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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:
November 02, 2016 - February 08, 2017
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:
17, July 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Species: Activated sludge, microorganisms from a domestic waste water treatment plant.
- Origin: Sewage plant for domestic sewage in Balatonfüred, Hungary (collection date: 27 October 2016).
- Storage length: 7 days
- Preparation: The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 5 g dry material per litre was mixed with mineral medium and then aerated under test conditions (for 7 days) until use. The pH of the activated sludge inoculum after preparation was 7.61, just before use the pH was: 7.3. A pH adjustment of activated sludge inoculum was not performed.
- Pre-conditioning: Consisted of aerating (2 L/minute) of activated sludge (in mineral medium, mineral medium ) for 7 days (from October 27 to November 03, 2016) at the test temperature (the actual temperature: 20.3 – 21.9 °C).
- Method for cell count of inoculum: The viability of the cultured sludge was determined by plating 0.1 mL of the different, usually 10-2, 10-3 and 10-4 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by these plating experiments by manual colony counting. The approximately cell count of aerated inoculum fell in the range of ~109/L; therefore on the day of the test this inoculum was diluted 10000 x with mineral medium to reach the necessary 105-106 cells/L cell concentration. After preparation the sludge was filtered through cotton wool. Pre-conditioning improves the precision of the method.
Pre-adaption: No. The inoculum was not pre-adapted to the test chemical.
Duration of test (contact time):
28 d
Initial conc.:
3 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:

Stock Solutions for Minreal Medium
In purified, deionised water analytical grade salts were added to prepare the following stock solutions:
A) Solution: KH2PO4 8.50 g
K2HPO4 21.75 g
Na2HPO4 x 12H2O 67.16 g
NH4Cl 0.50 g
Water ad 1000 mL
B) Solution: CaCl2 x 2 H2O 18.20 g
Water ad 500 mL
C) Solution: MgSO4 x 7 H2O 11.25 g
Water ad 500 mL
D) Solution: FeCl3 x 6 H2O 0.125 g
Water ad 500 mL

(The “D” stock solution was prepared on the day of the mineral medium preparation and was not further stored).

Preparation of Mineral Medium (Ratio of Ingredients): The mineral medium was prepared in the following ratio: 1 mL of the stock solutions A - D) were combined per 1000 mL total volume, filled with water (purified deionized) . The test medium was aerated for 20 minutes and allowed to stand for about 20 hours at the test temperature. The dissolved oxygen concentration was checked and found 8.76 mg/L. The pH of the mineral medium was 7.23.

- Additional substrate: No
- Solubilising agent: No
- Test temperature: 22 ± 2 °C (incubation: 20.0 - 20.3 °C; pre-conditioning: 20.3 - 21.9 °C)
- pH: 7.23
- pH adjusted: No
- Culturing apparatus: Incubator, climate chamber
- Number of culture flasks/concentration: 10 (+2 reserve)
- Method used to create aerobic conditions: Aeration system
- Test Units: Winkler bottles (300 mL, coded) with special neck and glass stoppers
- Continuous darkness: Yes

SAMPLING
- Sampling frequency: Temperature was measured continuously and recorded at least once a day; The oxygen concentration of test water (mineral medium) was measured at the start of the test.
- Sampling method: Temperature was measured using min/max thermometer (in controlled environment room) or built-in thermometer (in incubator); Oxygen and pH meter with appropriate O2 and pH electrode,
- Sample storage before analysis: No

CONTROL AND BLANK SYSTEM
- Inoculum blank: 10 (+2 reserve) bottles containing the filtered inoculum only)
- Procedure control: 10 (+2 reserve) bottles with the reference substance Sodium benzoate (at a concentration of 3.0 mg/L)
- Abiotic sterile control: Not performed
- Toxicity control: 10 (+2 reserve) bottles containing the test item, sodium benzoate and inoculum
Reference substance:
benzoic acid, sodium salt
Remarks:
ACROS
Preliminary study:
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 mg/L. No toxic effect of the test item was found at this investigated concentration. In the toxicity control containing both, the test item and the reference item, a mean of 36.6 % biodegradation was noted within 14 days.
Test performance:
The chosen test item concentration of 3.0 mg/L investigated in the main test was based on the results of the preliminary solubility and toxicity tests. The chemical oxygen demand (COD) of 1.99 mg O2/ mg test item of Leuco Sulfur Red 14 was determined at the start of the main experiment.

Under the test conditions ready biodegradation of this test item was not noticed. The percentage biodegradation of Leuco Sulfur Red 14 reached a mean of 5.9 % after 28 days based on its COD. Minimal biodegradation of the test item occurred in this study; therefore a biodegradability plateau was not determined. On the 7th day of the study 4.4 % biodegradation was obtained. From this day the slight changes were considered as being within the biological variability range of the applied test system.

The concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred (the changes in nitrite and nitrate concentration in the 14-day, 21-day and 28-day samples were caused likely by a technical effect: turbidity and/or discoloration). Therefore the biodegradability value of the test item was calculated based on its COD; any correction, based on the measured nitrite and/or nitrate content was not performed.

The reference item Sodium benzoate was sufficiently degraded to a mean of 79.0 % after 14 days, and to a mean of 88.7 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

In the toxicity control containing both, the test item and the reference item, a mean of 36.6 % biodegradation was noted within 14 days and 44.2 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days).
Key result
Parameter:
% degradation (O2 consumption)
Value:
5.9
Sampling time:
28 d
Details on results:
Under the test conditions the percentage biodegradation of Leuco Sulfur Red 14 reached a mean of 5.9 % after 28 days based on its COD. Minimal biodegradation of the test item occurred in this study; therefore a biodegradability plateau was not determined. On the 7th day of the study 4.4 % biodegradation was obtained. From this day the slight changes were considered as being within the biological variability range of the applied test system. The test item can be considered to be not readily biodegradable.
Key result
Parameter:
COD
Value:
1.99 mg O2/g test mat.
Results with reference substance:
The reference item Sodium benzoate was sufficiently degraded to a mean of 79.0 % after 14 days, and to a mean of 88.7 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum.

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

Treatment

Concentration

Bottle

mg O2/L after n days of exposure

[mg/L]

No.

0

7

14

21

28

Test item

 

1a

8.77

7.61

7.29

7.13

7.05

3.0

1b

8.75

7.59

7.27

7.07

7.02

 

mean

8.76

7.60

7.28

7.10

7.04

Reference item

 

2a

8.79

4.44

3.68

3.21

3.14

3.0

2b

8.78

4.12

3.51

2.99

2.81

 

mean

8.79

4.28

3.60

3.10

2.98

Inoculum control

3a

8.71

7.95

7.61

7.41

7.42

3b

8.76

7.72

7.38

7.36

7.30

mean

8.74

7.84

7.50

7.39

7.36

Toxicity control

Test item: 3.0
Reference item: 3.0

4a

8.81

4.01

3.55

2.79

2.64

4b

8.76

3.86

3.51

2.77

2.48

mean

8.79

3.94

3.53

2.78

2.56

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

Treatment

Concentration

Bottle

mg O2/L after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

3.0

1a

0.26

0.24

0.29

0.35

1b

0.26

0.24

0.33

0.36

Reference item

3.0

2a

3.45

3.87

4.23

4.28

2b

3.76

4.03

4.44

4.60

Toxicity control

Test item: 3.0
Reference item: 3.0

4a

3.90

4.02

4.67

4.80

4b

4.00

4.01

4.64

4.91

 

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 2)

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

mb0: oxygen concentration (mg/L) of inoculum blank on day 0 (mean of 3a and 3b from Table 2)

mbx: oxygen concentration (mg/L) of inoculum blank on day x (mean of 3a and 3b from Table 2)

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

Treatment

Concentration

Bottle

BOD after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

3.0

1a

0.09

0.08

0.10

0.12

1b

0.09

0.08

0.11

0.12

Reference item

3.0

2a

1.15

1.29

1.41

1.43

2b

1.25

1.34

1.48

1.53

Toxicity control

Test item: 3.0
Reference item: 3.0

4a

0.65

0.67

0.78

0.80

4b

0.67

0.67

0.77

0.82

 

BOD = = mg O2/mg T.i and/or R.i. where:

T.i.        =test item

R.i.       =reference item

i.control=inoculum control

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

Treatment

Concentration

Bottle

Percent of biodegradation after n days of exposure

[mg/L]

No.

7

14

21

28

Test item

 

1a

4.4

4.0

4.9

5.8

3.0

1b

4.4

4.0

5.5

5.9

 

mean

4.4

4.0

5.2

5.9

Reference item

 

2a

69.0

77.4

84.6

85.5

3.0

2b

75.2

80.6

88.8

91.9

 

mean

72.1

79.0

86.7

88.7

Toxicity control

Test item: 3.0
Reference item: 3.0

4a

35.6

36.6

42.6

43.7

4b

36.5

36.6

42.3

44.7

mean

36.0

36.6

42.4

44.2

Biodegradation % = where:

T.i.        =test item

R.i.       =reference item

i.control=inoculum control

CODof the test item = 1.99 mg O2/mg test item

ThODNH3of reference item = 1.67 mg O2/mg reference item

Nitrate Concentrations

Analytical occasions

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

1a

1b

3a

3b

4a

4b

0 day

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

7thday

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

14thday

<0.4

<0.4

<0.4

<0.4

<0.4

<0.4

21stday

0.6

0.6

<0.4

<0.4

0.6

0.5

28thday

0.6

0.7

0.6

0.6

0.7

0.7

Remarks:            LOQ of nitrate determination: 0.4 mg NO3/L

                              1a, 1b, 3a, 3b, 4a and 4b mean the bottle numbers.

 

 

 

Nitrite Concentrations

 

Analytical occasions

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

 

 

1a

1b

3a

3b

4a

4b

 

 

0 day

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

7thday

<0.03

<0.03

<0.03

<0.03

<0.03

<0.03

 

 

14thday

0.04

0.04

<0.03

<0.03

0.07

0.06

 

 

21stday

0.49

0.48

0.25

0.25

0.45

0.43

 

 

28thday

0.04

0.05

<0.03

<0.03

0.05

<0.03

 

Remarks:            LOQ of nitrite determination: 0.03 mg NO2/L

                              1a, 1b, 3a, 3b, 4a and 4b mean the bottle numbers.

 

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test item, Leuco Sulfur Red 14 was considered to be not readily biodegradable (5.9 % biodegradation on day 28). According to the test guidelines the pass level for ready biodegradability is 60 % of COD.
Executive summary:

The ready biodegradability of the test item was assessed under GLP conditions according to OECD Guideline OECD 301 D, EU Method C.4-E and US EPA OPPTS 835.3110. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item Sodium benzoate (at a concentration of 3.0 mg/L) was tested simultaneously under the same conditions as the test item, and functioned as a procedure control (reference control). Additionally inoculum (containing the filtered inoculum only) and toxicity (containing both the test item and reference item) controls were examined. The chosen test item concentration of 3.0 mg/L investigated in the main test was based on the results of the preliminary solubility and toxicity tests. The chemicaloxygen demand(COD) of 1.99 mg O2/ mg test item of the test item was determined at the start of the main experiment. In result, the percentage biodegradation ofLeuco Sulfur Red 14reached a mean of 5.9 % after 28 days based on its COD. Minimal biodegradation of the test item occurred in this study; therefore a biodegradability plateau was not determined. On the 7thday of the study 4.4 % biodegradation was obtained. From this day the slight changes were considered as being within the biological variability range of the applied test system. The concurrently conducted analytical determination of possible nitrite and nitrate development demonstrated that no nitrification occurred (the changes in nitrite and nitrate concentration in the 14-day, 21-day and 28-day samples were caused likely by a technical effect: turbidity and/or discoloration). Therefore the biodegradability value of the test item was calculated based on its COD; any correction, based on the measured nitrite and/or nitrate content was not performed. The reference item Sodium benzoate was sufficiently degraded to a mean of 79.0 % after 14 days, and to a mean of 88.7 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 36.6 % biodegradation was noted within 14 days and 44.2 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days). All validity criteria of the guidelines were fulfilled.

Description of key information

The substance was not readily biodegradable by OECD criteria (5.9 % biodegradation on day 28).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The ready biodegradability of the test item was assessed under GLP conditions according to OECD Guideline 301 D and GLP. The test item was exposed to activated sludge from the aeration tank of a domestic waste water treatment plant. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. The chosen test item concentration of 3.0 mg/L (10 bottles + 2 reserve; chemicaloxygen demand (COD) = 1.99 mg O2/ mg test item) investigated in the main test was based on the results of the preliminary solubility and toxicity tests. Besides analytical determination of possible nitrite and nitrate, a blank control (10 bottles + 2 reserve), a procedure control with the reference substance Sodium benzoate (at a concentration of 3.0 mg/L; 10 bottles + 2 reserve) and a toxicity control (containing both the test item and reference substance) were examined. In result, the percentage biodegradation of the test item reached a mean of 5.9 % after 28 days based on its COD. Minimal biodegradation of the test item occurred in this study; therefore a biodegradability plateau was not determined. On the 7thday of the study 4.4 % biodegradation was obtained. From this day the slight changes were considered as being within the biological variability range of the applied test system. As no nitrification occurred, biodegradability of the test item was calculated based on its COD; any correction, based on the measured nitrite and/or nitrate content was not performed. The reference item sodium benzoate was sufficiently degraded to a mean of 79.0 % after 14 days, and to a mean of 88.7 % after 28 days of incubation, based on ThODNH3, thus confirming the suitability of the used activated sludge inoculum. In the toxicity control containing both, the test item and the reference item, a mean of 36.6 % biodegradation was noted within 14 days and 44.2 % biodegradation after 28 days of incubation. Thus, the test item can be assumed to not inhibit the activated sludge microorganisms (higher than 25 % degradation occurred within 14 days). All validity criteria of the guidelines were fulfilled.