Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 January - 22 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidelines and GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Wareham Wastewater Treatment Plant, Wareham, Massachusetts, which receives primarily domestic waste
- Laboratory culture: N/A
- Method of cultivation: N/A
- Storage conditions: Aerated
- Storage length: 1 month
- Preparation of inoculum for exposure: The activated sludge used for this study was obtained from the Wareham Wastewater Treatment Plant, Wareham, Massachusetts, which receives primarily domestic waste. Approximately 4 L of activated sludge was collected on 16 January 2018 and transported to the testing laboratory. Upon arrival at the testing laboratory, the sludge was passed through a 2-mm sieve and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, the sludge was washed with mineral medium, the contents were centrifuged at least once again, and the supernatant was discarded. The moisture content of the activated sludge was determined, using an automated moisture analyzer (Sartorius MA-150), to be 96.14 % and the percent solids determined to be 3.86 %. A 15 mg solids/mL inoculum solution was prepared (77.72 g wet weight sludge brought to 200 mL with mineral media) and aerated until used. Each 2-L test vessel received 3.0 mL of the inoculum to produce an inoculum concentration of 30 mg/L. Each test vessel contained 1.5 L of mineral medium.
- Pretreatment: No
- Concentration of sludge: Described above
- Initial cell/biomass concentration: N/A
- Water filtered: No
- Type and size of filter used, if any: N/A
Duration of test (contact time):
35 d
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral Salts Medium - as per OECD 301A - prepared using purified water.
- Additional substrate: No
- Solubilising agent (type and concentration if used): No
- Test temperature: 22 ± 2 ºC
- pH: Not reported
- pH adjusted: No
- CEC (meq/100 g): Not reported
- Aeration of dilution water: Each test unit was loosely covered with perforated Parafilm to allow for free gas exchange.
- Suspended solids concentration: 3.86 g/L
- Continuous darkness: yes
- Other: N/A

TEST SYSTEM
- Culturing apparatus: Each test unit consisted of a 2-L glass Erlenmeyer flask which was loosely covered with perforated Parafilm to allow for free gas exchange. The flasks were placed on a mechanical shaker table set to 125 rpm in the dark.
- Number of culture flasks/concentration: Six test vessels were established: two for the test substance, two inoculum blanks, one sodium benzoate procedure control, and one toxicity control
- Method used to create aerobic conditions: Each test unit was loosely covered with perforated Parafilm to allow for free gas exchange.
- Method used to create anaerobic conditions: N/A
- Measuring equipment: Samples were analyzed using a Shimadzu TOC-L dissolved organic carbon analyzer.
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: Yes
- Details of trap for CO2 and volatile organics if used: N/A
- Other: N/A

SAMPLING
- Sampling frequency: On test days 0, 1, 4, 7, 11, 14, 18, 22, 25, 28, and 35, duplicate 20-mL samples were removed from each test vessel.
- Sampling method: Duplicate 20-mL samples were removed from each test vessel. Prior to sampling, the volume in the flasks was checked for losses by comparing the liquid level from the last interval, but no adjustments were necessary. The samples were analyzed using a Shimadzu TOC-L dissolved organic carbon analyzer.
- Sterility check if applicable: N/A
- Sample storage before analysis: Not reported
- Other: N/A

CONTROL AND BLANK SYSTEM
- Inoculum blank: 3 mL inoculum
- Abiotic sterile control: Not conducted
- Toxicity control: 10 mg C/L test item + 10 mg C/L sodium benzoate + 3 mL inoculum
- Other: Procedural control: 10 mg C/L sodium benzoate + 3 mL inoculum

STATISTICAL METHODS:

The DOC of each test substance and reference substance flask was corrected by subtracting the mean of the blank control values from the mean of the test and reference substances. The resulting net DOC was used to calculate biodegradability.

The percent biodegradability for each test system was calculated using the following equation and is expressed as cumulative percent biodegradation (or percent of theoretical DOC).

% biodeg. = (1 - ((DOC at time t - DOC in blank at time t) / (DOC at 0 h - DOC in blank at 0 h))) * 100
Reference substance:
other: sodium benzoate
Test performance:
The net DOC measured in the procedure control (sodium benzoate) system decreased from 10.24 mg/L (day 0) to 0.07 mg/L by day 4, confirming the almost total degradation of the reference substance. Degradation of 99.36% was recorded by day 4, which surpassed the OECD minimum criteria of 70% degradation by day 14.

The net DOC values observed in the toxicity control vessel decreased from 22.31 mg/L at day 0 to 12.00 mg/L at day 4 and 8.75 mg/L at day 28, indicating 46.20% and 60.80% degradation had occurred by day 4 and day 28, respectively. The OECD criterion of greater than 35% degradation (based on DOC) within 14 days was met by the toxicity control replicate. This indicated that the test substance was not toxic to the microbial community present in the test vessels.
Key result
Parameter:
% degradation (DOC removal)
Value:
32.5
Sampling time:
28 d
Key result
Parameter:
% degradation (DOC removal)
Value:
32.16
Sampling time:
35 d
Details on results:
Test item is not readily biodegradable.

The net DOC measured in the procedure control (sodium benzoate) system decreased from 10.24 mg/L (day 0) to 0.07 mg/L by day 4, confirming the almost total degradation of the reference substance. Degradation of 99.36% was recorded by day 4, which surpassed the OECD minimum criteria of 70% degradation by day 14.

The net DOC values observed in the toxicity control vessel decreased from 22.31 mg/L at day 0 to 12.00 mg/L at day 4 and 8.75 mg/L at day 28, indicating 46.20% and 60.80% degradation had occurred by day 4 and day 28, respectively. The OECD criterion of greater than 35% degradation (based on DOC) within 14 days was met by the toxicity control replicate. This indicated that the test substance was not toxic to the microbial community present in the test vessels.

Table2       Mean dissolved organic carbon (mg C/L) measured in the test vessels

 

Vessel

Rep.

Day

0

1

4

7

11

14

18

22

25

28

35

Test item

A

12.63

12.48

12.65

12.61

12.62

12.45

12.43

12.30

9.91

8.69

9.54

B

12.43

12.39

12.45

12.75

12.72

12.62

12.52

12.32

10.45

9.16

10.03

Mean

12.53

12.44

12.55

12.68

12.67

12.54

12.48

12.31

10.18

8.93

9.79

Blank control

A

0.56

0.62

0.69

0.70

0.78

0.76

0.80

0.83

0.75

0.83

1.87

B

0.53

0.49

0.62

0.62

0.76

0.73

0.75

0.86

0.72

0.85

1.45

Mean

0.55

0.56

0.66

0.66

0.77

0.75

0.77

0.84

0.73

0.84

1.66

Procedure control

 

10.79

7.23

0.72

0.76

0.86

0.77

0.80

0.94

0.86

0.88

1.40

Toxicity control

 

22.86

19.21

12.66

12.47

12.46

12.42

12.54

12.33

12.19

9.59

10.04

 

Table3       Mean net dissolved organic carbon (mg C/L) measured in the test vessels

 

Vessel

Rep.

Day

0

1

4

7

11

14

18

22

25

28

35

Test item

A

12.08

11.92

11.99

11.95

11.85

11.70

11.66

11.46

9.18

7.85

7.88

B

11.88

11.83

11.79

12.09

11.95

11.87

11.75

11.48

9.72

8.32

8.37

Mean

11.98

11.88

11.89

12.02

11.90

11.79

11.70

11.47

9.45

8.09

8.13

Procedure control

 

10.24

6.67

0.07

0.10

0.09

0.02

0.02

0.09

0.12

0.04

-0.26

Toxicity control

 

22.31

18.65

12.00

11.81

11.69

11.67

11.77

11.49

11.46

8.75

8.38

Table4       Biodegradation, expressed as a percent of the theoretical

 

Vessel

Rep.

Day

0

1

4

7

11

14

18

22

25

28

35

Test item

A

0

1.32

0.74

1.10

1.91

3.15

3.52

5.18

24.03

35.05

34.76

B

0

0.42

0.75

-1.74

-0.58

0.09

1.14

3.42

18.23

29.94

29.56

Mean

0

0.87

0.74

-0.32

0.67

1.62

2.33

4.30

21.13

32.50

32.16

Procedure control

 

0

34.88

99.36

99.00

99.08

99.76

99.78

99.09

98.80

99.64

102.51

Toxicity control

 

0

16.40

46.20

47.07

47.60

47.69

47.26

48.52

48.66

60.80

62.44

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Based on the results of this study, the test item cannot be classified as readily biodegradable since greater than 70% biodegradation was not observed.
Executive summary:

OECD 301A (2018) - The ready biodegradability of the test item was assessed in accordance with OECD Procedure 301A ‘DOC Die-Away Test’, adopted 17 July 1992.

 

The test was added to two bottles containing mineral salts medium inoculated with activated sludge (30 mg solids/L) to give a nominal test concentration of 10 mg C/L. Two control cultures contained inoculated mineral salts medium alone. Two cultures contained inoculated mineral salts medium plus the reference substance sodium benzoate (10 mg C/L) of which one also contained the test item (10 mg C/L) in order to assess the potential inhibitory effects of the test substance on the microbial inoculum. Duplicate 20 mL subsamples were taken from each of the bioreactor vessels on Day 0, 1, 4, 7, 11, 14, 18, 22, 25, 28 and 35 for analysis using a Shimadzu TOC analyser. The DOC of each vessel was calculated and from which the biodegradation determined.

 

In the test substance systems, the mean net DOC was 11.98 mg/L on day 0, 8.09 mg/L on day 28 and 8.13 mg/L on day 35. These data indicate that 32.50% and 32.16% degradation of the test substance occurred under the conditions of this study after correcting for the background DOC measured in the inoculum blanks at day 28 and day 35, respectively.

 

The net DOC measured in the procedure control (sodium benzoate) system decreased from 10.24 mg/L (day 0) to 0.07 mg/L by day 4, confirming the almost total degradation of the reference substance. Degradation of 99.36% was recorded by day 4, which surpassed the OECD minimum criteria of 70% degradation by day 14.

 

The net DOC values observed in the toxicity control vessel decreased from 22.31 mg/L at day 0 to 12.00 mg/L at day 4 and 8.75 mg/L at day 28, indicating 46.20% and 60.80% degradation had occurred by day 4 and day 28, respectively. The OECD criterion of greater than 35% degradation (based on DOC) within 14 days was met by the toxicity control replicate. This indicated that the test substance was not toxic to the microbial community present in the test vessels.

Based on the results of this study, the test item cannot be classified as readily biodegradable.

Description of key information

28 day biodegradation (based on DOC) = <70 % (mean actual = 32.5 %), not readily biodegradable; OECD 301A; McLaughlin, S. (2018)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

OECD 301A (2018) - The ready biodegradability of the test item was assessed in accordance with OECD Procedure 301A ‘DOC Die-Away Test’, adopted 17 July 1992.

 

The test was added to two bottles containing mineral salts medium inoculated with activated sludge (30 mg solids/L) to give a nominal test concentration of 10 mg C/L. Two control cultures contained inoculated mineral salts medium alone. Two cultures contained inoculated mineral salts medium plus the reference substance sodium benzoate (10 mg C/L) of which one also contained the test item (10 mg C/L) in order to assess the potential inhibitory effects of the test substance on the microbial inoculum. Duplicate 20 mL subsamples were taken from each of the bioreactor vessels on Day 0, 1, 4, 7, 11, 14, 18, 22, 25, 28 and 35 for analysis using a Shimadzu TOC analyser. The DOC of each vessel was calculated and from which the biodegradation determined.

 

In the test substance systems, the mean net DOC was 11.98 mg/L on day 0, 8.09 mg/L on day 28 and 8.13 mg/L on day 35. These data indicate that 32.50% and 32.16% degradation of the test substance occurred under the conditions of this study after correcting for the background DOC measured in the inoculum blanks at day 28 and day 35, respectively.

 

The net DOC measured in the procedure control (sodium benzoate) system decreased from 10.24 mg/L (day 0) to 0.07 mg/L by day 4, confirming the almost total degradation of the reference substance. Degradation of 99.36% was recorded by day 4, which surpassed the OECD minimum criteria of 70% degradation by day 14.

 

The net DOC values observed in the toxicity control vessel decreased from 22.31 mg/L at day 0 to 12.00 mg/L at day 4 and 8.75 mg/L at day 28, indicating 46.20% and 60.80% degradation had occurred by day 4 and day 28, respectively. The OECD criterion of greater than 35% degradation (based on DOC) within 14 days was met by the toxicity control replicate. This indicated that the test substance was not toxic to the microbial community present in the test vessels.

Based on the results of this study, the test item cannot be classified as readily biodegradable.