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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
no
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): The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP Bhandewadi, Nagpur, India, there is not history of contamination of test item, sampling depth 1-2 feet
- Laboratory culture and Method of cultivation: : Preparation of inoculum for exposure and pretreatment: Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º C test temperature
- Activated sludge details: Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport.
- Water filtered: yes
-Biomass concentration: Aliquot of the final sludge suspension weighed, dried and the ratio of wet sludge to its dry weight determined and concentration of sludge was 0.96 g dry material per litre final test medium.
- Method of cultivation: Aeration of incoming domestic sludge with micro-organisms by means of diffuser
- Preparation of inoculum for exposure and pre-treatment: Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º C test temperature.
Duration of test (contact time):
28 d
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
BOD
Details on study design:
TEST CONDITIONS
- Composition of medium: detailed in additional information
- Solubilising agent (type and concentration if used): Not applicable
- Test temperature: 20 °C
- pH: Blank control: 7.3, test chemical: 7.2 Procedure control: 7.4; Toxicity control: 7.2
- pH adjusted: No
- Aeration of dilution water: Not applicable
- Suspended solids concentration: 0.96 g dry material per litre final test medium.
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: BOD bottles
- Number of culture flasks/concentration: 2 flasks/ conentrations
- Measuring equipment: DO meter
- Test performed in closed vessels due to significant volatility of test substance: Not applicable, the BOD bottles were closed with glass lid

SAMPLING
- Sampling frequency: Sampling of all test vessels from each test group concentration were collected for analysis at zero-time (immediately after set-up), 3h, 7th, 14th, 21st, 28th 35 and 42 day during the incubation period.
- Sampling method: Oxygen consumption was measured directly by inserting oxygen probe in BOD bottles
- Sample storage before analysis: samples were analysed immediately after sampling

CONTROL AND BLANK SYSTEM
- Inoculum blank: Containing inoculum and mineral media;
- Toxicity control: Containing reference chemical (sodium benzoate), inoculum and mineral media
- Procedure control: Containing test chemical, reference compound (sodium benzoate), inoculum and mineral media
Reference substance:
benzoic acid, sodium salt
Test performance:
Test substance in the mineral medium, usually at 4 mg/L, is inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely full, closed bottles in the dark at a constant temperature. Degradation is followed by analysis of dissolved oxygen over a 28-d period. The amount of oxygen taken up by the microbial population during biodegradation of the test substance, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of ThOD. A reference compound is run in parallel to check the operation of the procedures. Normally, the test lasts for 28 d. Test however may be ended before 28 d, i.e. as soon as the biodegradation curve has reached a plateau for at least three determinations. Test may also be prolonged beyond 28 days when the curve shows that biodegradation has started but that the plateau has not been reached by day 28, but in such cases the chemical would not be classified as readily biodegradable.
Key result
Parameter:
% degradation (O2 consumption)
Value:
43.72
Sampling time:
28 d
Parameter:
% degradation (O2 consumption)
Value:
44.53
Sampling time:
42 d
Details on results:
The percent degradation of test chemical was found to be 43.72% based on the BOD (oxygen consumption).
Results with reference substance:
The reference compound degraded upto 74.85 % after 28 days of exposure.

BOD42, ThOD AND % BIODEGRADATION VALUES

Test System

BOD42

ThOD(mgO2/mg)

%
Biodegradation

Test Chemical

1.10

2.47

44.53

Procedure
Control(Reference
Chemical)

1.25

1.67

74.85

Toxicity control

1.23

2.47

49.80

Biodegradation for 42 days:

No. of days

Blank control

Procedure control (reference control)

Toxicity control

0

0

0

0

7

16.19

32.93

19.93

14

29.55

65.87

32.29

21

38.46

65.87

44.53

28

43.72

71.86

48.58

35

43.72

74.85

47.77

42

44.53

74.85

49.80

pH values:

No. of days

Blank control

Test chemical

Procedure control (reference control)

Toxicity control

0

7.3

7.2

7.4

7.2

7

7.2

7.2

7.3

7.3

14

7.2

7.2

7.3

7.3

21

7.1

7.1

7.1

7.0

28

7.1

7.1

6.8

6.8

35

7.0

6.9

6.9

6.8

42

7.1

6.8

7.0

6.8

BOD values

No. of days

Blank control

Procedure control (reference control)

Toxicity control

0

0

0

0

7

0.4

0.55

0.48

14

0.73

1.10

0.80

21

0.95

1.10

1.10

28

1.08

1.20

1.20

35

1.08

1.25

1.18

42

1.10

1.25

1.23

No. of days

Blank control

Test chemical

Procedure control (reference control)

Toxicity control

0

0

7.5

7.5

7.6

7

0.4

5.6

5.0

5.4

14

0.73

4.0

2.5

3.8

21

0.95

2.8

2.2

2.3

28

1.08

2.1

1.6

1.7

35

1.08

1.8

1.1

1.5

42

1.10

1.4

0.8

1.0

Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
The percent degradation of test chemical was found to be 43.72% based on the COD, After exposure period of 28 days. thus it has potential for inherently biodegrabale in water.
Executive summary:

The target test item was investigated for it’s readily biodegradability test by following OECD 301 D guideline. The test item was exposed to activated sludge (supernatant) from the aeration tank of a domestic waste water treatment plant for 28 days. The activated sludge was collected from SMS municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38±1º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. Inoculum was pre-conditioned for 1-2 days to the experimental conditions. The supernatant solution of the activated sludge was used in the study. 125 mL BOD vessels were used in the study. The test system includes an inoculum blank control group, a procedure control group, and a test item group a toxicity control group, each maintained in replicates. The biodegradation was determined by following the BOD, oxygen consumption of the test item in the incubation BOD vessels during exposure. Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. All test vessels were incubated at BOD incubator at a constant temperature of 20±1° C. Sampling of 0th h test vessels from each test group concentration were collected for analysis at zero-time (immediately after set-up). Samples were removed at regular intervals i.e., 7th, 14th, 21st, 28th, 35th and 42nd day which were measured for D.O and pH. The theoretical oxygen demand (ThOD) was calculated based on the chemical identifier. The procedure control Sodium benzoate was sufficiently degraded to 65.87% after 14 days, and to 74.85% after 28 days of incubation, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the procedure control sodium benzoate, 32.28% biodegradation was noted within 14 days and 48.58% biodegradation was determined after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the activated sludge microorganisms. Under the test conditions the percentage biodegradation of test item reached 44.53% after 28 days of incubation based on BOD consumption. As the final biodegradation is less than 70% in this test and all validity criteria were met, test item can be considered to be inherently biodegradablein nature.

Description of key information

Biodegradation in water:

The target test substance was investigated for its readily biodegradibilty nature by following OECD 301D guidelines. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38ºC) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º Ctest temperature. Aliquot of the final sludge suspension weighed, dried and the ratio of wet sludge to its dry weight determined and concentration of sludge was 0.96 g dry material per litre final test medium. The biodegradation was determined by following the BOD, oxygen consumption of the test item in the incubation vessels during exposure. Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.125 mL glass vessels (beakers) were used in the study. The test system includes an inoculum blank control group, a procedure control group, and a test item group a toxicitycontrol group, each maintained in replicates. All test vessels were incubated at BOD incubator at a constant temperature of 20°C. Sampling of all test vessels from each test group concentration were collected for analysis at zero-time (immediately after set-up), 3h, 7th, 14th, 21st, 28th, 35 th and 42nd day during the incubation period. Samples were removed at regular intervals, which were measured for BOD. The theoretical oxygen demand (THOD) was calculated based on the chemical identifier. The procedure control Sodium benzoate was sufficiently degraded to 65.87 % after 14 days, and to 74.85 % after 28 days of incubation, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the procedure control sodium benzoate, 32.28% biodegradation was noted within 14 days and 48.58% biodegradation was determined after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the activated sludge microorganisms. Under the test conditions the mean percentage biodegradation of test chemical reached 44.53% after 28 days of incubation based on Oxygen consumption. As the final biodegradation is less than 70% in this test and all validity criteria were met, test chemical can be considered to be potentially inherently biodegradable

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable
Type of water:
freshwater

Additional information

Experiment study of the test chemical and various supporting weight of evidence studies for its structurally and functionally similar read across chemicals were reviewed for the biodegradation in water end point which are summarized as below:

The target test substance was investigated for its readily biodegradibilty nature by following OECD 301D guidelines. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for 28 days. The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38ºC) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport. Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º Ctest temperature. Aliquot of the final sludge suspension weighed, dried and the ratio of wet sludge to its dry weight determined and concentration of sludge was 0.96 g dry material per litre final test medium. The biodegradation was determined by following the BOD, oxygen consumption of the test item in the incubation vessels during exposure. Sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control.125 mL glass vessels (beakers) were used in the study. The test system includes an inoculum blank control group, a procedure control group, and a test item group a toxicitycontrol group, each maintained in replicates. All test vessels were incubated at BOD incubator at a constant temperature of 20°C. Sampling of all test vessels from each test group concentration were collected for analysis at zero-time (immediately after set-up), 3h, 7th, 14th, 21st, 28th, 35 th and 42nd day during the incubation period. Samples were removed at regular intervals, which were measured for BOD. The theoretical oxygen demand (THOD) was calculated based on the chemical identifier. The procedure control Sodium benzoate was sufficiently degraded to 65.87 % after 14 days, and to 74.85 % after 28 days of incubation, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the procedure control sodium benzoate, 32.28% biodegradation was noted within 14 days and 48.58% biodegradation was determined after 28 days of incubation. Thus, the test item can be assumed to be not inhibitory to the activated sludge microorganisms. Under the test conditions the mean percentage biodegradation of test chemical reached 44.53% after 28 days of incubation based on Oxygen consumption. As the final biodegradation is less than 70% in this test and all validity criteria were met, test chemical can be considered to be potentially inherently biodegradable

 

In an experimental key study from study report (2018), biodegradation study was conducted for 35-days following the OECD guideline 301 D for determining the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C under aerobic conditions. Aerobic conditions was provided by means of mineral media which is aerated for 20 hours prior to start of the experiment. The test system included control, test chemical and reference substance. Polyseed capsule (mixed culture) was used as a test inoculum for the study. Test inoculum polyseed capsule was composed of blend of specialized microbial cultures and food grade gelatin made by International Laboratory Supply (InterLab), LTD. No pretreatment / preconditioning was given to the test inoculum as the polyseed capsule requires only one hour of stirring to activate it. 1 Polyseed capsule was added in 500 mL DI water and then stirred for 1 hour for proper mixing and functioning of inoculum & settled for 15 minutes to eliminate the bran. Decanted polyseed solution was used as mixed inoculum. Thus, concentration of test inoculum used for the study was 32 ml/l which corresponds to 10E7 to 10E8 CFU/ml. The concentration of test and reference substance (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference chemical was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test chemical and reference substance. The % degradation of procedure control (reference substance) was also calculated using BOD & ThOD and was determined to be 73.49 %. Degradation of Sodium Benzoate exceeds 39.15 % on 7 days & 70.48 % on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The BOD35 value of test chemical was observed to be 1.17 mgO2/mg. ThOD was calculated as 2.47 mgO2/mg. Accordingly, the % degradation of the test chemical after 35 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 47.36 %. Based on the results, the test chemical under the test conditions, was considered to be inherently biodegradable in nature.

 

Another biodegradation study was conducted for 28-days in accordance with the OECD guideline 301 D for determining the ready biodegradability of the test chemical (Study report, 2018). The study was performed at a temperature of 20°C under aerobic conditions and using the same procedure alongwith the test conditions as mentioned in the above study. The concentration of test and reference substance (Sodium Benzoate) chosen for both the study was 4 mg/L. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference chemical was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test chemical and reference substance. The % degradation of procedure control (reference substance) was also calculated using BOD & ThOD and was determined to be 79.51%. Degradation of Sodium Benzoate exceeds 64.46% on 7 days & 75.30% on 14th day. The activity of the inoculum is thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.22 mgO2/mg. ThOD was calculated as 2.47 mgO2/mg. Accordingly, the % degradation of the test chemical after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 49.39%. Based on the results, the test chemical under the test conditions, was considered to be inherently biodegradable in nature.

 

In a supporting weight of evidence study from peer reviewed journal (A.M. Api et. al., 2016), Biodegradation study was conducted for 28 days for evaluating the percentage biodegradability of test chemical. The study was performed in accordance with the OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test) under aerobic conditions. The percentage degradation of test chemical was determined to be 49% in 28 days. Thus, based on percentage degradation, test chemical was considered to be inherently biodegradable in water.

 

On the basis of above results of the test chemical, it can be concluded that the test chemical was considered to be inherently biodegradable in water.

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