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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):
42 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:
42.54
Sampling time:
42 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 BOD 42 value of test chemical was observed to be 0.77 mgO2/mg. ThOD was determined by calculation as 1.81 mgO2/mg. % Degradation was calculated using the values of BOD and ThOD for test item and was determined to be 42.54% at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 75.3%. Degradation of Sodium Benzoate exceeds 46.38 % on 7 days & 61.44 % 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 75.3%. Degradation of Sodium Benzoate exceeds 46.38 % on 7 days & 61.44 % 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

7.4

7.4

7.2

7

6.9

6

3.6

14

6.6

5

2.3

21

6.2

3.5

1.5

28

5.9

2.9

0.7

35

5.6

2.2

0.3

42

5.3

2.2

0.1

TABLE 2: BOD Values (mg O2/mg)

 

No. of Days

Test Suspension

Procedure Control

(Reference Item)

0

0

0

7

0.22

0.77

14

0.37

1.02

21

0.67

1.12

28

0.75

1.25

35

0.85

1.27

42

0.77

1.25

 

TABLE 3: PERCENT BIODEGRDATION RESULTS

No. of Days

Test Suspension

Procedure Control

(Reference Item)

0

0%

0%

7

12.15%

46.38%

14

20.44%

61.44%

21

37.01%

67.46%

28

41.63%

75.3%

35

46.96%

76.5%

42

42.54%

75.3%

 

Table 4: BOD42, THOD AND % BIODEGRADATION VALUES

 

Method details

BOD42(mgO2/mg)

ThOD (mgO2/mg)

%Biodegradation

Test Item

0.77

1.81

42.54

Reference Item

1.25

1.66

75.3

Validity criteria fulfilled:
yes
Interpretation of results:
other: ultimate inherently biodegradable
Conclusions:
The test chemical undergoes 42.54 % biodegradation after 42 days in the test condition. Thus, the test item was considered to be ultimate inherently biodegradable in nature.
Executive summary:

28-days Closed Bottle test following the OECD guideline 301 D to determine the ready biodegradability of the test item. 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 75.3%. Degradation of Sodium Benzoate exceeds 46.38 % on 7 days & 61.44 % 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 0.77 mgO2/mg. ThOD was calculated as 1.81 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 42.54%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherently 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 75.3%. Degradation of Sodium Benzoate exceeds 46.38 % on 7 days & 61.44 % 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 0.77 mgO2/mg. ThOD was calculated as 1.81 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 42.54%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherently biodegradable in nature.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

Various experimental key and supporting studies for the test chemical and supporting study for its structurally similar read across substance 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 item. 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 75.3%. Degradation of Sodium Benzoate exceeds 46.38 % on 7 days & 61.44 % 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 0.77 mgO2/mg. ThOD was calculated as 1.81 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 42.54%. Based on the results, the test item, under the test conditions, was considered to be ultimate inherently biodegradable in nature.

 

In a supporting study from peer reviewed journal (Masahiro Kawasaki, 1980) and authoritative database, biodegradation study was conducted for 14 days for evaluating the percentage biodegradability of test chemical. The study was performed according to OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I). Activated sludge was used as a test inoculum for the study. Concentration of inoculum i.e, sludge used was 30 mg/l and initial test substance conc. used in the study was 100 mg/l, respectively. The percentage degradation of test chemical was determined to be 0, 2.5 and 0.9% by BOD, TOC removal and UV-Vis parameter in 14 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

Another biodegradation study was conducted according to OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II)) for evaluating the percentage biodegradability of test chemical (from peer reviewed journal Kondo, M et. al., 1988 and secondary source, 2017). Initial test substance conc. used in the study was 20 mg/l. Namely, a water, acetone or DMSO solution (0.1 ml) of the test chemicals was added to a mixture of river/sea water (4.9 ml) from an unpolluted area and an autoclaved solution (5.0ml) of 0.2% peptone in a sterile test tube with a tight plug. After sealed with film and fixed at an angle of 30°in a dark box, the test tubes were incubated at 30°C and shaked at 120rpm. Inoculum used for the study was mixed culture obtained from different sources (Sea water from Mino river and River water from Akashi Beach).The percentage degradation of test chemical in river and sea water was determined to be 0 and 7% by BOD parameter after 14 days. Thus, based on percentage degradation, test chemical is considered to be not readily biodegradable in nature.

 

For the test chemical, biodegradation experiment was conducted for 28 days for evaluating the percentage biodegradability of test chemical. The study was performed according to simulation test method. Test inoculum i.e, micro-organisms was taken from river water. Initial test substance conc. used in the study was 36 mg/l. Artificial river water containing the test chemical as the sole carbon source with 20 mg/l of carbon was used as the growth medium for the study. Test vessel was shaken in the dark during the period of 28 days. Samples were drawn periodically and biodegradation was determined by measuring the remaining TOC. Although %degradation of test chemical was not known, but it was reported that chemical is difficult to undergo degradation. Thus, based on this, test chemical is considered to be not readily biodegradable in nature.

 

Although other studies from peer reviewed journal, authoritative database and secondary source indicates that the test chemical is not readily biodegradable, but on the basis of detailed experimental study result from study report (K1) and as per the OECD guideline, it has been concluded that the test chemical is considered to be ultimate inherently biodegradable in nature.

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