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Environmental fate & pathways

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:
2009-12-18 - 2010-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 7827 (Evaluation in an Aqueous Medium of the "Ultimate" Aerobic Biodegradability of Organic Compounds - Method by Analysis of Dissolved Organic Carbon (DOC))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
State Office for the Environment, Water and Trade Control, Mainz, Germany
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
- Name of test material (as cited in study report): Acetic acid,oxo-,sodium salt (FeNaEDDHA)
- Physical state: Solid / dark red
- Analytical purity: 100 % (UVCB)
- Storage condition of test material: Storage at room temperature under light exclusion
Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge from a wastewater treatment plant, treating municipal sewage
Details on inoculum:
- Source of inoculum/activated sludge: wastewater treatment plant of Mannheim, Germany.
- Laboratory culture: The inoculum was collected on 04.01.2010 from the aeration tank of the plant and aerated in the laboratory until use.
- Method of cultivation: A suitable aliquot of the activated sludge suspension was sieved by a finely woven mesh with a mesh size about 1 mm. The activated sludge suspension was pre-aerated for about 24 h at a temperature of 22 +/- 2 °C.
- Preparation of inoculum for exposure: On day of exposure the sludge was allowed to settle. After settling the supernatant was discarded and the remaining sludge suspension was filled up with tap water. The sludge of the suspension was adjusted to a concentration of 6.0 g/L dry weight.
- Concentration of sludge: Aliquots of 5 mL were added to the test vessels to obtain a sludge concentration of 30 mg/L dry substance.
Duration of test (contact time):
28 d
Initial conc.:
20 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST CONDITIONS
Mineral medium: The used medium complies with the test guideline OECD 301A
Solution A: 8.50 g KH2PO4, 21.75 g K2HPO4, 33.40 g Na2HPO4 × 2 H2O, 0.50 g NH4Cl
The compounds were dissolved with demineralised water to 1000 mL; the pH value was adjusted to 7.4.
Solution B: 36.40 g CaCl2 × 2 H2O - dissolved in demineralised water to 1000 mL
Solution C: 22.50 g MgSO4 × 7 H2O - dissolved in demineralised water to 1000 mL
Solution D: 0.25 g FeCl3 × 6 H2O - dissolved in demineralised water to 1000 mL
10 mL solution A, 1.0 mL solution B, 1.0 mL solution C and 1.0 mL solution D was used for the preparation of the test assays.

TEST SYSTEM
The DOC Die Away test was performed in 2L conical flaks filled up to a volume of 1000 mL. For preparation of the test assays, first the required volume of demineralized water and the solutions of mineral medium were dosed to the test vessels. The blank control assays (BC) contain only mineral medium. The test substance assays (TS) contain the test substance with concentration of 20 mg/L DOC; the reference substance assay (RS) contains aniline with a concentration of 20 mg/L DOC. The assay for inhibition control (IH) contains the test substance and the reference substance in the same concentration in relation to its dissolved organic carbon contents. The test of physical chemical elimination (PC) was prepared with test substance in the concentration of 20 mg/L DOC.
For avoidance of bacterial grow the assay was inhibited by adding of 10 mg/L mercury chloride. The test assay of adsorption control (AC) was prepared in the same way. The pH values of all test assays were measured and adjusted to 7.4 +/- 0.2 if necessary. Aliquots of the prepared inoculum suspension were added to all test vessels (exceptional PC- assay); to adjust a concentration of activated sludge of 30 mg/L dry weight. The test assays were
carefully mixed before sampling for DOC-measurement at begin of exposure. The sampling in the AC test assay was performed before adding of the inoculum. For aeration the test vessels were shaken at a temperature of 22 +/- 2°C using an orbital shaker. The DOC measurement was performed twice per week, these DOC-values were compared with the values from begin of exposure. The decrease of DOC concentration was expressed as biodegradation degree and plotted by a biodegradation curve.
Reference substance:
aniline
Preliminary study:
The test guideline recommends a test concentration of the test substance of 10 - 40 mg/L Dissolved Organic Carbon (DOC). A test concentration of 20 mg/L DOC was used. The selected test concentration of 20 mg DOC/L corresponds to approximately 62 mg/L Acetic acid, oxo-, sodium salt.
It is anticipated that the selected test concentration causes no toxic effects to the microorganisms. The selected test concentration was tested in an additional inhibition control test assay and no toxic effects to the microorganisms were observed.
Test performance:
No unusual observations during test or any other information affecting results were reported.
Parameter:
% degradation (DOC removal)
Value:
> 10 - < 20
Sampling time:
28 d
Remarks on result:
other: mean value of two test assays
Details on results:
The test substance is considered as not readily biodegradable according to the OECD criteria.
Results with reference substance:
The reference substance (aniline) attained 91 % degradation after 14 days, based on DOC.

ACCOUNT OF TEST RESULTS

Test substance: Acetic acid.oxo-.sodium salt

Stock Solution, initial weight: 1233.7 mg/L

Dissolved Organic Carbon (DOC): 403 mg/L

DOC conc. at the test (nominal): 20 mg/L

Reference substance: aniline

Stock Solution, initial weight: 400.5 mg/L

Theoretical Organic Carbon (ThOC): 310 mg/L (calculated by initial weight)

DOC conc. at the test (nominal): 20 mg/L

 

Sample

BC1

BC2

RS

IH

PC

AC

TS1

TS2

Test substance concentration [mg/L] |

-

-

-

61.7

61.7

61.7

61.7

61.7

Inoculum conc. [mg/L dry matter]

30

30

30

30

-

30

30

30

Demineralized water [mL/vessel]

982

982

917

867

936

931

932

932

Mineral

solution A

10

10

10

10

10

10

10

10

medium

solution B

1

1

1

1

1

1

1

1

[mL/L]

solution C

1

1

1

1

1

1

1

1

solution D

1

1

1

1

1

1

1

1

Stock Solution; test substance [mL]

-

-

-

50

50

50

50

50

Stock Solution; reference subst.[mL]

-

-

65

65

-

-

-

-

Mercury chloride sol.| [mL/vessel]

-

-

-

-

1

1

-

-

pH values before;
after correction

7.8

7.8

7.8

7.7

7.7

7.7

7.7

7.8

7.4

7.4

7.4

7.4

7.4

7.4

7.4

7.4

Inoculum (6 g/L) [mL/vessel]

5

5

5

5

-

5

5

5

Total test volume [mL]

1000

1000

1000

1000

1000

1000

1000

1000

 

Legend: BC = blank control; RS = reference substance; IH = inhibition control; PC = abiotic control; AC = adsorption control; TS = test substance

Removal of Dissolved Organic Carbon (DOC) [%]:

Test duration [days]

RS

IH

PC

AC

TS1

TS2

TS mv

0

0

0

0

0

0

0

0

1

-1

0

-5

3

-1

1

0

3

10

12

-3

7

7

5

6

7

96

53

1

7

9

10

10

10

92

52

-2

 

8

5

7

14

91

52

3

 

7

5

6

17

95

55

-3

 

12

12

12

21

93

54

1

 

8

6

7

24

95

57

6

 

13

13

13

27

98

59

3

 

17

16

17

28

97

57

4

 

11

10

11

Legend: BC = blank control; RS = reference substance; IH = inhibition control; PC = abiotic control; TS = test substance, AC = adsorption control; mv = mean value

Validity criteria fulfilled:
yes
Remarks:
All validity criteria are fulfilled according to the applied OECD Guideline.
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test material attained 10 - 20 % degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301A. The study was conducted under certificated GLP compliance and a well documented study report is available.
Executive summary:

The DOC Die-Away Test is a batch test method to determine the ultimate aerobic biodegradability of a test substance in water. Mixtures of the test substance, a defined inorganic medium and a non pre-adapted inoculum (e.g. activated sludge or effluent from a municipal wastewater treatment plant) are incubated and aerated at a temperature of 22 +/- 2 °C for up to 28 days (Schwarz, 2010). Samples are taken at regular intervals to determine the Dissolved Organic Carbon concentration (DOC). The mean value of two test assays was used to determine the final biodegradation rate after the regulary exposure time. Thereby the test substance attained 10 - 20 % degradation. Based on the co-incubation with the reference substance anilin, it could be demonstrated that the test concentration used (20 mg/L DOC) was not toxic (inhibitory effect) on the sewage treatment bacteria flora. Anilin attained 91 % degradation after 14 days, indicating that the test system was suitable. In this experiment, the test substance was poorly biodegradable and is therefore considered as "not readily biodegradable" according to OECD criteria.

Description of key information

Not readily biodegradable: 10 - 20 % degradation after 28 days (source substance Fe(Na)EDDHA: Biodegradation, OECD 301A).

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

No experimental investigations concerning the biodegradability of o-o-EDDHA (EC 240 -505-9) are available. Therefore, read-across from the structurally related substance Fe(Na)EDDHA (CAS 84539-55-9) was performed (BASF, 2010; Study No. 21G0753/093217). The DOC Die-Away Test is a batch test method to determine the ultimate aerobic biodegradability of the read-across substance Fe(Na)EDDHA in water. Mixtures of the test substance, a defined inorganic medium and a non pre-adapted inoculum (e.g. activated sludge or effluent from a municipal wastewater treatment plant) are incubated and aerated at a temperature of 22 +/- 2 °C for up to 28 days (Schwarz, 2010). Samples are taken at regular intervals to determine the Dissolved Organic Carbon concentration (DOC). The mean value of two test assays was used to determine the final biodegradation rate after the regular exposure time. Thereby the test substance attained 10 - 20 % degradation. Based on the co-incubation with the reference substance aniline, it could be demonstrated that the test concentration used (20 mg/L DOC) was not toxic (inhibitory effect) on the sewage treatment bacteria flora. Aniline attained 91 % degradation after 14 days, indicating that the test system was suitable. In this experiment, the test substance was poorly biodegradable and is therefore considered as "not readily biodegradable" according to OECD criteria.