Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 17 March 2008 and 17 April 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 21/07/2009 Date of Signature:15/10/2009

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Barium di(bistridecylsulfosuccinate) in mixture with Barium hydrogen phosphate
- Substance type: pale yellow solid
- Physical state: Solid
- Lot/batch No.: Y-T-1
- Storage condition of test material: room temperature in the dark
- Other:

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Severn trent Water Plc
- Laboratory culture: Not recorded
- Method of cultivation: Not recorded
- Storage conditions: Not recorded
- Storage length: Not recorded.
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the funnel three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 2.2 g/l prior to use.
* rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven
- Pretreatment: Not recorded
- Concentration of sludge: Not recorded
- Initial cell/biomass concentration: Not recorded
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper using Buchner funnel.

Culture medium:
The culture medium used in this study was that recommended in the OECD Guidelines.
Culture 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
1 ml of Solution d

Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
22.5 mg/L
Based on:
other: final concentration of 22.5 mg/l, equivalent to 10 mg carbon/l.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST SPECIES:
A mixed population of activated sewage sludge micro-organisms was obtained on from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage

TEST SYSTEM
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/l. The test was carried out in a temperature controlled room at approximately 21°C, in darkness.
Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 40.9 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.
The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.
The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.



SAMPLING
-CO2 analysis:
Samples (2 ml) were taken from the first CO2 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.
The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27, 28 and 29 were analysed for CO2 immediately. The samples taken on Days 12 and 18 were stored at approximately -20°C. However, these samples were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that the level of degradation of the test material did not significantly increase during this time and therefore additional analyses were considered to be unnecessary.
On Day 28, 1 ml of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.
The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-VCSH TOC analyser. Samples (300 or 50 µl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.


Dissolved Organic carbon (DOC) analysis:
Samples (20 ml) were removed from the test material and toxicity control vessels on Day 0 prior to the addition of the test material in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
DOC analysis of the test material dispersions after dosing was not possible due to the insoluble nature of the test material in water.
On Days 0 and 28 samples (20 ml) were removed from the control and standard material vessels and filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680°C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.


pH Measurement:

The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH 320 pH meter.

CONTROL AND BLANK SYSTEM
- Toxicity control:

For the purposes of the test a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge micro-organisms used in the test.
An amount of test material (67.5 mg) was dispersed in approximately 400 ml of culture medium and subjected to high shear mixing at approximately 7500 rpm for 15 minutes prior to dispersal in inoculated culture medium. An aliquot (51.4 ml) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 22.5 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.






Reference substance
Reference substance:
other: Sodium benzoate

Results and discussion

Preliminary study:
Not applicable
Test performance:
The total CO2 evolution in the control vessels on Day 28 was 35.51 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines

% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
56
Sampling time:
28 d
Remarks on result:
other: not readily biodegradable
Details on results:
Inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and standard materials and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1 (see attached background material). Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3, and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4. The pH values of the test preparations on Day 28 are given in Table 5.

The total CO2 evolution in the control vessels on Day 28 was 35.51 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material suspension in the mineral medium at the start of the test (see Table 3) was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was <20% and hence satisfied the validation criterion given in the OECD Test Guidelines.

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test material.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The test material attained 56% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
The toxicity control attained 41% degradation after 14 days and 73% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 76% degradation after 14 days and 104% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.
Analysis of the test media taken from the standard material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 98% for both Replicates R1 and R2.
Observations made throughout the test period (see Table 6) showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels to be light brown dispersions with no undissolved standard material visible. The contents of the test material vessels were slightly cloudy dispersions with particles of test material on the surface and small particles dispersed throughout. The contents of the toxicity control vessel was a slightly cloudy dispersion with particles of test material on the surface and small particles dispersed throughout, no undissolved standard material was visible.

Any other information on results incl. tables

Table1               Inorganic Carbon Values on Each Analysis Occasion

Day

Control (mg IC)

Sodium Benzoate (mg IC)

Test Material (mg IC)

Test Material
plus Sodium Benzoate Toxicity Control (mg IC)

R1

R2

R1

R2

R1

R2

R1

Abs1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

0

0.70

0.35

0.35

0.35

0.70

0.35

0.47

0.35

0.70

0.35

0.70

0.35

0.70

0.35

1

4.76

-

1.39

-

9.40

-

4.87

-

4.52

-

4.52

-

7.19

-

2

11.42

-

9.69

-

19.72

-

23.64

-

8.30

-

9.34

-

12.69

-

3

15.94

-

11.01

-

25.91

-

24.54

-

11.01

-

10.66

-

17.77

-

6

15.05

-

15.16

-

28.84

-

32.49

-

13.57

-

15.28

-

29.07

-

8

19.15

-

21.42

-

33.77

-

40.80

-

19.38

-

20.63

-

36.49

-

10

21.74

-

22.20

-

38.31

-

47.88

-

23.10

-

23.77

-

42.36

-

14

27.16

-

25.72

-

43.98

-

54.33

-

32.06

-

29.39

-

51.33

-

16

28.44

-

28.66

-

50.35

-

60.87

-

37.96

-

32.54

-

62.19

-

20

25.80

-

26.13

-

43.19

-

56.96

-

39.80

-

34.44

-

60.46

-

22

27.60

-

27.93

-

45.10

-

56.62

-

43.58

-

34.56

-

66.72

-

24

29.59

-

29.16

-

56.70

-

61.78

-

48.38

-

37.04

-

68.36

-

27

27.69

-

28.12

-

54.31

-

59.65

-

45.30

-

41.43

-

71.06

-

28

29.01

-

29.12

-

54.61

-

60.16

-

46.61

-

44.05

-

71.47

-

29

30.00

2.44

30.31

2.44

59.89

3.02

62.33

2.09

48.55

2.32

45.58

2.44

73.88

2.44

Table2               Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Material

% Degradation

Test Material plus Sodium Benzoate Toxicity Control

0

0

0

0

1

14

5

7

2

37

0

4

3

39

0

7

6

52

0

23

8

57

0

27

10

70

5

34

14

76

14

41

16

90

22

56

20

80

37

57

22

77

38

65

24

100

44

65

27

96

52

72

28

94

54

71

29*

104

56

73

 

 

Table3               Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel

Total Carbon*

(mg/l)

Inorganic Carbon*

(mg/l)

IC Content (% of TC)

Sodium Benzoate

10 mg C/lR1

10.18

0.17

2

Sodium Benzoate

10 mg C/l R2

10.07

0.04

0

Test Material

10 mg C/l R1

10.37**

0.15

1

Test Material

10 mg C/l R2

9.94**

-0.37

0

Test Material plus Sodium Benzoate Toxicity Control

20 mg C/l

20.40**

0.17

1

Table4               Dissolved Organic Carbon (DOC) Values in the Culture Vessels on Days 0 and 28

Test Vessel

DOC*Concentration

Day 0

Day 28

mg C/l

% of Nominal Carbon Content

mg C/l

% of Initial Carbon Concentration

% Degradation

Sodium Benzoate

10 mg C/lR1

10.03

100

0.19

2

98

Sodium Benzoate

10 mg C/l R2

10.05

101

0.18

2

98

Table5               pH Values of the Test Preparations on Day 28

Test Vessel

pH

ControlR1

7.5

Control R2

7.5

Sodium Benzoate

10 mg C/l R1

7.5

Sodium Benzoate

10 mg C/l R2

7.5

Test Material

10 mg C/l R1

7.5

Test Material

10 mg C/l R2

7.5

Test Material

plus Sodium Benzoate Toxicity Control 20 mg C/l

7.5

 


Table6               Observations on the Test Preparations Throughout the Test Period

Test Vessel

Observations on Test Preparations

Day 0

Day 6

Day 13

Day 20

Day 27

Control

R1

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

 

R2

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Standard Material

R1

Light brown dispersion, no undissolved standard material visible

Light brown dispersion, no undissolved standard material visible

Light brown dispersion, no undissolved standard material visible

Light brown dispersion, no undissolved standard material visible

Light brown dispersion, no undissolved standard material visible

 

R2

Light brown dispersion, no undissolved standard material visible

Light brown dispersion, no undissolved standard material visible

Light brown dispersion, no undissolved standard material visible

Light brown dispersion, no undissolved standard material visible

Light brown dispersion, no undissolved standard material visible

Test Material

R1

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout

 

R2

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout

Toxicity Control

 

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout. No undissolved standard material visible

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout. No undissolved standard material visible

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout. No undissolved standard material visible

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout. No undissolved standard material visible

Slightly cloudy dispersion, particles of test material on the surface and small particles dispersed throughout. No undissolved standard material visible


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test material attained 56% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B
Executive summary:

Introduction.

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).

Methods.

The test material, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

Results.

The test material attained 56% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.