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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:
23 April 2012 - 25 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-A (Determination of the "Ready" Biodegradability - Dissolved Organic Carbon (DOC) Die-Away 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 (adaptation not specified)
Details on inoculum:
-Test Species
A mixed population of sewage treatment micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
-Preparation of Inoculum
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed 2 times by settlement and resuspension in mineral medium to remove any excessive amounts of DOC that may have been present. A sub-sample of the washed sewage sludge was then removed and the suspended solids concentration determined.
Duration of test (contact time):
28 d
Initial conc.:
59 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
MINERAL MEDIUM
Solution a
KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H2O 33.40 g/L
NH4Cl 0.50 g/L
pH =7.4

Solution b
CaCl2 27.50 g/L

Solution c
MgSO4.7H2O 22.50 g/L

Solution d
FeCl3.6H2O 0.25 g/L

To 1 litre (final volume) of purified water* was added the following volumes of solutions a - d:
10 mL of Solution a, 1 mL of Solution b, 1 mL of Solution c and 1 mL of Solution d.

* Reverse osmosis purified and deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP)


PROCEDURE
-Preliminary Investigational Work
An amount of test item (59.0 mg) was dissolved in mineral medium (1 litre) to give a 59.0 mg/L stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter). A further amount of test item (59.0 mg) was dissolved in mineral medium and inoculated at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1 litre. Two samples were taken for DOC analysis; one after filtration through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and the other after centrifugation at 4000 x gravity for 15 minutes. Control samples were prepared by inoculating mineral medium (1 litre) at a suspended solids level of 30 mg ss/L and then filtering or centrifuging as per the test item samples.


EXPERIMENTAL PREPARATION
For the purpose of the test, the test item was prepared by dissolving directly in deionized reverse osmosis purified water.
An amount of test item (1000 mg) was dissolved in deionized reverse osmosis purified water with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 1000 mL to give a 1000 mg/L stock solution. An aliquot (59 mL) of this stock solution was dispersed in a final volume of 1 litre of inoculated mineral medium to give the final test concentration of 59.0 mg/L, equivalent to 40 mg carbon/L.

The volumetric flask containing the test item was inverted several times to ensure homogeneity of the solution.

REFERENCE ITEM
For the purpose of the test a reference item, sodium benzoate, was used. An initial stock solution of 1000 mg/L was prepared by direct solution in water and an aliquot (68.6 mL) added to a final volume of 1 litre of inoculated mineral medium to give a final test concentration of 68.6 mg/L, equivalent to 40 mg carbon/L.

TOXICITY CONTROL
For the purpose of the test, a toxicity control, containing the test item and sodium benzoate was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An aliquot (59.0 mL) of the 1000 mg/L test item stock solution along with an aliquot (68.6 mL) of the reference item stock solution were dispersed in a final volume of 1 litre of inoculated mineral medium to give a final concentration of 59.0 mg/L (as test item) plus 68.6 mg sodium benzoate/L, equivalent to a final concentration of 80 mg carbon/L.

PREPARATION OF TEST SYSTEM
The following test solutions were prepared and inoculated in 2 litre glass conical flasks each containing 1 litre of solution:
-An inoculum control, in duplicate, consisting of inoculated mineral medium.
-The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final test concentration of 40 mg carbon/L.
-The test item, in duplicate, in inoculated mineral medium to give a final test concentration of 40 mg carbon/L.
-The test item plus sodium benzoate, one replicate, in inoculated mineral medium to give a final test concentration of 80 mg carbon/L to act as a toxicity control.

Each test vessel was inoculated with prepared inoculum micro-organisms at a rate of 30 mg suspended solids/L.
The test vessels were covered with aluminium foil and incubated (INFORS TR-225 orbital platform shaker) at 22 ± 1 °C in darkness and constantly shaken at 100 rpm for 28 days.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge. Therefore, for the purpose of the test, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.
Parameter:
% degradation (DOC removal)
Value:
0
Sampling time:
28 d
Details on results:
DOC values for the test item, procedure control, toxicity control and inoculum control vessels are given in Table 1. The percentage degradation values are given in Table 2.

The test item attained 0 % degradation after 28 days.

The toxicity control (test item plus sodium benzoate) attained 49 % degradation after 14 days and 53 % degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms at the test concentration employed in the study.

The pH values did not fall below 7.2 in any test vessel after 28 days of the study. Dissolved oxygen concentrations remained at approximately 7.9 mg O2/L or above in all culture vessels.
Results with reference substance:
The reference item, sodium benzoate, attained 99 % degradation after 14 days and 100 % degradation after 28 days, thereby confirming the suitability of the inoculum and culture conditions.

Table 1 Dissolved Organic carbon (DOC) Values on Each Sampling Occasion

Vessel

DOC (mg C/L)

Day

0

1

3

6

10

14

21

28

Inoculum Control

R1

1.85

2.92

2.67

3.03

0.73

1.20

1.16

1.09

R2

1.79

2.34

2.38

2.55

0.57

1.02

0.99

0.85

Mean

1.82

2.63

2.53

2.79

0.65

1.11

1.08

0.97

Procedure Control

R1

40.96

35.56

29.17

6.46

0.91

1.37

1.39

0.99

R2

38.88

33.26

25.95

6.24

1.42

1.48

1.44

1.14

Mean

39.92

34.41

27.56

6.35

1.17

1.43

1.42

1.07

Test

Item

R1

33.32

32.93

35.16

39.04

36.65

36.49

35.36

34.23

R2

35.59

32.96

34.01

37.80

37.22

36.40

35.06

33.96

Mean

34.46

32.95

34.59

38.42

36.94

36.45

35.21

34.10

Toxicity Control

72.27

70.27

58.08

40.84

36.65

36.73

35.54

33.91

R1 - R2 = Replicates 1 and 2

 

Table 2 Percentage Degradation Values

Day

% Degradation*

Procedure Control

Test item

Toxicity Control

1

17

7

4

3

34

2

21

6

91

0

46

10

99

0

49

14

99

0

49

21

99

0

51

28

100

0

53

*Corrected for control levels of DOC

Validity criteria fulfilled:
yes
Interpretation of results:
other: Not readily biodegradable
Conclusions:
The test material attained 0 % degradation after 28 days. The test material is therefore not considered to be readily biodegradable.
Executive summary:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media in accordance with the OECD Guidelines for Testing of Chemicals (1992) No. 301A, "Assessment of Ready Biodegradability; DOC Die-Away Test" referenced as Method C.4-A of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (L)).

 

The test item was exposed to activated sewage sludge micro-organisms at a concentration of 40 mg C/L with mineral medium in the dark at 22 ± 1 °C for 28 days. The degradation of the test item was assessed by the determination of Dissolved Organic Carbon (DOC) removal. Control cultures with inoculum and the reference item, sodium benzoate, together with a toxicity control, abiotic control and abiotic test vessel were used for validation purposes.

 

The test material attained 0 % degradation after 28 days. The test material is therefore not considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301A.

Description of key information

The test material attained 0 % degradation after 28 days. The test material is therefore not considered to be readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

A study was performed to assess the ready biodegradability of the substance in an aerobic aqueous media in accordance with the OECD Guidelines for Testing of Chemicals (1992) No. 301A, "Assessment of Ready Biodegradability; DOC Die-Away Test" referenced as Method C.4-A of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (L)).

The test material was exposed to activated sewage sludge micro-organisms at a concentration of 40 mg C/L with mineral medium in the dark at 22 ± 1°C for 28 days. The degradation of the test item was assessed by the determination of Dissolved Organic Carbon (DOC) removal. Control cultures with inoculum and the reference item, sodium benzoate, together with a toxicity control, abiotic control and abiotic test vessel were used for validation purposes.

 

The substance attained 0% degradation after 28 days. The test item is therefore not considered to be readily biodegradable.

The study was performed to GLP and was assigned a reliability score of 1 in accordance with the criteria for assessing data quality by Klimisch (1997). It is considered suitable for assessment as an accurate reflection of the test material.  

Supporting information is available from a study which was conducted with the substance following a method similar to that which is outlined in OECD guideline 301C. However, not enough information is provided to determine the reliability of the study.

The results suggest that the material did not appear to be readily biodegradable attaining 0% degradation after 28 days by TOC analysis and up to 16% after 28 days by GC analysis. As such, the substance cannot be considered to be readily biodegradable.