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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:
2017-06-14 to 2017-07-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Principles of method if other than guideline:
There was no deviation from the above mentioned guidelines.
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
SOURCE OF ACTIVATED SLUDGE
- activated sludge from the aeration tank of a domestic waste water treatment plant in Balatonfüred, Hungary, on 08 June 2017 (six days before the main test)

PREPARATION OF INOCULUM FOR EXPOSURE
- washed twice by centrifugation
- supernatant liquid phase was decanted
- solid material re-suspended in isotonic saline solution and again centrifuged
- after washing and final centrifugation mixed with test water and then aerated under test conditions (for 6 days) until use
- Concentration of sludge: 5 g dry material per litre
- pH of the activated sludge: after preparation was 7.13, just before use: 7.18

PRE-CONDITIONING OF ACTIVATED SLUDGE INOCULUM
- period: 08 June 14 June 2017
- aerating (2 L/minute) for 6 days at the test temperature (the actual temperature: 20.0 – 21.6 °C)
- cell count during pre-conditioning: ~108/L
- filtering with cotton wool after preparation
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Surrounding type: The test (formulation, oxygen and pH measuring) was carried out in a controlled environment room at a temperature of 22 +/- 2 °C according to the guideline.
- Test temperature: During the preparation, aeration and incubation of the mineral medium the temperature was 20.1 - 21.4 °C. During the incubation (28 days) of the test units the temperature range was the following: 20.0 - 20.1 °C. Temperature was measured continuously using a built-in thermometer of the thermostat and noted at least once a day.
- Photoperiod: Darkness
- pH-value of the test water: The pH was checked prior study start and found to be 7.22. pH adjustment was considered as not necessary
- Oxygen concentration of the test water: About 8 - 9 mg/L (was measured at the start of the test and found to be 8.48 mg/L)
- Recording: Test conditions were measured with suitable instruments and documented in the raw data

TEST GROUPS
- Test item: bottles 1a and 1b
- Procedure control: bottkes 2a and 2b, with reference item Sodium benzoate
- Inoculum control: bottles 3a and 3b
- Toxicity control: bottles 4a and 4b
- General: Microbial inoculum (2.0 mL per litre test medium) was added to each preparation bottle

TEST BOTTLES
- 16 (+ 2 reserve) bottles containing the test item and inoculum
- 16 (+ 2 reserve) bottles containing the reference item and inoculum (procedure control)
- 16 (+ 2 reserve) bottles containing only inoculum (inoculum control)
- 16 (+ 2 reserve) bottles containing the test item, reference item and inoculum (toxicity control)

TEST PARAMETERS
- Measurement of dissolved oxygen concentration
- Measurement of total oxidized N: (nitrite and nitrate)
- Measurement of temperature


VALIDITY CRITERIA
- Inoculum control: Oxygen depletion in the inoculum control does not exceed 1.5 mg O2/L after 28 days
- Oxygen Concentration: The residual oxygen concentration in the test bottles does not drop below 0.5 mg O2/L at any time; the difference of duplicate values for the degradation at the plateau, at the end of the test or at the end of the 10-d window is not greater than 20 %
- Reference item: The percentage degradation of the reference item reaches the level for ready biodegradability (> 60 %) by exposure
day 14



Reference substance:
benzoic acid, sodium salt
Preliminary study:
Yes. The test item concentration was chosen based on the preliminary test results and based on its theoretical oxygen demand. The test item solubility, behavior, and toxicity were tested in a 14-day preliminary experiment. The test design was the same as described at the main experiment. In the preliminary experiment the test item was investigated at the concentration of 3.0 mg/L. No toxic effect of the test item was found at this investigated concentration.
Test performance:
There was no deviation from the above mentioned guidelines.
Key result
Parameter:
% degradation (O2 consumption)
Value:
76
Sampling time:
28 d
Details on results:
The biodegradation of the reference item was not inhibited by the test item. For detailed information on results please see tables in "Any other information on results incl. tables".

VALIDITY OF THE STUDY

- Inoculum control: The oxygen depletion in the inoculum control did not exceed 1.5 mg O2/L after 28 days. It was 1.44 mg O2/L in average
- Oxygen concentration: The residual oxygen concentration in the test bottles did not drop below 0.5 mg O2/L at any time. (The lowest value was 0.54 mg O2/L, it was measured on the 14th and 21st days in the toxicity control.)
- Parallels: The difference of duplicate values for the degradation at the plateau, at the end of the test or at the end of the 10-d window was not greater than 20 %
- Reference item: The percentage degradation of the reference item reached the level for ready biodegradability (> 60 %) by exposure day 14. (The percentage degradation of the reference item was 71.1 % on the 14th day.)

Results with reference substance:
The reference item was sufficiently degraded to a mean of 71.1 % after 14 days, and to a mean of 79.6 % after 28 days of incubation, based on its ThODNH4.

Biodegradation of Toxicity Control

In the toxicity control containing both, the test item and the reference item, a mean of 65.1 % biodegradation was noted within 14 days and a mean of 62.8 % biodegradation was determined after 28 days of incubation.

Table 1: Dissolved Oxygen Concentrations at Different Time Intervals during the Exposure Period of 28 Days

Treatment 

Concentration

[mg/L]

 

Flask

No.

 

mg O2/L after n days of exposure         

0

1

2

5

7

14

21

28

Test item   

3.0

   

1a

8,62

7.99

7.90

5.22

4.61

3.41

2.99

2.88

1b

8,66

8.17

7.93

5.00

4.46

3.64

3.15

3.10

mean

8.61

8.08

7.92

5.11

4.54

3.53

3.07

2.99

Reference item   

3.0

   

2a

8.69

6.13

5.67

4.39

4.32

4.01

3.64

3.46

2b

8.69

6.07

5.3

4.48

4.15

3.76

3.37

3.07

mean

8.69

6.1

5.49

4.44

4.24

3.89

3.51

3.27

Inoculum control   

   

3a

8.68

8.13

8.09

7.62

7.58

7.41

7.32

7.25

3b

8.66

8.19

8.08

7.54

7.46

7.43

7.4

7.2

mean

8.67

8.16

8.09

7.58

7.52

7.42

7.36

7.23

Toxicity control   

Test item: 3.0

Reference item: 3.0

   

4a

8.64

5.62

5.03

1.02

0.62

0.54

0.54

0.57

4b

8.62

5.27

4.83

0.77

0.62

0.55

0.60

0.61

mean

8.63

5.45

4.93

0.90

0.62

0.55

0.57

0.59

Table 2: Oxygen Depletion at Different Time Intervals during the Exposure Period of 28 Days

Treatment 

Concentration [mg/L]

 

Flask No.

 

mg O2/L after n days of exposure

1

2

5

7

14

21

28

Test item 

3.0 

1a

0.12

0.14

2.31

2.86

3.96

4.32

4.30

1b

-0.08

0.09

2.51

2.99

3.71

4.14

4.06

Reference item 

3.0 

2a

2.05

2.44

3.21

3.22

3.43

3.74

3.79

2b

2.11

2.81

3.12

3.39

3.68

4.01

4.18

Toxicity control 

Test item: 3.0

Reference item: 3.0

 

4a

2.51

3.03

6.53

6.87

6.85

6.79

6.63

4b

2.84

3.21

6.76

6.85

6.82

6.71

6.57

oxygen depletion : (mt0- mtx) - (mbo- mbx), where:

mt0: oxygen concentration (mg/L) of test group on day 0 (1a, 2a, 4a and 1b, 2b, 4b from Table 1)

mtx: oxygen concentration (mg/L) of test group on day x (1a, 2a, 4a and 1b, 2b, 4b from Table 1)

mb0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b from Table 1)

mbx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b from Table 1)

Table 3: BOD at Different Time Intervals during the Exposure Period of 28 Days

Treatment  Concentration[mg/L]   FlaskNo.   BOD after n days of exposure       
1 2 5 7 14 21 28
Test item 

3.0

1a

0.04

0.04

0.77

0.95

1.32

1.44

1.43

1b

-0.03

0.03

0.84

1.00

1.24

1.38

1.35

Reference item 

3.0 

2a

0.68

0.81

1.07

1.07

1.14

1.25

1.26

2b

0.7

0.94

1.04

1.13

1.23

1.34

1.39

Toxicity control 

Test item: 10.0

Reference item: 3.0  

4a

0,42

0.50

1.09

1.15

1.14

1.13

1.10

4b

0.47

0.53

1.13

1.14

1.14

1.12

1.09

Table 4: Percentage Biodegradation at Different Time Intervals during the Exposure Period of 28 Days

Treatment

Concentration [mg/L]

Flask No.

Percent of biodegradation after n days of exposure

 

1

2

5

7

14

21

28

Test item

 

 

3.0

 

1a

2.2

2.5

42.0

52.0

72.1

78.6

78.1

1b

-1.5

1.5

45.7

54.4

67.5

75.3

73.8

mean

0.4

2.0

43.8

53.2

69.8

77.0

76.0

Reference item

 

3.0

 

2a

41.0

48.7

64.2

64.4

68.6

74.8

75.7

2b

42.2

56.1

62.4

67.8

73.6

80.2

83.5

mean

41.6

52.4

63.3

66.1

71.1

77.5

79.6

Toxicity control

Test item: 3.0

Reference item: 3.0

 

 

4a

23.9

28.8

62.2

65.5

65.3

64.7

63.1

4b

27.1

30.5

64.4

65.3

65.0

63.9

62.5

mean

25.5

29.7

63.3

65.4

65.1

64.3

62.8

Table 5: Nitrate Concentrations

Analytical measurements of the xth day samples

 

Measured nitrate concentration (mg/L) in the test bottles

1A

1B

3A

3B

4A

4B

Day 0

< 0.4

 ---

 ---

 ---

< 0.4

 ---

7th day

 ---

< 0.4

 ---

< 0.4

< 0.4

 ---

14th day

< 0.4

< 0.4

 ---

 ---

 ---

 ---

21st day

0.7

1.2

0.6

0.7

---

---

28th day

0.9

1.4

0.9

0.7

---

---

Remark: 1A, 1B, 3A, 3B, 4A and 4B are the Bottle numbers.

Limit of Quantification (LOQ) = 0.4 mg/L

--- : nitrate concentrations were not detectable.

Table 6.: Nitrite Concentrations

Analytical measurements of the xth day samples

 

Measured nitrite concentration (mg/L) in the test bottles

1A

1B

3A

3B

4A

4B

Day 0

---

---

---

---

---

---

7th day

---

---

---

---

---

---

14th day

0.05

0.08

0.03

---

---

---

21st day

0.39

0.41

0.38

0.39

---

---

28th day

0.71

0.61

0.81

0.76

---

---

Remark: 1A, 1B, 3A, 3B, 4A and 4B are the bottle numbers.

Limit of Quantification (LOQ) = 0.03 mg/L

--- : nitrite concentrations were not detectable.

Table 7.: Quality Control Samples

Analytical occasions

 

 

 

Nitrite

 

 

Nitrate

 

Nominal concentration 

 

Nominal concentration 

0.05 mg/L

0.5 mg/L

2 mg/L

0.5 mg/L

2 mg/L

 (Measured concentrations mg/L)

7th day

0.05

0.50

2.00

0.5

2.2

21th day

0.05

0.49

2.00

0.5

2.0

28th day

0.05

0.51

2.01

0.5

2.1

Measurements of the QC samples were performed to provide information about the method applicability.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The ready biodegradability of the test item was investigated according to the guidelines OECD TG 301 D, EU Method C.4.E and OPPTS 835.3110. The test item is classified as ready biodegradable as the pass level of 60% degradation was reached in a 10-d-window within the 28 days period of the study (76.0 % biodegradation in 28 days). The toxicity control showed that the test item is not inhibitory at the tested concentration of 3.0 mg/L.
Executive summary:

The study was conducted under GLP-conditions, according to the guidelines OECD TG 301 D (Closed Bottle Test), EU Method C.4.E and OPPTS 835.3110, in order to check the rate of biodegradation [%] of the test item by determination of oxygen uptake of the microorganisms during exposure. The test was carried out over a period of 28 days with non-adapted activated sludge from a municipal sewage treatment plant. The activated sludge was pretreated by washing with isotonic saline solution, resuspension in mineral salts medium and aeration for 6 days at the test temperature. 2.0 mL/L test medium were used for inoculation (cell concentration of pre-conditioned inoculum: 104‑105cells/L). The test item was tested in duplicates at a concentration of 3.0 mg/L, which corresponds to a ThODNH4 of 5.49 mg O2/L. A procedure control with the reference substance sodium benzoate and also a toxicity control, containing the test item as well as the reference item was performed simultaneously. In result, the test item replicates exceeded the 10 % level at the third day of the test and passed the level of 60 % on about the tenth day. Futhermore, after 28 days a biodegradation of 76.0 % % (based on its ThODNH4) was reached. The biodegradation of the procedure control passed the level of 60 % after 5 days and after 28 days a biodegradation of 79.6% was determined. In the toxicity control the biodegradation came to 62.8 % after 28 days, thus no inhibtion of biodegradation by the test item was observed. In conclusion, all validity criteria were fulfilled. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. The test item is regarded to be in the 10-d-window and after 28 days readily biodegradable.

Description of key information

In a Closed Bottle Test conducted according to OECD TG 301 D, EU Method C.4.E and OPPTS 835.3110, the test item was determined to be readily biodegradable (5.2.1 -1).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A Closed Bottle Test was conducted under GLP-conditions, according to the guidelines OECD TG 301 D, EU Method C.4.E and OPPTS 835.3110, in order to check the rate of biodegradation [%] of the test item 6-Aminocaproic acid by determination of oxygen uptake of the microorganisms during exposure (5.2.1 -1). The test was carried out over a period of 28 days with non-adapted activated sludge from a municipal sewage treatment plant. The activated sludge was pretreated by washing with isotonic saline solution, resuspension in mineral salts medium and aeration for 6 days at the test temperature. 2.0 mL/L test medium were used for inoculation (cell concentration of pre-conditioned inoculum: 104‑105cells/L). The test item was tested in duplicates at a concentration of 3.0 mg/L, which corresponds to a ThODNH4of 5.49 mg O2/L. A procedure control with the reference substance sodium benzoate and also a toxicity control, containing the test item as well as the reference item was performed simultaneously. In result, the test item replicates exceeded the 10 % level after three days and passed the level of 60 % on day 10. Futhermore, after 28 days a biodegradation of 76.0 % (based on its ThODNH4) was reached. The biodegradation of the procedure control passed the level of 60 % after 5 days and after 28 days a biodegradation of 79.6% was determined. In the toxicity control the biodegradation came to 62.8 % after 28 days, thus no inhibtion of biodegradation by the test item was observed. In conclusion, all validity criteria were fulfilled. The test item is regarded to be in the 10-d-window and after 28 days readily biodegradable.