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Environmental fate & pathways

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 substance.
GLP compliance:
no
Oxygen conditions:
aerobic
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.

OR

Mixed Inoculum Preparation:Domestic waste water, surface soil and soil samples from polluted sites receiving predominantly domestic waste was collected and were mixed to get diluted suspension. The inoculum was kept aerobic until being used for experiment by supplying organic and inorganic sources required by micro flora to sustain at controlled laboratory conditions. This gave the bacterial count as 107 to 108 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:
benzoic acid, sodium salt
Parameter:
% degradation (O2 consumption)
Value:
88.19
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 are reported in Tables 2 and 3, respectively. The BOD28 value of test chemical was observed to be 1.27 mgO2/mg. ThOD was determined by calculation as 1.44 mgO2/mg. % degradation was calculated using the values of BOD and ThOD for test item and was determined to be 88.19 % at 20 ± 1°C. The % degradation of procedure control (reference item) was also calculated using BOD & ThOD and was determined to be 70.48 %. Degradation of Sodium Benzoate exceeds 46.38 % on 7 days & 60.24 % on 14th day. The activity of the inoculum is 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 70.48 %. Degradation of Sodium Benzoate exceeds 46.38 % on 7 days & 60.24 % on 14th day. The activity of the inoculum is 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

6.7

6.5

 

7

6.3

4

3.1

14

6.1

1.8

2

21

5.8

0.9

1.2

28

5.5

0.5

0.7

 

 

TABLE 2

BOD values (mgO2/mg)

No. of Days

Test Suspension

Procedure Control

(Reference Item)

 

0

0

0

7

0.6

0.77

14

1.1

1

21

1.25

1.12

28

1.27

1.17

 

TABLE 3

PERCENT BIODEGRDATION RESULTS

No. of Days

Test Suspension

Procedure Control

(Reference Item)

 

0

0%

0%

7

41.66%

46.38%

14

76.38%

60.24%

21

86.8%

67.46%

28

88.19%

70.48%

 

 

Table 4

BOD28, THOD AND % BIODEGRADATION VALUES

Method details

BOD28 (mgO2/mg)

ThOD (mgO2/mg)

% Biodegradation

 

Test Item

1.27

1.44

88.19

Reference Item

1.17

1.66

70.48

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test chemical undergoes 88.19 % biodegradation after 28 days in the test condition. Thus, the test chemical was considered to be readily biodegradable.
Executive summary:

28-days Closed Bottle test following the OECD guideline 301 D was performed 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. 1 polyseed capsule were 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.The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32ml/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 70.48 %. Degradation of Sodium Benzoate exceeds 46.38 % on 7 days & 60.24 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 1.27 mgO2/mg. ThOD was calculated as 1.44 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 88.19 %. 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 was performed 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. 1 polyseed capsule were 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.The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32ml/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 70.48 %. Degradation of Sodium Benzoate exceeds 46.38 % on 7 days & 60.24 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 1.27 mgO2/mg. ThOD was calculated as 1.44 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 88.19 %. 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

Experimental studies for target chemical from different sources have been done for biodegradation in water endpoint and results of all the studies are summarized below.

 

In first study the 28-days Closed Bottle test following the OECD guideline 301 D was performed 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. 1 polyseed capsule were 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.The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 4 mg/L, while that of inoculum was 32ml/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 70.48 %. Degradation of Sodium Benzoate exceeds 46.38 % on 7 days & 60.24 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 1.27 mgO2/mg. ThOD was calculated as 1.44 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 88.19 %. Based on the results, the test item, under the test conditions, was considered to be readily biodegradable in nature.

 

Another study was reviewed from peer reviewed journal in this study the Biodegradation experiment of test chemical was conducted by using packed bed bioreactor. The inoculum used was Pseudomonas. putida BCRC 14365. The freeze-dried Pseudomonas putida culture embedded in a tube was stored at 4°C in the laboratory until use. Then, 0.5 mL of nutrient medium was added to the tube to dissolve the frozen stock culture for activation and then form suspended cells. The suspended P. putida cells with nutrient medium were incubated at 30°C for 3–4 days to ensure the P. putida cells’ viability and usefulness as fresh stock culture for further experiments.  The packed bed bioreactor was filled with 140 mL of glass beads with diameters of 7 mm and 198 mL of mineral salt medium The reactor porosity was approximately 58.6%. The feed solution was mixing using a magnetic stirrer and then pumped into the reactor using a peristaltic pump. The liquid broth was recirculated from the top left-hand side to the bottom right-hand side of the reactor using a second peristaltic pump. In the reactor, the recirculation rate was maintained at 12.3 mL=min and the superficial up flow velocity was 7 m=h. The feed solution was pumped into the reactor from the bottom of the reactor. Test chemical was fed into reactor with mineral salt medium at a flow rate of 1,188 cm3=day. The effective working volume was 338 cm3, which yielded a hydraulic retention time of 6.8 h. The reactor temperature was maintained at 30 oC. Samples for analysis were taken from the feed and the effluent. The samples from effluent were taken at time intervals of 2–10 min in the beginning of test, then 1–2 h, and subsequently 0.5–1.0 and 1–4 days after a steady-state condition. Then samples were analysed by using HPLC apparatus. The HPLC apparatus consisted of a Waters 2707 autosampler and a Waters 2487 UV/Vis detector, and equipped with a Symmetry (Waters, Miford, Massachusetts) C18 column (particle size of 5 μm). The test chemical undergoes 100 % degradation in less than 60 hrs when initial concentration of test chemical taken was less than 0.72 mM whereas when initial concentration of test chemical was grater then 0.72mM it takes 66-70 hrs. On the basis of time required for 100 % degradation it is concluded that test chemical is readily biodegradable.

 

Next experimental study was done by Ruey-Shin Juang , (2006) in this study an experiment was conducted to determine biodegradability of test chemical by using Pseudomonas putida CCRC 14365 as inoculums. The stock cultures of Pseudomonas putida were stored at 4 ◦C. P. putida cells were activated at 30 ◦C in the nutrient medium, into which 1.06 mM of test chemical was added for enzyme adaptation for 24 h. Activated cells in the late-exponential phase were harvested as inoculums. The cells collected after centrifugation (6000 rpm) for 10 min were re-suspended in phosphate buffer and re-centrifuged. After cleaning, the activated cells were inoculated into the culture medium (250 mL) in 500-mL Erlenmeyer flasks to give an initial cell concentration of about 1.3×108 cells/mL. After inoculation, the flask was capped with cotton plugs and placed in a shaker controlled at 120 rpm and 30 ◦C. When the OD value of the pre-cultured cells reached 2.6–3.1, an aliquot of the culture was centrifuged at 6000 rpm and 4 ◦C for 10 min. To clean the cells, it was re-suspended in phosphate buffer and centrifuged. The cells were then transferred and inoculated in a 250-mL conical flask, to which 100 mL of the solution containing MSM and substrates was poured to give an initial OD of 0.064. The cells were cultivated at 30 ◦C and 100 rpm. The samples were withdrawn at suitable time intervals and, the cell density and the concentrations of test chemical were measured y using HPLC on a Phenomenex C18 column. The test chemical undergoes 100 % degradation in 15. On the basis percent degradability it concluded that test chemical is readily degradable.

 

Another experimental study was reviewed from peer reviewed Journal in this study the Biodegradation experiment was conducted by taking Pseudomonas putida CCRC 14365 as inoculums for degradation of test chemical P. putida cells were activated at 30 ◦C in the nutrient medium, into which 1.06 mM of test chemical was added for enzyme adaptation for 24 h. The activated cells in the late exponential phase were harvested as inoculums. The activated cells were inoculated into the culture medium (250 mL) in 500-mL Erlenmeyer flasks to give an initial cell concentration of around 1.3×108 cells/mL. After inoculation, the flask was capped with cotton plugs and placed in a shaker controlled at 120 rpm and 30 ◦C. As the OD value of adapted cells reached 2.6–3.1, an aliquot of the culture was centrifuged at 6000 rpm and 4 ◦C for 10 min.

To clean the biomass, it was re-suspended in phosphate buffer and centrifuged. The cells were then transferred and inoculated in a 250-mL conical flask, to which 100mL of solution containing MSM and the test chemical was poured to yield an initial OD of 0.064. The cells were cultivated at 30 ◦C and 100 rpm. Samples were withdrawn at suitable time intervals, and the concentrations of cells, test chemical were measured by using HPLC. The samples were subject to filtration through a Millipore filter (0.2[1]m) before the analysis of test chemical by HPLC on a Phenomenex C18 column (particle size, 5[1]m).A mixture of methanol (49%, v/v), acetic acid (1%, v/v), and water was used as the mobile phase, and the flow rate was 1.0 mL/min. An aliquot of 10µL of the sample was injected and analyzed using an UV detector (Jasco 975, Japan). The wavelength for test chemical was set at 240 nm. 

The test chemical undergoes 100 % degradation in 9 hrs 15 hrs and 56 hrs at different concentration of test chemical 0.53, 1.06, and 3.18 mM. On the basis percent degradability and time required for completed degradation of test chemical it concluded that test chemical is readily degradable.

 

 On the basis of results of all the experimental studies for test chemical it is concluded that test chemical is readily biodegradable