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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
Study period:
03-03-2020 to 21-08-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met with acceptable deviations.
Qualifier:
according to guideline
Guideline:
other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals, 301D Closed Bottle Test. Second edition. Beijing: China Environmental Science Press. 2013. 44-50.
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Deviations:
yes
Remarks:
Temperature exceeded guideline specified range of 22 ± 2°C. Applied range: 20 ± 1°C : 19.2 - 21.8°C. Not considered to impact reliability of the study.
Qualifier:
according to guideline
Guideline:
other: GB/T 21831-2008 Chemicals-Ready Biodegradability: Closed Bottle Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals (HJ/T 153-2004). Beijing: China Environmental Science Press. 2004.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: HJ 506-2009: Water quality - Determination of dissolved oxygen - Electrochemical probe method.
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Secondary effluent (batch number: IEw2020031 0-1) collected from the Datansha Sewage Treatment Plant of Guangzhou, The People’s Republic of China. Full details of the inoculum is included in the full study report. The secondary effluent of waste water treatment plant is recommended by test guideline.
- Storage conditions: See pretreatment field.
- Storage length: less than 7 days from sampling to test initiation.
- Preparation of inoculum for exposure: See pretreatment field.
- Pretreatment: The inoculum was filtered through a coarse filter paper to remove any coarse particles and impurities on the surface, then the filtrate was kept aerobic until required. When testing, 1.0 mL of secondary effluent per litre of test medium was added into the test system to give a final bacteria concentration of 10^4 – 10^6 CFU/L. The aerobic plate count presented in inoculum was 3.4 x 10^5 CFU per millilitre. Respectively, 7900 mL, 7900 mL, 7900 mL and 7900 mL of the test medium which had been fully-aerated for 2 h were added into flasks 1 to 4, and then stood for approximately 20 h. The test solutions were prepared according to Table 1. The reference substance stock solution and secondary effluent were respectively added into each flask and filled up to the final volume with test medium, and then these solutions were mixed uniformly. The pH values were measured and then the solutions were dispensed immediately. The prepared solutions were dispensed into the respective group of BOD bottles by hose from the lower quarter of the appropriate large bottle, and all the BOD bottles were completely filled, then tapped gently to remove any air bubbles, finally they were made airtight.
- Concentration of sludge: Not applicable.
- Initial cell/biomass concentration: 1.0 mL of secondary effluent per litre of test medium was added into the test system to give a final bacteria concentration of 10^4 – 10^6 CFU/L. The aerobic plate count presented in inoculum was 3.4 x 10^5 CFU per millilitre.
- Water filtered: Yes.
- Type and size of filter used, if any: The water for preparing medium and solution was made by the Water Purifier through deionisation.
Duration of test (contact time):
28 d
Initial conc.:
2.1 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Solution A [KH2PO4: 8.50 g; K2HPO4.3H2O: 28.50 g; Na2HPO4.12H2O: 67.15 g; NH4Cl: 0.50 g - 1L water – pH = 7.43]; Solution B [CaCl2: 13.75 g in 0.5L water]; Solution C [MgSO4.7H2O: 11.25 g in 0.5L water]; Solution D [FeCl3,6H2O: 0.125 g in 0.5L water]. 1 mL of solution A, B, C and D was mixed with appropriate deionised water then made up to 1 L with deionised water.
- Solubilising agent (type and concentration if used): None.
- Test temperature: 20 ±1 °C (actual: 19.2 - 21.8°C)
- pH: 7.4 (medium)
- pH adjusted: No. pH values of all vessels was to be: pH 7.4 ± 0.2 at the start of the test ; at the end of the test pH = 6.62 to 6.78 (test vessels) ; 6.87 to 6.88 (inoculum blank), 6.79 to 6.87 (procedure control) and 6.51 to 6.52 (toxicity control).
- Aeration of dilution water: Not reported
- Suspended solids concentration: Not applicable.
- Continuous darkness: Yes.

TEST SYSTEM
- Culturing apparatus: BOD bottles with continuous stirring (magnetic stirrer)
- Number of culture flasks/concentration: 22 BOD bottles (Test suspension: test item, silica gel and Inoculum) 18 BOD bottles (inoculum blank : silica gel only); 18 BOD bottles (Procedure control : reference item silica gel and inoculum) ; 22 BOD bottles (Toxicity control : silica gel, test item, reference item and inoculum)
- Test performed in closed vessels due to significant volatility of test substance: Yes, suspected volatile.
- Test performed in open system: No.

SAMPLING
- Sampling frequency: Duplicate bottles of all series were withdrawn for dissolved oxygen analysis at time intervals (0d, 4d, 7d, 11d, 14d, 21d, 28d) over the 28 days incubation. Procedure and toxicity controls were similarly analysed until day 28.
- Sampling method: Analyses of the dissolved oxygen concentration and/or test item determination (day 0 and day 28). The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter. The pH was measured using a pH meter. The temperature was measured and recorded with a thermometer connected to a data logger. Test item concentration at beginning and end of the test period was determined by HPLC.
- Sterility check if applicable: No.
- Sample storage before analysis: Not applicable.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes.
- Abiotic sterile control: No.
- Toxicity control: Yes.
- Other: Positive reference control (Sodium Benzoate).
Reference substance:
benzoic acid, sodium salt
Remarks:
2.0 mg/L
Test performance:
1. The endogenous respiration was 0.66 mg/L at day 28 (i.e. < 1.5 mgO2/L after 28 days)
2. The minimum concentration of dissolved oxygen during the test was measured as 0.65 mg/L, which met the condition that the residual concentration of oxygen in the test bottles should not fall below 0.5 mg/L.
3. The pH at day 28 was in the range of 6.0 to 8.5. Actual for controls and test item vessels: pH = 6.62 to 6.78 (test vessels) ; 6.87 to 6.88 (inoculum blank), 6.79 to 6.87 (procedure control) and 6.51 to 6.52 (toxicity control).
4. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item at the end of the 10/14-day window and/or on day 28.
5. The procedure control (Sodium Acetate) attained 81.0% degradation at 14 days which had reached the pass level (60% and 25% of ThOD), thereby confirming the suitability of the inoculum and test conditions.
6. The percentage biodegradation of the toxicity control was 61.5% at 14 days, which had reached the pass level (60% and 25% of ThOD).
Parameter:
% degradation (O2 consumption)
Value:
64.8
Sampling time:
28 d
Remarks on result:
other: mean degradation (n=2); 10-day window met
Details on results:
Results:
1. The two replicates of the test suspensions attained 68.3% and 61.4% degradation in 28 days, the mean degradation was 64.8%. The 10-day window criteria was met (start day 9 = 10% and end between day 12 and day 19. End day 19: 61.0%).
2. Toxicity control attained 61.5% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment microorganisms used in the study.
3. Reference item (Sodium Benzoate) attained 81.0% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
Validity Criteria:
1. The endogenous respiration was 0.66 mg/L at day 28 (i.e. < 1.5 mgO2/L after 28 days)
2. The minimum concentration of dissolved oxygen during the test was measured as 0.65 mg/L, which met the condition that the residual concentration of oxygen in the test bottles should not fall below 0.5 mg/L.
3. The pH at day 28 was in the range of 6.0 to 8.5. Actual for controls and test item vessels: pH = 6.62 to 6.78 (test vessels) ; 6.87 to 6.88 (inoculum blank), 6.79 to 6.87 (procedure control) and 6.51 to 6.52 (toxicity control).
4. The repeatability validity criterion (not more than 20% difference between replicates) is fulfilled for the flasks containing test item at the end of the 10/14-day window and/or on day 28.
5. The procedure control (Sodium Acetate) attained 81.0% degradation at 14 days which had reached the pass level (60% and 25% of ThOD), thereby confirming the suitability of the inoculum and test conditions.
6. The percentage biodegradation of the toxicity control was 61.5% at 14 days, which had reached the pass level (60% and 25% of ThOD).
7. The test temperature variation range was 19.2 - 21.8°C, which exceeded the OECD TG 301D normal range (20°C ± 2°C) although was within the test specified range of 20 ±1 °C. It was considered the test results met the quality requirements and the deviation did not affect the validity of the test results.
Results with reference substance:
Degradation of sodium benzoate exceeded 80 % after 14 days; actual mean (n=2) = 81.0 %: the activity of the inoculum was thus verified (validity criterion).

Table 2.0 : Replicate dissolved oxygen concentration (mg O2/L) and and (mean) calculated % biodegradation for test suspension, inoculum blank, reference control and toxicity control

 

0 days

1 days

4 days

7 days

11 days

14 days

21 days

28 days

Test Group

mgO2/L

% biodeg.

(mean)

mgO2/L

% biodeg.

mgO2/L

% biodeg.

mgO2/L

% biodeg.

mgO2/L

% biodeg.

mgO2/L

% biodeg.

mgO2/L

% biodeg.

mgO2/L

% biodeg.

Test Suspension

(T)

8.94

9.04

0.0

0.0

(0.0)

8.96

8.97

-0.1

-0.3

(0.0)

8.81

8.82

0.6

0.5

(0.6)

8.57

8.63

1.9

1.0

(1.4)

7.40

7.66

18.8

15.0

(16.9)

4.34

4.82

64.2

57.2

(60.7)

3.86

4.23

65.4

60.0

(62.7)

3.61

4.09

68.3

61.4

(64.8)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inoculum Blank

(B)

9.01

8.98

-

8.97

8.94

 

-

8.86

8.86

-

8.73

8.68

-

8.67

8.74

-

8.79

8.77

-

8.36

8.40

-

8.32

8.35

-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Procedure Control

(R)

9.01

9.01

0.0

0.0

(0.0)

7.10

7.06

56.0

57.2

(56.6)

6.38

6.53

74.7

70.2

(72.5)

6.24

6.21

74.2

752

(74.7)

6.10

6.03

78.4

80.5

(79.5)

6.09

6.09

81.0

81.0

(81.0)

6.05

5.96

70.2

72.9

(71.6)

5.59

5.69

82.6

79.6

(81.1)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Toxicity Control

(T)

9.00

8.94

0.0

0.0

(0.0)

8.75

8.81

1.8

1.2

(1.5)

6.60

6.47

21.8

23.1

(22.4)

5.98

6.21

26.3

24.1

(25.2)

4.08

4.42

44.9

41.6

(43.2)

2.61

2.30

60.0

63.0

(61.5)

1.22

1.14

69.6

70.4

(70.0)

0.68

0.65

74.4

74.7

(74.6)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Where biodegradation was reported as negative, this was reported as 0%

- = not applicable

( ) = mean value

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The mean biodegradation in duplicate was 64.8 % at day 28. The 10-day window criteria was met.
Executive summary:

The ready biodegradability test was carried out according to OECD TG 301D and China Guidelines for the Testing of Chemicals, Effects on Degradation and Accumulation, 301D Closed Bottle Test (2013) under GLP. The test item, at a concentration of 2.1 mg/L was exposed to secondary effluent micro-organisms obtained from the Datansha Sewage Treatment Plant of Guangzhou, China, with culture medium in sealed culture vessels in the dark at approximately 20°C ± 2°C (actual temperature: 19.2 to 21.8°C) for 28 days. The temperature deviation was not considered to impact the study. The secondary effluent of waste water treatment plant is recommended by test guideline. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The inoculum was filtered through a coarse filter paper to remove any coarse particles and impurities on the surface, then the filtrate was kept aerobic until required. When testing, 1.0 mL of secondary effluent per litre of test medium was added into the test system to give a final bacteria concentration of 10^4 – 10^6 CFU/L. The aerobic plate count presented in inoculum was 3.4 x 10^5 CFU per millilitre. Respectively, 7900 mL, 7900 mL, 7900 mL and 7900 mL of the test medium which had been fully-aerated for 2 h were added into flasks 1 to 4, and then stood for approximately 20 h. The test solutions were prepared. Reference substance stock solution and secondary effluent were respectively added into each flask and filled up to the final volume with test medium, and then these solutions were mixed uniformly. The pH values were measured and then the solutions were dispensed immediately. The prepared solutions were dispensed into the respective group of BOD bottles by hose from the lower quarter of the appropriate large bottle, and all the BOD bottles were completely filled, then tapped gently to remove any air bubbles, finally they were made airtight. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter. The pH was measured using a pH meter. The temperature was measured and recorded with a thermometer connected to a data logger. At the beginning of the study, the test item concentrations of the test suspension and the toxicity control were measured by HPLC. After the solution of the test suspension and the toxicity control were dispensed into BOD bottles, the first and the last BOD bottles of each group were taken out to stir for about 15 min, and then an appropriate amount of each solution was filtered through 0.45µm filter membrane, and the filtrate was determined after dilution twice with acetonitrile. At the end of the study, randomly chosen duplicate bottles of the test suspension and the toxicity control were fetched to do the same operation steps as day 0 to analyse the test item concentration. On day 14 of the test, percentage biodegradation of the reference substance (procedure control) and the toxicity control were 81.0% and 61.5%, respectively, reaching the pass level of 60% and 25% of theoretical oxygen demand (ThOD). The average oxygen depletion in the inoculum blank was 0.66 mg/L, which was less than 1.5 mg O2/L after 28 days. The minimum concentration of dissolved oxygen during the test was measured as 0.65 mg/L, meeting the condition that the residual concentration of oxygen in the test bottles should not fall below 0.5 mg/L. The difference of extremes of replicate values of the biodegradation of the test item during the test was less than 20%. All relevant validity criteria were met. The percentage biodegradation of the test item on day 28 were 68.3% and 61.4%, with a calculated mean of 64.8%. At the beginning and end of the test, the actual concentrations of the test item in the test suspension and the toxicity control were measured as 2.05 mg/L, 1.99 mg/L and 0.834 mg/L, 1.18 mg/L, respectively. The percentage decrease of the test item at the end of the test was 59.3% and 40.8%, respectively. The mean biodegradation for duplicate test flasks at 28 days for the test item was 64.8% (the 10-day window was met). Under the conditions of the study, test item is considered as readily biodegradable.

Description of key information

Biodegradation: readily biodegradable, mean biodegradation 64.8 % at 28 -days (10-day window met), OECD TG 301D, 2020

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable
Type of water:
freshwater

Additional information

Key study : OECD TG 301D, 2020 : The ready biodegradability test was carried out according to OECD TG 301D and China Guidelines for the Testing of Chemicals, Effects on Degradation and Accumulation, 301D Closed Bottle Test (2013) under GLP. The test item, at a concentration of 2.1 mg/L was exposed to secondary effluent micro-organisms obtained from the Datansha Sewage Treatment Plant of Guangzhou, China, with culture medium in sealed culture vessels in the dark at approximately 20°C ± 2°C (actual temperature: 19.2 to 21.8°C) for 28 days. The temperature deviation was not considered to impact the study. The secondary effluent of waste water treatment plant is recommended by test guideline. Control solutions with inoculum and the reference substance, sodium benzoate, together with a toxicity control were used for validation purposes. The inoculum was filtered through a coarse filter paper to remove any coarse particles and impurities on the surface, then the filtrate was kept aerobic until required. When testing, 1.0 mL of secondary effluent per litre of test medium was added into the test system to give a final bacteria concentration of 10^4 – 10^6 CFU/L. The aerobic plate count presented in inoculum was 3.4 x 10^5 CFU per millilitre. Respectively, 7900 mL, 7900 mL, 7900 mL and 7900 mL of the test medium which had been fully-aerated for 2 h were added into flasks 1 to 4, and then stood for approximately 20 h. The test solutions were prepared. Reference substance stock solution and secondary effluent were respectively added into each flask and filled up to the final volume with test medium, and then these solutions were mixed uniformly. The pH values were measured and then the solutions were dispensed immediately. The prepared solutions were dispensed into the respective group of BOD bottles by hose from the lower quarter of the appropriate large bottle, and all the BOD bottles were completely filled, then tapped gently to remove any air bubbles, finally they were made airtight. The dissolved oxygen concentrations were determined electrochemically using an oxygen electrode and meter. The pH was measured using a pH meter. The temperature was measured and recorded with a thermometer connected to a data logger. At the beginning of the study, the test item concentrations of the test suspension and the toxicity control were measured by HPLC. After the solution of the test suspension and the toxicity control were dispensed into BOD bottles, the first and the last BOD bottles of each group were taken out to stir for about 15 min, and then an appropriate amount of each solution was filtered through 0.45µm filter membrane, and the filtrate was determined after dilution twice with acetonitrile. At the end of the study, randomly chosen duplicate bottles of the test suspension and the toxicity control were fetched to do the same operation steps as day 0 to analyse the test item concentration. On day 14 of the test, percentage biodegradation of the reference substance (procedure control) and the toxicity control were 81.0% and 61.5%, respectively, reaching the pass level of 60% and 25% of theoretical oxygen demand (ThOD). The average oxygen depletion in the inoculum blank was 0.66 mg/L, which was less than 1.5 mg O2/L after 28 days. The minimum concentration of dissolved oxygen during the test was measured as 0.65 mg/L, meeting the condition that the residual concentration of oxygen in the test bottles should not fall below 0.5 mg/L. The difference of extremes of replicate values of the biodegradation of the test item during the test was less than 20%. All relevant validity criteria were met. The percentage biodegradation of the test item on day 28 were 68.3% and 61.4%, with a calculated mean of 64.8%. At the beginning and end of the test, the actual concentrations of the test item in the test suspension and the toxicity control were measured as 2.05 mg/L, 1.99 mg/L and 0.834 mg/L, 1.18 mg/L, respectively. The percentage decrease of the test item at the end of the test was 59.3% and 40.8%, respectively. The mean biodegradation for duplicate test flasks at 28 days for the test item was 64.8% (the 10-day window was met). Under the conditions of the study, test item is considered as readily biodegradable.