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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:
key study
Study period:
From April 23, 1992 to May 2, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A) The study was conducted according to OECD Guideline 301D and EEC Directive 67/548 Annex V C.6 as published in 84/449/EEC. B) Well-defined information were provided on: 1. Test system (inoculum and composition) 2. Compositional information of the test and reference material (identity and purity) 3. Dosing procedure information (Controls and their composition) 4. Test results and observations (Kinetics of % biodegradation, oxygen concentration and depletion) 5. Definite conclusion
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.6 (Degradation: Chemical Oxygen Demand)
Version / remarks:
EEC Directive 67/548 Annex V C.6 as published in 84/449/EEC
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
anaerobic
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): Aeration stage of HRC Limited sewage treatment plant treating predominantly domestic sewage
- Laboratory culture: No
- Method of cultivation: Not applicable
- Storage conditions: Allowed to settle at room temperature
- Storage length: No data
- Preparation of inoculum for exposure: The supernatant of the settled sludge was filtered after the first 250 mL was discarded.
- Pretreatment: No
- Concentration of sludge: No data
- Initial cell/biomass concentration: No data
- Water filtered: Yes
- Type and size of filter used, if any: Whatman GF filter paper
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 45 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Analytical grade salts and buffers were dissolved in osmosis purified and deionised water to prepare the nutrient medium.
- Additional substrate: None
- Solubilising agent (type and concentration if used): None
- Test temperature: 20 ±1 °C
- pH: No data
- pH adjusted: Yes
- CEC (meq/100 g): No data
- Aeration of dilution water: No
- Suspended solids concentration: No data
- Continuous darkness: Yes, darkened bottles used
- Other: None

TEST SYSTEM
- Culturing apparatus: 280 mL darkened glass Biological Oxygen Demand (BOD) bottles
- Number of culture flasks/concentration: Two per sampling time
- Method used to create aerobic conditions: Test performed under anerobic conditions
- Method used to create anaerobic conditions: Bottles firmly stoppered to exclude all air bubbles
- Measuring equipment: Yellow Springs oxygen meter and BOD Probe
- Test performed in closed vessels due to significant volatility of test substance: Not applicable
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: No

SAMPLING
- Sampling frequency: Dissolved oxygen concentrations for each test medium were determined, in duplicate at Days 0, 5, 15 and 28
- Sampling method: By means of a Yellow Springs BOD Probe (Model 50B)
- Sterility check if applicable: Not applicable
- Sample storage before analysis: No data

CONTROL AND BLANK SYSTEM
- Inoculum blank: Inoculated nutrient medium
- Abiotic sterile control: Non-inoculated nutrient medium
- Toxicity control: Containing both test item (45 mg/L) and reference material (3 mg/L) in test medium
- Procedure control: Containing reference material without test item

STATISTICAL METHODS: No data
Reference substance:
benzoic acid, sodium salt
Remarks:
Batch No. 2923430L
Preliminary study:
Not applicable
Test performance:
No data
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 83
Sampling time:
28 d
Details on results:
% Degradation after 28 d:
Test item - 83 %
Toxicity control - 72 %
Results with reference substance:
% Degradation after 28 d:
Reference material - 92 %

ThOD value (mg O2/mg):
Reference material - 1.67
Estimated oxygen depletion for 3 mg/L is 5.01 mg O2/L.

Dissolved oxygen measurement (mg O2/L) after 28 d:

Test series

          Dissolved oxygen (mg O2/L)

                             Days

              0          5     15      28

Dilution water without inoculum

R1

8.90

8.78

8.74

8.80

R2

8.90

8.81

8.80

8.78

Dilution water with inoculum

R1

8.90

8.80

8.63

8.54

R2

8.90

8.84

8.67

8.78

Test substance

R1

8.90

7.81

5.62

5.38

R2

8.90

8.15

5.73

5.25

Sodium Benzoate in test medium (procedure control)

R1

8.90

5.03

4.36

4.16

R2

8.90

4.93

4.10

3.92

Test substance and reference material (toxicity control)

R1

8.90

NA

NA

2.87

R2

8.90

NA

NA

2.91

NA - Not applicable % degradation after 28 d:

Test series

% degradation value   

                            Days                  7 15           28

Test substance

21

73

81

Sodium Benzoate in test medium (procedure control)

77

88

92

Validity criteria fulfilled:
yes
Remarks:
1. Reference material yielded ≥ 60 % degradation by Day 14; 2. Oxygen depletion of the control did not exceed 30 % of the oxygen in the test bottle; 3. Residual oxygen concentration in the test bottles did not fall below 0.5 mg/L
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions the test substance attained 83 % degradation within 28 days and the pass level of 60 % was reached within 10 days of exceeding the 10 % level, therefore the test substance can be considered to be readily degradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the test substance in an aerobic aqueous medium according to OECD Guideline 301D and EEC Directive 67/548 Annex V C.6 as published in 84/449/EEC, in compliance with GLP. The test substance with the standard nutrient medium was kept in sealed Biological Oxygen Demand (BOD) bottles (darkened glass) at 20°C for 28 days. Non-inoculated nutrient medium (vehicle control), inoculated nutrient medium (inoculum control), toxicity control consisting of test substance (45 mg/L) and the reference material (3 mg/L) in nutrient medium (procedure control) were run in parallel for validation purposes. Dissolved oxygen concentrations for each test medium were determined, in duplicate on Days 0, 5, 15 and 28 by means of a Yellow Springs oxygen meter and BOD Probe. The reference material and the inhibition check attained 92 and 72% biodegradation, respectively, after 28 days. Under the study conditions, the test substance attained 83% degradation within 28 days and the pass level of 60% was reached within 10 days of exceeding the 10% level, therefore the test substance can be considered to be readily degradable (Douglas, 1992).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 2, 2008 to August 11, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SEPA, the Guidelines for the Testing of Chemical No. 301D “Closed Bottle Test” (May 2004)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
anaerobic
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): Aeration tank of Shenyang Fairy River Sewage Treatment Center (treats predominantly domestic waste)
- Laboratory culture: No
- Method of cultivation: Not applicable
- Storage conditions: Kept under aeration till the test
- Storage length: 5 d
- Preparation of inoculum for exposure: After passing the sample through a fine 20-mesh sieve, 1 L of the sub-sample was removed and the remaining centrifuged at 3000 rpm for 1 min. The concentrated sludge, after removing the supernatant, was resuspended in mineral test medium.
- Pretreatment: No
- Concentration of sludge: No data
- Initial cell/biomass concentration: No data
- Water filtered: No data
- Type and size of filter used, if any: No data
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 47.12 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
Preparation of mineral nutrient medium

The nutrient medium was composed of four stock solutions the composition of which are as follows:
Solution A (pH 7.4):
Ingredient weight (g/L)
KH2PO4 8.5
K2HPO4 21.75
Na2HPO4.2H2O 33.4
NH4Cl 0.5

Solution B:
27.50 g anhydrous CaCl2 in 1 L water

Solution C:
22.50 g MgSO4.7H2O in 1 L water

Solution D:
0.25 g FeCl3.6H2O in 1 L water

For each liter of mineral medium, 1 mL stock solution A was added to approximately 800 mL deionized water and the mixture stirred before adding, sequentially, 1 mL each of stock solutions B, C and D. After mixing, the mixture was made to 1 L with deionised water. The pH was maintained in the range of 7.4±0.2.


- Additional substrate: None
- Solubilising agent (type and concentration if used): None
- Test temperature: 20 ± 2 °C
- pH: 7.4± 0.2
- pH adjusted: Yes
- CEC (meq/100 g): No data
- Aeration of dilution water: Strongly aerated for 4 h and then allowed to stand for 20 h
- Suspended solids concentration: 3-5 g/L
- Continuous darkness: Yes, test performed in dark and nutrient medium prepared in brown glass bottle


TEST SYSTEM
- Culturing apparatus: BOD flasks (100~125mL) with glass stopper
- Number of culture flasks/concentration:
35 replicates for each concentration in test suspension, inoculum control and process control
21 replicates for the toxicity control

- Method used to create aerobic conditions: Test performed under anerobic condition
- Method used to create anaerobic conditions:
1. The BOD flasks were firmly stoppered with glass stoppers.
2. All transfer and filling operations of the air-saturated medium were done by siphons and the outlet maintained below the surface of the medium.

- Measuring equipment: O2-electrode and detecting instrument
- Test performed in closed vessels due to significant volatility of test substance: Not applicable
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Not applicable


SAMPLING
- Sampling frequency: 0, 2, 4, 7, 9, 11, 14, 16, 18, 21, 24, and 28 d after treatment
- Sampling method: Dissolved oxygen concentration for each test medium were determined in triplicate by the Oxygen Electrode method at each sampling frequency
- Sterility check if applicable: Not applicable
- Sample storage before analysis: No data


CONTROL AND BLANK SYSTEM
- Inoculum blank: Test system with the inoculum (added at 0.05 mL/L to all test flasks) and without the test and reference substances
- Abiotic sterile control: No data
- Toxicity control: 283.3 mg/L of the test substance and 5 mL of the stock solution of reference substance in the nutrient medium with inoculum
- Other:
Procedure/Process control - Reference substance added at 2.024 mg/L in the inoculated medium from a stock solution of 2024 mg/L.


STATISTICAL METHODS: Not applicable
Reference substance:
benzoic acid, sodium salt
Remarks:
used at a concentration of 2.024 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 79
Sampling time:
28 d
Details on results:
The accumulated percentage biodegradation in activated sludge on 0, 2, 4, 7, 9, 11, 14, 16, 18, 21, 24, and 28 d after treatment were 0, 13.0, 26.7, 44.7, 60.8, 63.2, 66.8, 75.8, 76.4, 77.9, 78.9 and 79.0 respectively.
Results with reference substance:
The % biodegradation values of the reference material were as follows:
Day 2 - 32.0
Day 9 - 74.3
Day 28 - 87.7

Dissolved Oxygen Concentration in the test media:

Day

IC-1

IC-2

IC-3

TS-1

TS-2

TS-3

BOD

% Degradation

0

8.63

8.63

8.62

8.49

8.53

8.48

-

0.0

2

8.40

8.42

8.45

7.89

7.85

7.85

0.433

13.0

4

8.21

8.25

8.23

7.20

7.22

7.21

0.893

26.7

7

8.05

7.86

7.91

6.31

6.32

6.32

1.497

44.7

9

7.86

7.88

7.80

5.69

5.72

5.65

2.033

60.8

11

7.68

7.70

7.68

5.45

5.42

5.47

2.113

63.2

14

7.61

7.62

7.62

5.27

5.24

5.26

2.233

66.8

16

7.54

7.58

7.56

4.98

4.89

4.82

2.537

75.8

18

7.50

7.52

7.50

4.88

4.76

4.83

2.557

76.4

21

7.46

4.47

7.45

4.71

4.71

4.76

2.607

77.9

24

7.42

7.45

7.46

4.68

4.65

4.70

2.640

78.9

28

7.41

7.40

7.38

4.65

4.62

4.61

2.643

79.0

Note: IC means Inoculum Control; TS means Test Substance.

Dissolved Oxygen Concentration in the process control:

Day

IC-1

IC-2

IC-3

PC-1

PC-2

PC-3

BOD

% Degradation

0

8.63

8.63

8.62

8.43

8.38

8.36

-

0.0

2

8.40

8.42

8.45

7.00

7.15

7.15

1.087

32.2

4

8.21

8.25

8.23

6.47

6.47

6.48

1.520

45.1

7

8.05

7.86

7.91

5.45

5.40

5.51

2.250

66.7

9

7.86

7.88

7.80

5.12

5.10

5.10

2.503

74.3

11

7.68

7.70

7.68

4.82

4.78

4.87

2.627

77.9

14

7.61

7.62

7.62

4.69

4.65

4.64

2.720

80.7

16

7.54

7.58

7.56

4.52

4.54

4.51

2.800

83.1

18

7.50

7.52

7.50

4.48

4.42

4.40

2.837

84.1

21

7.46

4.47

7.45

4.35

4.36

4.38

2.860

84.8

24

7.42

7.45

7.46

4.25

4.31

4.32

2.913

86.4

28

7.41

7.40

7.38

4.22

4.21

4.18

2.957

87.7

Note: IC means Inoculum Control; PC means Process Control.

Dissolved Oxygen Determination in Toxicity Control:

Day

IC-1

IC-2

IC-3

TC-1

TC-2

TC-3

BOD

% Degradation

0

8.63

8.63

8.62

8.87

8.88

8.88

-

0.0

2

8.40

8.42

8.45

6.64

6.65

6.62

2.037

27.5

4

8.21

8.25

8.23

5.69

5.76

5.69

2.767

37.4

7

8.05

7.86

7.91

4.66

4.66

4.67

3.527

47.7

9

7.86

7.88

7.80

3.52

3.51

3.51

4.583

62.0

11

7.68

7.70

7.68

2.37

2.33

2.28

5.610

75.9

14

7.61

7.62

7.62

2.17

2.20

2.22

5.670

76.7

Note: IC means Inoculum Control; TC means Toxicity Control.

Calculated ThOD values:

For test substance - 0.071 mg O2/mg

For reference substance - 1.67 mg O2/mg

Validity criteria fulfilled:
yes
Remarks:
1. Dissolved oxygen did not exceed 1.5 mg/L after 28 d 2. Residual concentration of oxygen did not fall below 0.5 mg/L after 28 d 3. Percentage degradation in the toxicity control was 76.7 % after 14 d
Interpretation of results:
readily biodegradable
Conclusions:
Under the study conditions the test substance attained 79 % degradation within 28 days and the pass level of 60 % was reached within 10 days of exceeding the 10 % level, therefore the test substance can be considered to be readily degradable.
Executive summary:

A study was conducted to determine the ready biodegradability of the test substance in an aerobic aqueous medium according to OECD Guideline 301D and SEPA Guideline 301D (closed bottle test), in compliance with GLP. The test substance with the standard nutrient medium was kept in sealed Biological Oxygen Demand (BOD) bottles (darkened glass) at 20°C for 28 days. Non-inoculated nutrient medium (vehicle control), inoculated nutrient medium (inoculum control), toxicity control consisting of test substance (45 mg/L) and the reference material (3 mg/L) in nutrient medium (procedure control) were run in parallel for validation purposes. Dissolved oxygen concentrations for each test medium were determined, in duplicate on Days 0, 2, 4, 7, 9, 11, 14, 16, 18, 21, 24 and 28 by means of a Yellow Springs oxygen meter and BOD Probe. The reference material and the inhibition check attained 87.7 and 76.7% biodegradation, respectively, after 28 days. Under the study conditions, the test substance attained 79% degradation within 28 days and the pass level of 60% was reached within 10 days of exceeding the 10% level, therefore the test substance can be considered to be readily degradable (Zhao, 2008).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals (1992) No. 306
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA Fate, Transport and Transformation Test Guidelines OPPTS 835.3160
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
anaerobic
Inoculum or test system:
natural water
Details on inoculum:
Test Species:
Mixed population of marine micro-organisms in sea water was used as test species (obtained from Huttoft on the East Coast of the United Kingdom).
Treatment: The sea water after filtration was maintained under continuous aeration in a temperature-controlled room at 21 °C for 8 d prior to the start of the study in order to reduce the basal BOD.
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Sea water obtained from Huttoft on the East Coast of the United Kingdom
- Laboratory culture: Yes
- Method of cultivation: The sea water after collection, was filtered and was aerated continuously under constant temperature.
- Storage conditions: Maintained under continuous aeration in a temperature controlled room at 21 °C
- Storage length: 8 d
- Preparation of inoculum for exposure: No data
- Pretreatment: 1 mL of buffered solutions and salt solutions were added to 1 L of aerated sea water
- Concentration of sludge: No data
- Initial cell/biomass concentration: No data
- Water filtered: Yes
- Type and size of filter used, if any: Coarse filter paper
Duration of test (contact time):
>= 28 d
Initial conc.:
ca. 35 mg/L
Based on:
act. ingr.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
The composition of different solutions are given below:

Solution a
pH = 7.4 KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H20 33.40 g/L
NH4Cl 0.50 g/L

Solution b CaCl2 27.50 g/L
Solution c MgSO4.7H2O 22.50 g/L
Solution d FeC13.6H2O 0.25 g/L

1 ml aliquot of each the above solutions was added to each liter of aerated sea water to prepare the test medium.

Test item concentration:
1000 mg of test item was dissolved in test medium and the volume adjusted to 1 L to give a 1000 mg/L stock solution. An aliquot (210 mL) stock solution was dispersed in a final volume of 6 L of test medium to give a concentration of 35 mg/L. The Chemical Oxygen Demand (COD) of the test material was determined to be 0.098 mg 02/mg. Hence if complete degradation of the test material occurred the oxygen depletion in the test vessels would be 3.43 mg 02/mg and as such deoxygenation of the test media would not occur. Therefore, a test concentration of 35 mg/L was employed in the study.

- Test temperature: 20 °C
- pH: 7.4
- pH adjusted: Yes
- CEC (meq/100 g): No data
- Aeration of dilution water: Yes
- Suspended solids concentration: No data
- Continuous darkness: Yes


TEST SYSTEM
- Culturing apparatus: 250-300 mL Biological Oxygen Demand (BOD) bottles (darkened glass)
- Number of culture flasks/concentration: Two per concentration
- Method used to create aerobic conditions: Carried under anerobic condition
- Method used to create anaerobic conditions: BOD bottles were stoppered tightly to exclude all air bubbles
- Measuring equipment: Yellow Springs oxygen meter and BOD Probe.
- Test performed in closed vessels due to significant volatility of test substance: Not applicable
- Test performed in open system: Not applicable
- Details of trap for CO2 and volatile organics if used: None


SAMPLING
- Sampling frequency: Dissolved oxygen concentrations for each test medium were determined, in duplicate on Days 0, 3, 6, 9, 12, 15, 18, 21, 24 and 28
- Sampling method: Samples were analysed at said intervals by means of a Yellow Springs oxygen meter and BOD Probe.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Test medium alone
- Toxicity control: The test material (35 mg/L) and the reference material (1mg/L) in test medium



STATISTICAL METHODS: No data
Reference substance:
benzoic acid, sodium salt
Remarks:
Sodium Benzoate (Lot no. 77H05005) at a concentration of 2 mg/L
Preliminary study:
No
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 79
Sampling time:
28 d
Details on results:
% Degradation after 28 d:

Dissolved Oxygen measurement (mg O2/L) after 28 d
1. Dissolved oxygen of test medium (control) was 7.20 and 7.10 at Day 0 in replicate 1 and 2 respectively and was reduced to 6.20 at Day 28 in both the replicates.

2. Dissolved oxygen of reference material in test medium was 7.15 and 7.10 at Day 0 in replicate 1 and 2 respectively and was reduced to 3.15 and 3.10 in replicate 1 and 2 respectively at Day 28.

3. Dissolved oxygen of test material in test medium was 7.15 at Day 0 in both replicates and was reduced to 3.55 and 3.45 at Day 28 in replicate 1 and 2 respectively.

4. Dissolved oxygen of reference material and test item in test medium was 7.10 at Day 0 in both replicates and was reduced to 1.80 and 1.75 at Day 28 in replicate 1 and 2 respectively.
Results with reference substance:
% Degradation after 28 d:
91 % degradation of reference material

Dissolved oxygen of reference material in the test medium was 7.15 and 7.10 at Day 0 in replicate 1 and 2 respectively and was reduced to 3.15 and 3.10 in replicate 1 and 2 respectively at Day 28.

% Degradation after 28 d

Test substance - 79 % degradation

Reference material - 91% degradation

Toxicity control - 86 % degradation

The oxygen depletion in the control vessels was 13 % of the initial dissolved oxygen concentration.

Oxygen concentration remained at 1.75 mg O2/L or greater in all test vessels and thus the validation criteria for oxygen depletion was satisfied.

ThOD value (mg O2/mg)

Reference material: 1.67

Estimated oxygen depletion for 2 mg/L is 3.34 mg O2/L

COD values (mg O2/mg)

Test material : 0.098

Reference material: 1.69

Reference material (Sodium Benzoate) attained 91% degradation after 28 d. This confirms the suitability of the test method and culture conditions.

Dissolved Oxygen measurement (mg O2/L) after 28 d:

Test series

     Dissolved oxygen (mg O2/L)

                             Days

               0       9     18       28

Test medium

R1

7.20

6.80

6,40

6.20

R2

7.10

6.60

6.45

6.20

Sodium Benzoate in test medium (procedure control)

R1

7.15

4.00

3.75

3.15

R2

7.10

4.00

3.80

3.10

Test material in test medium

R1

7.15

5.80

5.20

3.55

R2

7.15

5.75

5.00

3.54

Test material and Sodium Benzoate in test medium (toxicity control)

R1

7.10

3.70

2.60

1.80

R2

7.10

3.40

2.60

1.75

Mean % degradation value after 28 d:

Test series

            Mean % degradation value

                             Days

3               9            18         28

Sodium Benzoate in test medium (procedure control)

43

30

79

91

Test material in test medium

21

27

39

79

Test material and Sodium Benzoate in test medium (toxicity control)

41

61

74

86

Validity criteria fulfilled:
yes
Remarks:
1. Standard material yields ≥ 60% degradation by day 14 2. Oxygen depletion of the control did not exceed 30% of the oxygen in the test bottle after 28 days
Interpretation of results:
other: potential for biodegradation in the marine environment
Conclusions:
Under the test conditions the test substance attained 79 % degradation after 28 days and therefore can be considered to have the potential for biodegradation in marine environment.

Executive summary:

A study was conduted to determine the ready biodegradability of the test substance in an aerobic aqueous medium according to OECD Guideline 306 and US EPA Fate, Transport and Transformation Test Guidelines OPPTS 835.3160, in compliance with GLP. The test substance, at a concentration of 35 mg/L, was exposed to marine microorganisms with test medium in sealed culture vessels in the dark at 20°C for 28 days. The degradation of the test substance was assessed by the determination of the amount of oxygen consumed. Control solutions containing the standard material, sodium benzoate, together with a toxicity control were used for validation purposes. Under the test conditions the test substance attained 79% degradation after 28 days and therefore can be considered to have the potential for biodegradation in marine environment (Mead, 2000).

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 4, 2000 to October 2, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals (1992) No. 306, "ReadyBiodegradability in Sea Water: Closed Bottle Test"
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3160
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Oxygen conditions:
anaerobic
Inoculum or test system:
natural water
Details on inoculum:
Treatment: The sea water was filtered and maintained under continuous aeration in temperature controlled room at 21 °C for 10 d in order to reduce the basal BOD.
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Mixed population of marine micro-organisms in sea water was used as test species (obtained from Huttoft on the East Coast of the United Kingdom).
- Laboratory culture: No
- Method of cultivation: Not applicable
- Storage conditions: Under continuous aeration at a constant temperature of 21 °C
- Storage length: 10 d
- Preparation of inoculum for exposure: The sea water after collection was filtered.
- Pretreatment: No data
- Concentration of sludge: No data
- Initial cell/biomass concentration: No data
- Water filtered: Yes
- Type and size of filter used, if any: Coarse filter paper
Duration of test (contact time):
ca. 28 d
Initial conc.:
ca. 40 mg/L
Based on:
COD
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium:
Solution a
pH = 7.4 KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H20 33.40 g/L
NH4Cl 0.50 g/L

Solution b CaCl2 27.50 g/L
Solution c MgSO4.7H2O 22.50 g/L
Solution d FeC13.6H2O 0.25 g/L
1 mL aliquot of each the above solutions was added to each liter of aerated sea water to prepare the test medium.

- Concentration of test chemical:
500 mg of test item was dissolved in test medium and the volume adjusted to 1 L to give a 500 mg/l stock solution. An aliquot (480 Ll)of stock solution was dispersed in a final volume of 6 L of test medium to give a concentration of 40 mg/L.
The Chemical Oxygen Demand (COD) of the test material was determined to be 0.081 mg 02/mg. Hence if complete degradation of the test material occurred the oxygen depletion in the test vessels would be 3.20 mg 02/mg and as such deoxygenation of the test media would not occur. Therefore a test concentration of 40 mg/L was employed in the study.

Reference material:Sodium Benzoate (Sigma Lot No. 77H05005) at a concentration of 2 mg/L

- Dosing procedure: Following test solutions were inoculated in 250-300 mL Biological Oxygen Demand (BOD) bottles (darkened glass) in duplicate:
1.Test medium acting as control
2. Sodium Benzoate in test medium to give a concentration of 2 mg/L
3. The test item in test medium to give a concentration of 40 mg/L
4. The test item (40 mg/L) plus reference material (1mg/L) in test medium to act as toxicity control
The BOD bottles were firmly stoppered to exclude all air bubbles after transferring the test solutions and incubated in a temperature controlled water bath at 20 °C.
- Test temperature: 20 °C


- Test performed in open system:
- Details of trap for CO2 and volatile organics if used: No data


SAMPLING
- Sampling frequency: In duplicate, on Days 0, 3, 6, 9, 12, 15, 18, 21, 24 and 28
- Sampling method: Yellow Springs oxygen meter and BOD Probe


CONTROL AND BLANK SYSTEM
- Toxicity control: The test item (40 mg/L) and the reference material (1 mg/L) in test medium
- Vehicle control: Test medium alone
- Procedure control: With the reference material without test item in test medium


STATISTICAL METHODS: No data
Reference substance:
benzoic acid, sodium salt
Remarks:
(Sigma Lot No. 77H05005) at a concentration of 2 mg/L
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 66
Sampling time:
28 d
Details on results:
% Degradation after 28 d:
Test substance - 66 % degradation
Toxicity control - 66 % degradation
The oxygen depletion in the control vessels was 22 % of the initial dissolved oxygen concentration.
Oxygen concentration remained at 2.25 mg O2/L or greater in all test vessels and thus validation criteria for oxygen depletion was satisfied.

Key result
Parameter:
COD
Value:
ca. 0.1 other:
Results with reference substance:
% Degradation after 28 d:
Reference material - 75 % degradation

ThOD value (mg O2/mg):
Reference material: 1.67
Estimated oxygen depletion for 2 mg/L is 3.34 mg O2/L.

COD values (mg O2/mg):
Reference material: 1.69
Reference material (Sodium Benzoate) attained 75 % degradation after 28 d. This confirms the suitability of the test method and culture conditions.

Dissolved oxygen measurement (mg O2/L) during the study period

Test series

   Dissolved oxygen (mg O2/L)

                             Days

   0      9        18    28

Test medium

R1

8.0

8.0

8.0

7.8

R2

8.0

8.0

8.0

7.9

Sodium Benzoate in test medium (procedure control)

R1

8.0

7.9

8.0

7.8

R2

8.0

7.9

7.9

7.8

Test material in test medium

R1

8.1

8.0

8.0

7.9

R2

8.1

8.0

7.9

7.8

Test material and Sodium Benzoate in test medium (toxicity control)

R1

8.1

8.0

7.9

7.8

R2

8.1

7.9

8.0

7.8

Mean % degradation value after during the study period

Test series

   Mean % degradation value

                  Days

3          9        18     28

Sodium Benzoate in test medium (procedure control)

63

72

74

75

Test material in test medium

14

20

58

66

Test material and Sodium Benzoate in test medium (toxicity control)

37

56

63

66

Validity criteria fulfilled:
yes
Remarks:
1. Reference material yielded ≥ 60 % degradation by Day 14; 2. Oxygen depletion of the control did not exceed 30 % of the oxygen in the test bottle; 3. Residual oxygen concentration in the test bottles did not fall below 0.5 mg/L
Interpretation of results:
other: potential for biodegradation in the marine environment
Conclusions:
Under the study conditions the test substance attained 66% degradation after 28 days and therefore can be considered to have the potential for biodegradation in the marine environment.
Executive summary:

A study was conducted to determine the biodegradability of the test substance in an aerobic aqueous medium according to OECD Guideline 306 and US EPA Fate, Transport and Transformation Test Guideline OPPTS 835.3160, in compliance with GLP. The test substance, at a concentration of 40 mg/L, was exposed to marine microorganisms with test medium in sealed culture vessels in the dark at 20°C for 28 days. The degradation of the test substance was assessed by the determination of the amount of oxygen consumed. Control solutions containing the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. Under the study conditions, the test substance attained 66% degradation after 28 days and therefore can be considered to have the potential for biodegradation in the marine environment (Clark, 2000).

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Biodegradation in seawater:

A study was conducted to determine the biodegradability of the test substance in an aerobic aqueous medium according to OECD Guideline 306 and US EPA Fate, Transport and Transformation Test Guideline OPPTS 835.3160, in compliance with GLP. The test substance, at a concentration of 40 mg/L, was exposed to marine microorganisms with test medium in sealed culture vessels in the dark at 20°C for 28 days. The degradation of the test substance was assessed by the determination of the amount of oxygen consumed. Control solutions containing the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. Under the study conditions, the test substance attained 66% degradation after 28 days and therefore can be considered to have the potential for biodegradation in the marine environment (Clark, 2000).

A study was conducted to determine the ready biodegradability of the test substance in an aerobic aqueous medium according to OECD Guideline 306 and US EPA Fate, Transport and Transformation Test Guidelines OPPTS 835.3160, in compliance with GLP. The test substance, at a concentration of 35 mg/L, was exposed to marine microorganisms with test medium in sealed culture vessels in the dark at 20 °C for 28 days. The degradation of the test substance was assessed by the determination of the amount of oxygen consumed. Control solutions containing the standard material, sodium benzoate, together with a toxicity control were used for validation purposes. Under the test conditions, the test substance attained 79% degradation after 28 days and therefore can be considered to have the potential for biodegradation in marine environment (Mead, 2000).

Biodegradation in freshwater:

A study was conducted to determine the ready biodegradability of the test substance in an aerobic aqueous medium according to OECD Guideline 301D and EEC Directive 67/548 Annex V C.6 as published in 84/449/EEC, in compliance with GLP. The test substance with the standard nutrient medium was kept in sealed Biological Oxygen Demand (BOD) bottles (darkened glass) at 20°C for 28 days. Non-inoculated nutrient medium (vehicle control), inoculated nutrient medium (inoculum control), toxicity control consisting of test substance (45 mg/L) and the reference material (3 mg/L) in nutrient medium (procedure control) were run in parallel for validation purposes. Dissolved oxygen concentrations for each test medium were determined, in duplicate on Days 0, 5, 15 and 28 by means of a Yellow Springs oxygen meter and BOD Probe. The reference material and the inhibition check attained 92 and 72% biodegradation, respectively, after 28 days.Under the study conditions, the test substance attained 83% degradation within 28 days and the pass level of 60% was reached within 10 days of exceeding the 10% level, therefore the test substance can be considered to be readily degradable (Douglas, 1992).

A study was conducted to determine the ready biodegradability of the test substance in an aerobic aqueous medium according to OECD Guideline 301D and SEPA Guideline 301D (closed bottle test), in compliance with GLP.The test substance with the standard nutrient medium was kept in sealed Biological Oxygen Demand (BOD) bottles (darkened glass) at 20°C for 28 days. Non-inoculated nutrient medium (vehicle control), inoculated nutrient medium (inoculum control), toxicity control consisting of test substance (45 mg/L) and the reference material (3 mg/L) in nutrient medium (procedure control) were run in parallel for validation purposes. Dissolved oxygen concentrations for each test medium were determined, in duplicate on Days 0, 2, 4, 7, 9, 11, 14, 16, 18, 21, 24 and 28 by means of a Yellow Springs oxygen meter and BOD Probe.The reference material and the inhibition check attained 87.7 and 76.7% biodegradation, respectively, after 28 days. Under the study conditions, the test substance attained 79% degradation within 28 days and the pass level of 60% was reached within 10 days of exceeding the 10% level, therefore the test substance can be considered to be readily degradable (Zhao, 2008).