-

-

-

-

-

-

Test substance stock solution 2 (mL)

-

-

-

-

-

-

-

1.0

-

Reference substance (mg)

-

-

-

-

-

-

12.0

8.2

-

n-hexane (mL)

-

-

-

-

-

-

-

-

1.0

Inoculated mixture (mL)

365

365

365

432

432

432

365

250

432

Final volume (mL)

365

365

365

432

432

432

365

250

432

Reserved cavity volume (mL)

135

135

-

68

68

-

135

250

68

Test substance concentration (mg/L)

22

22

-

-

-

-

-

22

-

Reference substance concentration

-

-

-

-

-

-

33

33

-

Inoculum concentration (mg/L)

28

28

-

28

28

-

28

28

28

ThOD/COD (mg/L)

54

54

-

-

-

-

55

109

-

Note: The test substance stock solution and the cosolvent were added into the corresponding bottles before the test and put in the fume hood to make n-hexane volatilize completely.

 

pH Values

Test bottles

Bottle 1

Bottle 2

Bottle 3

Bottle 4

Bottle 5

Bottle 6

Bottle 7

At the start of the test

7.47

9.47

8.46

7.46

7.46

7.47

7.46

At the end of the test

7.53

7.52

7.54

7.56

7.71

7.66

7.54

 

Biochemical oxygen demand (BOD) of each test bottle during the test (mg/L)

Time (d)

Test suspension

Inoculum blank

Procedure control

Toxicity control

Cosolvent control

Bottle 1

Bottle 2

Bottle 3

Bottle 4

Mean

Bottle 5

Bottle 6

Bottle 7

1

5.7

6.2

5.3

3.9

4.6

29.4

36.6

5.9

2

7.9

8.5

8.4

6.7

7.6

35.6

42.3

8.7

3

11.3

10.7

10.4

8.4

9.4

39.6

49.3

10.1

4

11.9

11.3

11.2

9.0

10.1

44.1

53.5

10.4

5

12.4

11.3

12.1

8.7

10.4

47.5

56.4

10.9

6

13.0

12.4

12.9

9.5

11.2

49.7

59.2

11.2

7

12.4

14.1

11.2

9.8

10.5

52.6

69.0

11.2

8

14.7

16.4

12.1

10.1

11.1

53.7

71.9

11.5

9

16.4

17.0

12.6

10.1

11.4

53.7

69.0

11.8

10

19.2

19.8

13.8

10.9

12.4

56.0

74.7

12.4

11

20.9

20.9

14.3

11.2

12.8

57.1

76.1

12.6

12

22.0

22.0

15.2

11.5

13.4

57.7

77.5

12.9

13

23.7

23.2

15.4

11.5

13.5

58.2

78.9

13.2

14

24.9

26.6

16.0

11.5

13.8

58.8

80.3

13.5

15

27.1

28.8

16.6

11.5

14.1

58.8

80.3

13.5

16

30.0

31.1

17.1

12.1

14.6

59.9

83.1

13.8

17

32.2

32.8

17.4

12.1

14.8

59.9

84.5

14.0

18

34.5

34.5

18.2

12.6

15.4

61.0

87.4

14.3

19

35.6

35.6

18.5

12.6

15.5

61.0

88.8

14.6

20

36.7

36.2

19.1

12.6

15.9

61.0

88.8

14.6

21

37.9

37.3

19.4

12.6

16.0

61.0

90.2

14.9

22

38.4

37.9

20.2

12.9

16.6

61.6

91.6

15.2

23

37.9

37.3

20.2

12.4

16.3

59.9

86.0

14.9

24

39.6

39.0

20.8

12.6

16.7

61.0

93.0

15.2

25

40.7

39.6

22.2

12.4

17.3

61.6

94.4

15.2

26

40.7

40.1

22.5

12.4

17.5

61.6

94.4

15.2

27

41.3

40.7

23.0

12.4

17.7

61.6

95.8

15.4

28

42.4

41.3

23.9

12.6

18.3

62.2

97.2

15.4

 

The percentage biodegradation calculated by BOD (%)

Time (d)

Test groups

Test suspension

Procedure control

Toxicity control

Bottle 1

Bottle 2

Average

Difference

Bottle 5

Bottle 6

1

-0.4

0.6

0.3

0.6

45.1

28.2

2

-1.5

-0.4

-0.9

1.1

51.0

30.8

3

2.2

1.1

1.7

1.1

54.9

36.0

4

2.8

1.7

2.2

1.1

61.8

39.5

5

2.8

0.7

1.8

2.0

67.4

41.7

6

3.3

2.2

2.8

1.1

70.0

44.0

7

2.2

5.4

3.8

3.1

76.6

53.0

8

5.9

9.1

7.5

3.1

77.4

55.4

9

8.5

9.6

9.1

1.1

77.0

52.5

10

12.6

13.7

13.2

1.1

79.4

57.2

11

15.4

15.4

15.4

0

80.6

58.3

12

16.8

16.8

16.8

0.9

80.6

59.3

13

19.4

18.5

19.0

0

81.4

60.3

14

21.1

24.3

22.7

3.1

81.9

61.3

15

25.2

28.3

26.8

3.1

81.4

61.3

16

30.0

32.0

31.0

2.0

82.4

63.6

17

33.7

34.8

34.3

1.1

82.1

64.7

18

37.4

37.4

37.4

0

82.9

67.1

19

38.9

38.9

38.9

0

82.6

68.1

20

40.9

40.0

40.5

0.9

82.1

68.1

21

42.6

41.5

42.0

1.1

81.8

69.1

22

43.0

42.0

42.5

0.9

81.9

70.1

23

42.6

41.5

42.0

1.1

79.3

65.2

24

45.2

44.1

44.6

1.1

80.6

71.4

25

47.2

45.2

46.2

2.0

80.6

72.7

26

47.2

46.1

46.7

1.1

80.3

72.7

27

48.0

46.8

47.4

1.1

79.8

73.8

28

50.0

48.0

49.0

2.0

79.9

75.0

Note: when one of the percentage biodegradation in the two parallel was negative, the negative was regarded as 0% for the calculation of the average and the difference.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The percentage biodegradation of the test substance did not reach 60% at 10d-window under the conditions of the study presently performed, so the test substance can not considered to be readily biodegradable.
Executive summary:

Purpose of the study: Determine the ready biodegradability of the test substance in the dark at 22±1°C over 28 days.

 

Guidelines

[1] Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals Degradation and Accumulation, 301F Ready Biodegradability: Manometric Respirometry Test. Second Edition. Beijing: China Environmental Science Press. 2013: 56-61.

[2] Dangerous Chemicals Administration. GB/T 21801-2008 Chemicals—Ready Biodegradability: Manometric Respirometry Test. Beijing: Standards Press of China, 2008.

[3] OECD.OECD Guidelines for Testing of Chemicals, 301F Manometric Respirometry Test. Paris: OECD. Adopted 17 th July, 1992.

[4] State Environmental Protection Administration of China. HJ/T 153-2004 The Guidelines for the test of chemical. Beijing: China Environmental Science Press. 2004.

[5] Xi Danli, Sun Yusheng, Liu Xiuying. Environmental monitoring. Third Edition. Beijing: Higher Education Press, 2004: 142.

[6] China National standardizing committee. GB/T 11914-1989 Chemicals-Degradation screening test - Chemical oxygen demand. China Standards Press, 1989.

 

Principle: A measured volume of inoculated mineral medium, containing a known concentration of the test substance (100mg test substance/L giving at least 50-100 mg ThOD/L) as the nominal sole source of organic carbon, is stirred in a closed bottle at 22±1°C for up to 28 days. The consumption of oxygen is determined by measuring the change of pressure in the apparatus.

Evolved carbon dioxide is absorbed by sodium hydroxide. The amount of oxygen taken up by the microbial population during biodegradation of the test substance (corrected for uptake by inoculum blank, run in parallel) is expressed as a percentage of ThOD or COD.

 

Test substance: NovaSpec Base Oil

Effective concentration of the test substance: 22mg/L

Preparation of the test substance stock solution: 800mg of the test substance was weighted into a beaker and dissolved with some n-hexane, then transferred to 100mL volumetric flask and diluted with n-hexane to 100mL, and the test substance stock solution 1 with the concentration of 8000mg/L was obtained.

55.0mg of the test substance was weighted into a beaker and dissolved with some n-hexane, then transferred to 10mL volumetric flask and diluted with n-hexane to 10mL, and the test substance stock solution 2 with the concentration of 5500mg/L was obtained.

The purity of the test substance is 100%.

Addition of the test substance stock solution: 1.0mL of the test substance stock solution 1 were added to bottles 1, 2 and A respectively; 1.0mL of the test substance stock solution 2 was added to bottle 6.

The above bottles were placed into fume hood to make n-hexane volatilized completely.

Test groups: Test suspension (bottles 1 & 2), inoculum blank (bottles 3 & 4), procedure control (bottle 5), toxicity control (bottle 6), cosolvent control (bottle 7)

Reference substance concentration: 33mg/L

Inoculum: Activated sludge

Inoculum concentration: 28mg/L

Total volume: 365mL (test suspension and procedure control), 432mL (inoculum blank and cosolvent control), 250mL (toxicity control)

Test temperature: 21.8°C - 22.2°C

Test period: 28d (in the dark)

 

Analysis and determination: The consumption of oxygen of each test bottle was determined and recorded automatically by OxiTop® Control BOD Test System.

 

Results

Percentage biodegradation

Procedure control (14d): 81.9%

Procedure control (28d): 79.9%

Toxicity control (14d): 61.3%

Toxicity control (28d): 75.0%

Test suspension (28d): 50.0% 48.0% (two replicates); 49.0% (average)

 

Determination of the recovery: The recovery test was set to verify whether the test substance loss during the volatilization of the cosolvent. Three replicates were set in the recovery tests and the theoretical concentration of them were 32mg/L. The recovery concentrations of the three replicates were 31.80mg, 32.23mg and 31.51mg, respectively. The percent recovery were 99.37%, 100.7% and 98.46%, with the mean of 99.5%. It revealed that the test substance had no obvious loss during the volatilization of the cosolvent.

 

Conclusion: The percentage biodegradation of the test substance did not reach 60% at 10d-window under the conditions of the study presently performed, so the test substance cannot be considered to be readily biodegradable. However it demonstrates significant degradation under the conditions of the study, and is considered to show degradation within the test.

Description of key information

Ready biodegradation

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

OECD 301B (in the presense of silica gel)

The test substance achieved the requirements for Ready Biodegradability by the OECD 301B standard, achieving 86.7% bidegradation. 60% biodegradation was achieved at 8.9 days; thus passing the 10 day window. The test material can be considered to be readily biodegradable under the conditions of the test.

 

OECD 301B (in the absence of silica gel)

The biodegradability - based on CO2 evolution - of TS2235 was calculated to be 78% and 88% of the theoretical value (ThCO2) after an incubation time of 28 and 56 days, respectively. The biodegradation of TS2235 reached 52% at the end of the 10-d window. Significant biodegradation of the test substance was observed after a lag phase of about 1 day. Whilst TS2235 did not reach the pass level of 60% for ready biodegradability in the CO2 Evolution Test within the 10-d window and cannot be termed as readily biodegradable it did reach the pass level of > 60% after 28 days.

 

OECD 301 C (Ready Biodegradability: Modified MITI Test (I))

Under the test conditions of this study, some of the test item was biodegraded and four converted products were produced. The converted products and the rest of the test item were not biodegraded.

Toc removal 26-27% in 28 days

Test material analysis 24-28% in 28 days (determined by HPLC)

 

OECD 302 C (Inherent biodegradability: Modified MITI Test (II))

Under the conditions of the study, the percentage biodegradation of the test substance on day 28 which was calculated by BOD was 11.0%, the primary biodegradation which was calculated by the residual test substance was 11.1%.

 

OECD 301 F (Ready Biodegradability: Manometric Respirometry Test)

The percentage biodegradation of the test substance did not reach 60% at 10d-window under the conditions of the study presently performed, so the test substance cannot be considered to be readily biodegradable.

O2 consumption 49% in 28 days (mean %)

 

It is considered on the basis of these results that the substance shows significant biodegradation, and can be considered to be biodegradable under normal conditions. The lighter fractions are expected to be readily biodegradable. Heavier fractions (majority by mass) are predicted to be inherently biodegradable.

 

Tests are assigned a Klimish rating of 1 or 2. However all are conducted to a recognised current OECD or alternative Guidelines, in accordance with ISO 17025. The results are considered to be appropriate and reliable.