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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 December 2010 to 18 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD test guidance in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Reactive Red F08-0146
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Name: Activated sludge filtrate
Source: The secondary of Liede Sewage Treatment Plant of Guangzhou
Selection reason: The inoculum is recommended by "The Guidelines for the Testing of Chemicals"
Treatment: The sludge was removed any coarse particles and impurities on the surface, then was washed with the mineral medium (1100g, 10min, repeated for 5 times), the supernatant liquid phase was decanted and the solid material resuspended in a mineral medium. The sludge was mixed by vortex for 5min and filtered by qualitative filter paper. The filtrate was kept aerobic until use. 1mL of filtrate per liter of medium was inoculated in the test system
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
3.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test Medium
The test medium was prepared with the deionised water.

Instruments and equipments
Electronic balance: BSA223S Sartorius Scientific Instruments (Beijing) Co. Ltd.
Electronic balance: AG245 METTLER TOLEDO
High speed refrigerated: BECKMAN
Centrifuge: Avanti J-301
Magnetic stirrer: S21-1 Shanghai Sile Instrument Co. Ltd. Aeration pump IPX4 ACO-9610 Guangdong Haili Grpoup Co. Ltd.
Dissolved oxygen analyzer: WTW
Oxi 730

Test methods
Selection of the test method
The test substance is easily soluble in water, while the relevant physical and chemical properties of the test substance including the vapour pressure is unknown, so the ready biodegradability: closed bottle test was adopted.

Addition of the test substance
49.0mg test substance was dissolved in some test medium. The solution was transferred to 200ml volumetric flask. The stock solution was diluted with test medium to 200mL, and the stock solution with the concentration of 245mg/L was obtained.

Preparation of reference substance stock solution
50.0mg sodium benzoate was dissolved in some test medium, then transferred to 50mL volumetric flask. The stock solution was diluted with test medium to 50mL, and the stock solution with the concentration of 1000mg/L was obtained.

Test grouping
Test suspension: 20 BOD bottles containing test substance and inoculum
Inoculum blank: 20 BOD bottles containing only inoculum
Procedure control: 10 BOD bottles containing reference compound and inoculum
Toxicity control: 10 BOD bottles containing test substance, reference compound and inoculum

Preparation of test system
Addition of the test medium firstly: 6800mL, 6800mL, 4100mL and 4100mL of fully-aerated mineral medium was added to flask 1, flask 2, flask 3 and flask 4 respectively. The medium was ready for use after standing for 20h at 20°C
Addition of test substance stock solution: 100mL and 60mL test substance stock solution was added to flask 1 and flask 4 respectively
Addition of reference substance stock solution: 8.4mL reference substance stock solution was added to flask 3 and flask 4 respectively
Addition of activated sludge filtrate: 7.0mL, 7.0mL, 4.2mL and 4.2mL of activated sludge filtrate were added to flask 1, flask 2, flask 3 and flask 4
Addition of the test medium lastly: The final volumes of suspensions in flask 1, flask 2, flask 3 and flask 4 were made up to 7000mL, 7000mL, 4200mL and 4200mL respectively, by the addition of fully-aerated mineral medium. The suspensions were mixed uniformly.
Dispense reaction solutions: Reaction solutions were dispensed immediately into BOD bottles by hose from the 3/4 quarter under solution surface. All the BOD bottles were completely filled and were taped gently to remove any air bubbles.
Test substance concentration: 3.5mg/L
Reference substance concentration: 2.0mg/L
Concentration of inoculums: 1ml filtrate/L medium

Test conditions
All the BOD bottles were incubated at 20°C±2°C in the dark.

Analysis and determination
Duplicate bottles of all series were withdrawn for dissolved oxygen analysis firstly, and then for NO2¯-N and NO3¯-N analysis at time intervals (0d, 3d, 7d, 10d, 14d, 17d, 21d, 24d and 28d) over the 28 days incubation. Procedure and toxicity controls were analysed until 14d.
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
Not applicable.
Test performance:
The oxygen depletion of the inoculum blank (0.72mg/L) was lesser than 1.5mg/L on the 28th day.
The residual concentration of oxygen in the test bottles did not fall below 0.5 mg/L at any time.
The difference of replicate values of the removal of the test chemical at the end of the test was less than 20%.
The percentage degradation of the reference compound (73.2%) had reached the pass levels (60%) by day 14.
The percentage degradation of the toxicity control was 28.9% by day 14, greater than 25%. So the test was valid.
% Degradation
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
During the test period, the dissolved oxygen depletion in test suspensions was lower than the blank control, so there was no need to correct the dissolved oxygen concentration by nitrification.
During the test period, the measured minimum dissolved oxygen concentration was 6.16mg/L, which revealed that the residual concentration of oxygen in the test bottles were greater than 0.5mg/L. The oxygen depletion of the inoculum blank (0.72mg/L) was lesser than 1.5mg/L on the 28th day, which met the test requirement.
The percentage biodegradation of reference substance and the toxicity control was 73.2% and 28.9% respectively on the 14th day, which showed that the activity of the inoculum met the requirement of the test. Statistical analysis was carried out using t-test on the oxygen depletion at the end of the test for the control and test substance vessels to determine any significant differences between the test and control groups. The analysis results revealed that there were no statistically significant differences (P≥0.05) between the oxygen depletion for the test and group groups. So the test substance was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study.
The percentage biodegradation of parallel of the test substance were 0%, so the test substance was not degradable during the test.

BOD5 / COD results

Results with reference substance:
The percentage biodegradation of reference substance and the toxicity control was 73.2% and 28.9% respectively on the 14th day, which showed that the activity of the inoculum met the requirement of the test.

Any other information on results incl. tables

Table 1 – Composition of test systems in flasks

Test groups

Test suspension

Inoculum blank

Reference control

Toxicity control

Flask 1

Flask 2

Flask 3

Flask 4

Test medium added firstly (mL)

6800

6800

4100

4100

Test substance stock solution (mL)

100

-

-

60

Reference substance stock solution (mL)

-

-

8.4

8.4

Activated sludge filter (mL)

7.0

7.0

4.2

4.2

Test medium added lastly (mL)

93.0

193.0

87.4

27.4

Final volume (mL)

7000

7000

4200

4200

Test substance concentration (mL)

3.5

-

-

3.5

Reference substance concentration (mg/L)

-

-

2.0

2.0

 

Table 2 – NO2+-N, NO3+-N contents of the test suspension during the test

Time (d)

Test suspension (T)

Inoculum blank (B)

T1

T2

B1

B2

NO3¯-N

NO2¯-N

NO3¯-N

NO2¯-N

NO3¯-N

NO2¯-N

NO3¯-N

NO2¯-N

0

0.132

0.170

0.049

0.156

0.012

0.054

0.012

0.054

3

0.011

0.055

0.009

0.075

0.012

0.055

0.013

0.074

7

0.011

0.085

0.012

0.081

0.012

0.091

0.012

0.090

28

0.011

0.085

0.012

0.081

0.012

0.091

0.012

0.090

 

Table 3 – DO of the test suspensions during the test

Time (d)

Test suspension (T)

Inoculum blank (B)

Reference control (R)

Toxicity control (C)

T1

T2

B1

B2

R1

R2

C1

C2

0

8.55

8.55

8.65

8.62

8.57

8.65

8.61

8.60

3

8.92

8.97

9.09

9.08

7.09

7.13

7.14

7.06

7

8.89

8.92

8.97

8.96

6.84

6.86

6.57

6.58

10

8.91

8.99

8.78

8.73

6.46

6.41

6.46

6.48

14

9.54

9.63

8.65

8.68

6.16

6.17

6.87

6.77

21

9.40

9.42

8.89

8.66

-

-

-

-

28

8.54

8.47

8.31

8.25

-

-

-

-

 

Table 4 – Percentage biodegradation during the test

Test groups

Time (d)

7

14

21

28

Test substance

Percentage biodegradation (%)

-12.0

-13.1

-34.9

-38.1

-12.2

-9.7

-7.4

-3.5

Reference substance

Percentage biodegradation (%)

51.8

53.6

72.2

74.3

-

-

-

-

Average (%)

52.7

73.2

-

-

Toxicity control

Percentage biodegradation (%)

33.0

32.7

28.2

29.6

-

-

-

-

Average (%)

32.9

28.9

-

-

Note: The percentage was regarded as 0% when the calculated value was negative.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance cannot be considered to be readily biodegradable under the conditions of the study presently performed.
Executive summary:

Ready Biodegradability: Closed Bottle Test of Reactive Red F08-0146 was performed in compliance with the Good Laboratory Practice (GLP), the test was conducted according to the study plan and OECD Guidelines for the Testing of Chemicals, 301D Closed Bottle Test. Paris: OECD, Adopted 17 July 1992.

 

Analysis and determination

The dissolved oxygen, NO2¯-N and NO3¯-N concentrations of the test solution was measured at intervals.

 

Results

Consumed oxygen of inoculum blank (28d): 0.72mg/L

 

Percentage biodegradation

Reference substance (14d): 73.2%

Toxicity control (14d): 28.9%

Test substance (28d): 0%

 

Conclusion

The test substance cannot be considered to be readily biodegradable under the conditions of the study presently performed.