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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental result using OECD guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Principles of method if other than guideline:
Biodegradation study was conducted according to OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test) for evaluating the percentage biodegradability of test chemical.
GLP compliance:
no
Oxygen conditions:
not specified
Inoculum or test system:
other: Mixed inoculum
Details on inoculum:
Mixed Inoculum Preparation:Polyseed were used for this study. 1 polyseed capsule was added in 500 ml D.I water and then stirred for 1 hour for proper mixing and functioning of inoculum. This gave the bacterial count as 10E7 to 10E8 CFU/ml. At the regular interval microbial plating was also performed on agar to confirm the vitality and CFU count of microorganism.
Duration of test (contact time):
28 d
Initial conc.:
4 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: OECD mineral medium was used for the study
- Test temperature: 20°C
- Continuous darkness: Yes
- Other: The water used in this study is deionized water.

TEST SYSTEM
- Culturing apparatus: The apparatus used in this study is BOD bottles; with glass stoppers (125 ml), BOD incubator & oxygen electrode and meter.

CONTROL AND BLANK SYSTEM
- Inoculum blank: it contains only test inoculum
- Procedure control: contains reference compound and inoculum
Reference substance:
other: Sodium Benzoate
Key result
Parameter:
% degradation (O2 consumption)
Value:
66.87
Sampling time:
28 d
Remarks on result:
other: Other details not known
Details on results:
The oxygen consumed by the test systems was corrected for oxygen consumption occurring in the blank test systems.The BOD Values (mgO2/mg) and percent biodegradation results for each test system arereported in Tables 2 and 3, respectively. The BOD28 value of test chemical was observed to be 1.07 mgO2/mg. ThOD was determined by calculation as 1.6 mgO2/mg. % Degradation was calculated using the values of BOD and ThOD for test item and was determined to be 66.87% at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 79.51%. Degradation of Sodium Benzoate exceeds 64.45 % on 7 days & 75.30 % on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid.
Results with reference substance:
The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 79.51%. Degradation of Sodium Benzoate exceeds 64.45 % on 7 days & 75.30 % on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid.

TABLE 1: D.O Values (mg/L)

 

No. of Days

Inoculum Blank

(Control)

Test Suspension

Procedure Control

(Reference Item)

0

6.6

6.4

6.5

7

6.4

3.4

2

14

6.1

1.9

1

21

5.8

1.6

0.4

28

5.6

1.1

0.2

TABLE 2: BOD Values (mg O2/mg)

 

No. of Days

Test Suspension

Procedure Control

(Reference Item)

0

0

0

7

0.7

1.07

14

0.92

1.25

21

1

1.32

28

1.07

1.32

 

TABLE 3: PERCENT BIODEGRDATION RESULTS

No. of Days

Test Suspension

Procedure Control

(Reference Item)

0

0%

0%

7

43.75%

64.45%

14

57.5%

75.3%

21

62.5%

79.52%

28

66.87%

79.51%

 

Table 4: BOD28, THOD AND % BIODEGRADATION VALUES

 

Method details

BOD28(mgO2/mg)

ThOD (mgO2/mg)

%Biodegradation

Test Item

1.07

1.6

66.87

Reference Item

1.32

1.66

79.51

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Test chemical undergoes 66.87 % biodegradation after 28 days in the test condition. Thus, the test chemical was considered to be readily biodegradable in nature.
Executive summary:

28-days Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C. The test system included control, test item and reference item. Polyseed were used for this study. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/l. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 79.51%. Degradation of Sodium Benzoate exceeds 64.45 % on 7 days & 75.30 % on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.07 mgO2/mg. ThOD was calculated as 1.60 mgO2/mg. Accordingly, the % degradation of the test item after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 66.87%. Based on the results, the test item, under the test conditions, was considered to be readily biodegradable in nature.

Description of key information

28-days Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test chemical (Experimental study report, 2018). The study was performed at a temperature of 20°C. The test system included control, test item and reference item. Polyseed were used for this study. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/l. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 79.51%. Degradation of Sodium Benzoate exceeds 64.45 % on 7 days & 75.30 % on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.07 mgO2/mg. ThOD was calculated as 1.60 mgO2/mg. Accordingly, the % degradation of the test item after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 66.87%. Based on the results, the test item, under the test conditions, was considered to be readily biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Various experimental key and supporting studies of the test chemical were reviewed for the biodegradation end point which are summarized as below:

 

In an experimental key study from study report (2018),28-days Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test chemical. The study was performed at a temperature of 20°C. The test system included control, test item and reference item. Polyseed were used for this study. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32 ml/l. OECD mineral medium was used for the study. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % degradation was calculated using the values of BOD and ThOD for test item and reference item. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 79.51%. Degradation of Sodium Benzoate exceeds 64.45 % on 7 days & 75.30 % on 14th day. The activity of the inoculum was thus verified and the test can be considered as valid. The BOD28 value of test chemical was observed to be 1.07 mgO2/mg. ThOD was calculated as 1.60 mgO2/mg. Accordingly, the % degradation of the test item after 28 days of incubation at 20 ± 1°C according to Closed Bottle test was determined to be 66.87%. Based on the results, the test item, under the test conditions, was considered to be readily biodegradable in nature.

 

Another biodegradation study was conducted for 35 days for evaluating the percentage biodegradability of test chemical (Experimental study report, 1991). The study was performed in accordance with the OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test) under aerobic conditions. Activated sludge obtained from a laboratory plant operated with concomitant wastewater was used as a test inoculum. Conc. of the inoculum used was 1 g/l dry substance in the test batch. Test inoculum was first pre-adapted with the test substance. After completion of this test, the pre-adapted inoculum was separated and washed twice. With the pre-adapted inoculum, the Zahn-Wellens test was performed. Test chemical conc. used for the study was 765 mg/l based on test material analysis / 400 mg/l based on DOC removal. Measuring equipment used during the DOC / TOC analysis with the analyzer was Shimadzu. Diethylene glycol was used as a reference substance for the study. The percentage degradation of test chemical was determined to be 71% based on DOC removal parameter after a period of 35 days. Thus, based on the percentage degradation, test chemical can be considered to be readily biodegradable in nature.

 

In a supporting study,biodegradation experiment was conducted for 28 days for evaluating the percentage biodegradability of test chemical (Experimental study report, 1991). The study was performed in accordance with the OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test) under aerobic conditions. Municipal wastewater obtained from a laboratory treatment plant operated with concomitant wastewater was used as a test inoculum. Test chemical conc. used for the study was 37 mg/l based on test material analysis. Sodium benzoate was used as a reference substance for the study. Reference substance sodium benzoate undergoes 100% degradation after 4 days. The percentage degradation of test chemical was determined to be 7% based on DOC removal parameter after a period of 28 days. Thus, based on the percentage degradation, test chemical can be considered to be not readily biodegradable in nature.

 

For the test chemical from peer reviewed journal (Rita Beel et. Al, 2013), biodegradation study was conducted for 20 days for evaluating the percentage biodegradability of test chemical. The study was performed under aerobic conditions. Activated sludge was used as a test inoculums for the study. Initial test substance conc. used in the study was 20 mg/l. Analytical methods involve the use of both MS and HPLC. The percentage degradation of test chemical was determined to be 100% by after 7 days. Thus, based on percentage degradation, test chemical is considered to be readily biodegradable in nature.

 

Although result from one screening test (performed as per the OECD Guideline 301 E (Ready biodegradability: Modified OECD Screening Test)) indicates that the test chemical is not readily biodegradable; to confirm this another study as per the OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test) was carried out indicating that the test chemical is readily biodegradable which is supported further by the results of other screening test (from study reports (K1) performed as per the OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test))) and peer reviewed journal. On the basis of this, it has been concluded that the test chemical is considered to be readily biodegradable in nature.