Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-06-21 to 2004-07-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Reference item:
The reference item and the following information were supplied by Fluke (BioChemika)
Identity: Sodium benzoate
Product No.: 71295
Lot No,: 432992/1
Purity: 99.8%
Expiration date: 2005-09-30
Storage conditions: In tightly closed original container, at room temperature at about 20°C
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenised aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

Based on this ratio, calculated amounts of wet sludge were suspended in test water to obtain a concentration equivalent to 4 g (±10%) dry material per litre. During holding, the sludge was aerated at room temperature until use. Prior to use, the sludge was diluted with test water to a concentration of 1 g/L (dry weight basis). This diluted activated sludge was used as inoculum to give a final concentration of 30 mgdry material/L.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Test water:
The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified water to obtain the following stock solutions:

a)
KH2PO4: 8.50 g/L
K2HPO4: 21.75 g/L
Na2HPO4x2H2O: 33.40 g/L
NH4Cl: 0.50 g/L
The pH of this solution was 7.4

b)
MgSO4x7H2O: 22.50 g/L

c)
CaCl2x2H2O: 36.40 g/L

d)
FeCl3x6H2O: 0.25g/L, stabilised with one drop of concentrated HCl/L

To obtain the final test water, 10 mL of stock solution a) and 1 mL each of stock solutions b) to d) were combined and made up to 1000 mL with purified water. The pH was adjusted from 7.8 to 7.4 with a diluted hydrochloric acid solution.

Test concentrations:
The test item was directly weighed into the test flasks. No emulsifiers or solvents were used.

The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 2.5 g sodium benzoate/L test water was prepared by dissolving 250 mg sodium benzoate completely in 100 mL of test water. From this, aliquots of 10 mL were added to the corresponding test flasks.

To each flask (with the exception of the abiotic control), activated sludge was added. Finally, the flasks were made up to a volume of 250 mL with test water (see Table 1 for test concentrations).

Conditions of the test:
Apparatus:
The test flasks (500 mL Erlenmeyer flasks, labelled with all necessary information to ensure unmistakable identification) were incubated under continuous stirring in a SAPROMAT D12 (Voith, GmbH, Heidenheim, Germany). Oxygen consumption was recorded manually by taking a daily reading at least each working day.

Principle:
Electro-chemical analysis process:
The biodegradation process consumes the dissolved oxygen in the liquid and generates CO2. The CO2 is adsorbed by soda lime and the total pressure decreases in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate solution.

Test duration: 28 days

Light conditions: Darkness

Test temperature: 22°C, maintained with a built-in-thermostat and checked once per week

pH: Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum (see Table 4). At the end of incubation, the pH was measured again in each test flask.
Reference substance:
benzoic acid, sodium salt
Key result
Parameter:
% degradation (O2 consumption)
Value:
ca. 2 - ca. 4
Sampling time:
28 d
Remarks on result:
other: The test substance was found not to be biodegradable under the conditions of the study within 28 days
Details on results:
The biochemical oxygen demand (BOD) of the test item in the test media was in the range of the biochemical oxygen demand of the inoculum controls throughout the study period of 28 days.

The test substance was consequently not found to be biodegradable under the conditions of the test within 28 days.

Abiotic control:
No degradation of the test item occurred in the abiotic control under the conditions of the test.
Results with reference substance:
For sodium benzoate, 87% and 85% biodegradation was attained in 14 and 28 days respectively.

Biodegradation of the reference item:

The results are presented in Tables 2 and 3.

The % biodegradation of the reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg O2/mg (ThOD).

In the procedure controls, the reference item was degraded by an average of 87% by exposure Day 14, confirming suitability of the activated sludge.

Biodegradation in the toxicity control:

In the toxicity control, containing both test item and the reference item, the course of biodegradation over 28 day exposure period was identical to that in the two procedure controls containing the reference item only (See Table 2).

Thus, the test item has obviously no inhibitory effect on activated sludge microorganisms.

Measurement of pH:

The results are presented in Table 4.

The pH measured in all flasks at the start of the test was 7.4. At the end of exposure (Day 28), pH values were between 7.3 and 7.8.

The tabulated values represent rounded results obtained by calculation using exact raw data

Table 2: Oxygen consumption in the test flasks

Time (days)

Cumulative oxygen consumption (mg/L)

Test item (flask No.)

Inoculum control (flask No.)

Procedure control (flask No.)

Abiotic control (flask No.)

Toxicity control (flask No.)

1

2

7

8

5

6

3

4

0

0

0

0

0

0

0

0

0

1

0

1

0

0

8

6

0

10

2

0

2

0

0

77

78

0

77

3

0

3

1

0

102

103

0

100

4

-

-

-

-

-

-

-

-

5

-

-

-

-

-

-

-

-

6

0

5

3

0

130

131

0

131

7

4

8

6

0

134

135

0

136

8

4

8

6

0

138

139

0

139

9

4

8

6

0

141

141

0

141

10

4

8

6

0

143

143

0

144

11

4

8

6

0

145

145

0

146

12

4

8

6

0

146

147

0

147

13

4

8

6

0

148

148

0

148

14

4

8

6

0

149

149

0

149

15

4

8

6

0

150

150

0

150

16

4

8

6

0

151

150

0

151

17

4

8

6

0

151

151

0

152

18

4

8

7

0

152

152

0

152

19

4

8

7

0

153

153

0

153

20

4

8

7

0

154

153

0

154

21

4

8

7

6

154

154

0

154

22

4

8

7

6

155

154

0

155

23

4

8

7

6

155

155

0

155

24

4

8

8

6

155

155

0

155

25

4

8

8

6

155

155

0

155

26

4

8

8

6

156

155

0

156

27

4

8

8

6

156

155

0

156

28

4

8

11

16

156

156

0

156

-No biodegradation

Table 3: Biodegradation of the reference item in the procedure and toxicity controls

Time (days)

Biodegradation*

Procedure control (Flask No.)

Toxicity control (Flask No.)

5

6

4

0

0

0

0

1

5

4

6

2

46

47

46

3

61

61

60

4

-

-

-

5

-

-

-

6

77

78

78

7

78

79

80

8

81

81

81

9

83

83

83

10

84

84

84

11

85

85

86

12

86

86

86

13

87

87

87

14

87

87

87

15

88

88

88

16

89

88

89

17

89

89

89

18

89

89

89

19

90

90

90

20

90

90

90

21

88

88

88

22

89

88

89

23

89

89

89

24

89

89

89

25

89

89

89

26

89

89

89

27

89

89

89

28

85

85

85

Mean (Day 28)

28

 

*Corrected for the mean oxygen uptake of the inoculum controls

-Not determined

Table 4: pH values at the start and at the end of the test

Flask No.

Identification

pH

Start

End

1

Test item

7.7

7.4

2

Test item

7.4

7.3

7

Inoculum control

7.4

7.4

8

Inoculum control

7.4

7.4

5

Procedure control

7.4

7.8

6

Procedure control

7.4

7.8

3

Abiotic control

7.4

7.4

4

Toxicity control

7.4

7.8

 

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
% Biodegradation of 2-4% in 28 days were obtained for the substance using a relevant test method and in compliance with GLP. The result is considered to be reliable.

Description of key information

Biodegradation in water: screening tests: 2 - 4% in 28 days (O2 consumption, OECD 301F)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

% Biodegradation of 2 - 4% in 28 days (O2 consumption) was obtained for the submission substance in accordance with OECD Test Guideline 301F and in compliance with GLP. The result is considered to be reliable.