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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:
2017-10-10 to 2017-11-07
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)
Version / remarks:
adopted on 17 July 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
January 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Activated sludge, microorganisms from a domestic waste water treatment plant in Balatonfüred, Hungary, on 03 October 2017 (seven days before the main test).
- Preparation of inoculum for exposure: washed by centrifugation, supernatant liquid phase decanted, solid material re-suspended in isotonic saline solution and again centrifuged (procedure repeated twice); aliquot of final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined; washed sludge suspension, corresponding to 5 g dry material per litre mixed with test water (mineral medium) and then aerated under test conditions (for 7 days) until use; pH adjustment of activated sludge inoculum was not performed.
- Pre-conditioning (03-10 October 2017): Aeration (2 L/minute) of activated sludge (in mineral medium) for 7 days at the test temperature.
- Concentration of sludge: 2.0 mL per litre test medium
- Initial cell/biomass concentration: 104 - 106 cells/L
- Sludge filtered: Yes (after preparation)
- Type of filter used: Cotton wool
Duration of test (contact time):
28 d
Initial conc.:
7 mg/L
Based on:
ThOD
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: In water (purified deionised) analytical grade salts were added to prepare the following stock solutions

a)
KH2PO4, 8.50 g
K2HPO4, 21.75 g
Na2HPO4 x 12H2O 67.16 g1
NH4Cl 0.50 g
Water ad. 1000 mL

b)
CaCl2 x 2H2O 36.40 g
Water ad. 1000 mL

c)
MgSO4 x 7 H2O 22.50 g
Water ad. 1000 mL

d)
FeCl3 x 6H2O 0.125 g
Water ad. 500 mL2

- Additional substrate: No
- Test temperature: 20.0 - 20.1 °C
- pH: 7.38 at start of the test
- pH adjusted: No
- Aeration of mineral medium: Yes
- Continuous darkness: Yes
- Oxygen concentration of the test water: 8-9 mg/L

TEST SYSTEM
- Culturing apparatus: Incubator with thermometer with exclusion of light
- Number of culture flasks/concentration: 16 (+2 reserve)
- Method used to create aerobic conditions: Aeration of mineral medium at preparation test solutions
- Measuring equipment: Built-in thermometer, O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method], pH meter with appropriate pH electrode
- Test performed in closed vessels: Yes, closed with glass stoppers

SAMPLING
- Sampling frequency: Oxygen measurements were performed in all duplicate bottles in all groups on days 0, 2, 5, 7, 12, 14, 21 and 28.
- Sampling method: The oxygen concentration was measured with an O2 electrode [working based on LDO (Luminescent Dissolved Oxygen) method].

CONTROL AND BLANK SYSTEM
- Inoculum blank: 16 replicates (+ 2 reserve)
- Positive control: 16 replicates (+ 2 reserve)
- Toxicity control: 16 replicates (+ 2 reserve)
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Not performed.
Test performance:
VALIDITY OF THE TEST

- 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.

The test is valid. There was no deviation from the guidelines and the study plan in this study.
Key result
Parameter:
% degradation (O2 consumption)
Value:
81.6
Sampling time:
28 d
Details on results:
The highest biodegradation value for the test item of 83.1 % was obtained on the 21st day of the test. The biodegradation curve reached its plateau about on the 7th day, from that day slight increases in biodegradability values occurred; however for informative reason the test was not stopped before the 28th day. The test item biodegradation of 66.6 % was above the pass level of 60 % of ThODNH4 already on the fifth day of the test consequently fulfilled the criterion for ready biodegradability since it surely reached the pass level of 60 % of ThODNH4 in a 10-day window within the 28-d period of the test.
Results with reference substance:
The percentage degradation of the reference item reaches the level for ready biodegradability (> 60 %) by exposure day 14.

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

Treatment

Concentration

[mg/L]

Flask

mg O2/L after n days of exposure

No.

0

2

5

7

12

14

21

28

Test item

7.0

1a

9.29

6.37

5.08

3.90

3.75

3.71

3.68

3.72

1b

9.30

6.42

4.91

4.14

3.85

3.90

3.87

3.84

mean

9.30

6.40

5.00

4.02

3.80

3.81

3.78

3.78

Reference item

3.0

2a

9.27

6.43

4.90

4.69

3.88

3.65

3.57

3.12

2b

9.29

6.18

5.14

4.23

3.95

3.78

3.71

3.65

mean

9.28

6.31

5.02

4.46

3.92

3.72

3.64

3.39

Inoculum control

3a

9.21

8.78

8.37

7.96

7.91

7.96

7.91

7.81

3b

9.24

8.76

8.26

7.88

7.92

7.76

7.96

7.92

mean

9.23

8.77

8.32

7.92

7.92

7.86

7.94

7.87

Toxicity control

Test item: 7.0
Reference item: 3.0

4a

9.31

4.66

2.94

1.78

1.58

1.39

1.38

1.29

4b

9.29

4.52

3.07

1.93

1.31

1.34

1.35

1.31

mean

9.30

4.59

3.01

1.86

1.45

1.37

1.37

1.30

 

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

Treatment

Concentration

[mg/L]

Flask

mg O2/L after n days of exposure

No.

2

5

7

12

14

21

28

Test item

7.0

1a

2.47

3.30

4.09

4.23

4.22

4.32

4.21

1b

2.43

3.48

3.86

4.14

4.04

4.14

4.10

Reference item

3.0

2a

2.39

3.46

3.28

4.08

4.26

4.41

4.79

2b

2.66

3.24

3.76

4.03

4.15

4.29

4.28

Toxicity control

Test item: 7.0
Reference item: 3.0

4a

4.20

5.46

6.23

6.42

6.56

6.64

6.66

4b

4.32

5.31

6.06

6.67

6.59

6.65

6.62

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]

Flask

BOD after n days of exposure

No.

2

5

7

12

14

21

28

Test item

7.0

1a

0.35

0.47

0.58

0.60

0.60

0.62

0.60

1b

0.35

0.50

0.55

0.59

0.58

0.59

0.59

Reference item

3.0

2a

0.79

1.15

1.09

1.36

1.42

1.47

1.60

2b

0.88

1.08

1.25

1.34

1.38

1.43

1.43

Toxicity control

Test item: 7.0
Reference item: 3.0

4a

0.42

0.55

0.62

0.64

0.66

0.66

0.67

4b

0.43

0.53

0.61

0.67

0.66

0.67

0.66

 

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

Treatment

Concentration

[mg/L]

Flask

Percent of biodegradation after n days of exposure

No.

2

5

7

12

14

21

28

Test item

7.0

1a

48.4

64.8

80.3

83.1

82.8

84.9

82.7

1b

47.7

68.4

75.8

81.4

79.3

81.4

80.6

mean

48.0

66.6

78.0

82.2

81.1

83.1

81.6

Reference item

3.0

2a

47.7

69.2

65.5

81.6

85.1

88.2

95.8

2b

53.1

64.8

75.1

80.6

82.9

85.8

85.6

mean

50.4

67.0

70.3

81.1

84.0

87.0

90.7

Toxicity control

Test item: 7.0
Reference item: 3.0

4a

35.1

45.6

52.0

53.6

54.8

55.5

55.6

4b

36.1

44.4

50.6

55.7

55.0

55.6

55.3

mean

35.6

45.0

51.3

54.7

54.9

55.5

55.5

 

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item is considered to be ready biodegradable, since a mean biodegradation of 81.6 % was reached after 28 days based on its ThODNH4. The 10-day criterion was fulfilled and all validity criteria of the guidelines were met.
Executive summary:

To determine the ready biodegradability of the test item, a GLP-compliant Closed Bottle Test according to OECD Guideline 301 D, EU Method C.4-E and EPA OPPTS 835-3110 was performed. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 oC) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item was investigated at the concentration of 7.0 mg/L, chosen based on its theoretical oxygen demand (ThODNH4, assuming that no nitrification occurs) of 0.73mg O2/mg. In parallel (under the same conditions as the test item), positive reference item, sodium benzoate at the concentration of 3.0 mg/L (as procedure control), inoculum control and toxicity control were investigated. In result, the percentage biodegradation of the test item reached a mean of 81.6 % after 28 days based on its ThODNH4. The 10-day window criterion for ready biodegradation was unequivocally fulfilled; more than 60 % biodegradation of the test item (66.6 %, based on its ThODNH4) was observed on the fifth day of the test. The reference item sodium benzoate was sufficiently degraded to a mean of 84.0 % after 14 days, and to a mean of 90.7 % after 28 days of incubation, based on ThODNH4. In the toxicity control containing both, the test item and the reference item, a mean of 54.9 % biodegradation was noted within 14 days and 55.5 % biodegradation after 28 days of incubation. In conclusion, the test item is considered to be ready biodegradable, since fulfilled the pass level for ready biodegradability that is the removal of 60 % ThODNH4in a 10-day window. The percentage biodegradation of the reference item confirms the suitability of the used activated sludge inoculum. According to the test guidelines, the test item can be assumed as not inhibitory at the applied concentration level on the activated sludge microorganisms because the degradation in the toxicity control group was higher than 25 % within 14 days. All validity criteria of the guidelines were met.

Description of key information

The substance is readily biodegradable (according to OECD criteria).

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

To determine the ready biodegradability of the test item, a GLP-compliant Closed Bottle Test according to OECD Guideline 301 D, EU Method C.4-E and EPA OPPTS 835-3110 was performed. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant in completely full and closed bottles in the dark at controlled temperature (22 ± 2 oC) for 28 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The test item was investigated at the concentration of 7.0 mg/L. In parallel, a procedure control, inoculum control and toxicity control were conducted. In result, the percentage biodegradation of the test item reached a mean of 81.6 % after 28 days based on its ThODNH4. The 10-day window criterion for ready biodegradation was unequivocally fulfilled; more than 60 % biodegradation of the test item (66.6 %, based on its ThODNH4) was observed on the fifth day of the test. The procedure confirmed the suitability of the used activated sludge inoculum and no indications for inhibitory at the applied concentration level on the activated sludge microorganisms were noted. In conclusion, the test item is considered to be ready biodegradable, since fulfilled the pass level for ready biodegradability that is the removal of 60 % ThODNH4in a 10-day window. All validity criteria of the guidelines were met (reference 5.2.1 -1).