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:
28 May 2008 and 15 September 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect 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:
The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 11 August 2008 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Laboratory culture: Not applicable
- Method of cultivation: Not applicable
- Storage conditions: The washed sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC
- Storage length: Used on the day of collection.
- 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 2.6 g/l prior to use.
- Pretreatment: As above.
- Concentration of sludge: Not stated
- Initial cell/biomass concentration: As above.
- Water filtered: yes
- Type and size of filter used, if any: Not applicable
Duration of test (contact time):
28 d
Details on study design:
TEST CONDITIONS
- Composition of 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

- Additional substrate: None
- Solubilising agent (type and concentration if used):None used
- Test temperature: 21°C
- pH: 7.4
- pH adjusted:No
- CEC (meq/100 g): Not available
- Aeration of dilution water:
- Suspended solids concentration: 2.6 g/l
- Continuous darkness: yes
- Other:


TEST SYSTEM
- Culturing apparatus:
- Number of culture flasks/concentration: 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 34.6 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.

- Method used to create aerobic conditions: 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.
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: 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.
- Test performed in closed vessels due to significant volatility of test substance: No
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: 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.
- Other:


SAMPLING
- Sampling frequency: 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.
- Sampling method: 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 degradation of the test material had met the 10-Day window validation criterion given in the OECD Guidelines 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
On Days 0 and 28 samples (20 ml) were removed from all culture 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.

On Days 0 and 28 samples (20 ml) were removed from all culture 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.


- Sterility check if applicable: Not available
- Sample storage before analysis: The samples taken on Days 12 and 18 were stored at approximately 20°C.
- Other:


CONTROL AND BLANK SYSTEM
- Inoculum blank:
- Abiotic sterile control:
- 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 aliquot (44.1 ml) of the test material stock solution was dispersed in inoculated culture medium along with an aliquot (51.4 ml) of the sodium benzoate stock solution. The volume was adjusted to 3 litres to give a final concentration of 14.7 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.



STATISTICAL METHODS: None
Reference substance
Reference substance:
benzoic acid, sodium salt

Results and discussion

Preliminary study:
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test material did not absorb to filter matrices or to activated sewage sludge (see Appendix 1). Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test material.
Test performance:
Analysis of the test media from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 12% and 15% respectively for the test material Replicates R1 and R2 and 76% for the toxicity control. Sodium benzoate attained 100% degradation for Replicates R1 and R2 calculated from the results of the DOC analyses. The degradation rates calculated from the results of the DOC analyses for sodium benzoate and the toxicity control vessels were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring. The degradation rates calculated from the results of the DOC analyses for the test material vessels were lower than those calculated from inorganic carbon analysis. This was considered to be due to sampling/analytical variation.
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 light brown dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a light brown dispersion with no undissolved standard material or test material visible.
% Degradation
Parameter:
% degradation (test mat. analysis)
Value:
55
Sampling time:
28 d
Remarks on result:
other: Cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Details on results:
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 light brown dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a light brown dispersion with no undissolved test material or standard material visible.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 97% degradation for both Replicates R1 and R2 calculated from the results of the DOC analyses. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring.
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 light brown dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a light brown dispersion with no undissolved test material or standard material visible.

Any other information on results incl. tables

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. 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 CO2evolution in the control vessels on Day 28 was 26.38 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 CO2production 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 CO2present in the test vessels. Therefore any additional CO2detected in the Day 29 samples originated from dissolved CO2that 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 a decrease in all replicate vessels with the exception of test material Replicate R1. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2into the second absorber vessels occurred.

The test material attained 55% 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 71% degradation after 14 days and 61% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test. The decrease in degradation obtained for the toxicity control between Day 14 and Day 28 was due to a slightly greater increase in CO2evolution in the control vessels compared to that in the toxicity control vessel during this time period.

Sodium benzoate attained 64% degradation after 14 days and 66% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Analysis of the test media from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 92% and 98% respectively for the test material Replicates R1and R2and 97% for the toxicity control. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to adsorption of the test material to the glassware and/or activated sewage sludge and the subsequent removal by filtration prior to DOC analysis of the test samples. The results obtained from the preliminary investigational work indicated that the test material did not absorb to the activated sewage sludge (see Appendix 1). However, the samples taken from this work were sampled immediately after the test material stock solution had been inoculated and therefore, it was considered that over the test period of 28 days the test material could have adsorbed to the activated sewage sludge and/or the glassware.

Sodium benzoate attained 97% degradation for both Replicates R1and R2calculated from the results of the DOC analyses. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2evolution occurring.

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 light brown dispersions with no undissolved test material visible and the contents of the toxicity control vessel was a light brown dispersion with no undissolved test material or standard material visible.

Table 1 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

0.35

0.47

0.35

1.40

0.35

0.47

1.52

0.35

0.35

0.70

0.47

0.58

0.35

0.70

1

6.03

-

5.22

-

10.79

-

9.16

-

3.37

-

4.18

-

7.31

-

2

7.73

-

6.81

-

19.14

-

10.03

-

4.38

-

4.84

-

14.30

-

3

8.37

-

6.99

-

21.79

-

10.66

-

6.08

-

6.19

-

19.61

-

6

12.08

-

10.49

-

27.93

-

28.50

-

10.37

-

13.34

-

38.88

-

8

15.64

-

14.96

-

29.81

-

40.35

-

19.04

-

30.15

-

53.61

-

10

14.20

-

14.87

-

28.28

-

39.10

-

22.42

-

31.88

-

50.59

-

 14

17.48

-

16.48

-

34.63

-

37.74

-

26.83

-

38.97

-

59.79

-

16

17.15

-

14.72

-

34.31

-

38.51

-

25.90

-

37.85

-

56.00

-

 20

19.57

-

17.38

-

35.53

-

42.86

-

25.69

-

38.48

-

57.51

-

22

19.13

-

18.69

-

36.84

-

42.92

-

26.73

-

36.29

-

56.62

-

24

20.63

-

20.41

-

38.99

-

41.90

-

27.86

-

41.47

-

59.29

-

27

22.43

-

21.25

-

40.68

-

44.65

-

30.27

-

40.89

-

60.75

-

28

21.97

-

21.23

-

40.85

-

43.63

-

34.45

-

40.96

-

59.52

-

29

20.99

2.44

20.77

2.09

39.43

2.09

42.19

1.74

35.08

2.09

39.85

1.74

57.34

1.85

 


Table2               Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Material

% Degradation

Test Material plus Sodium Benzoate Toxicity Control

0

0

0

0

1

15

0

3

2

24

0

12

3

29

0

20

6

56

2

46

8

66

31

64

10

64

42

60

14

64

53

71

16

68

53

67

20

69

45

65

22

70

42

63

24

66

47

65

27

69

46

65

28

69

54

63

29*

66

55

61

 


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

10.37

-0.52

0

Sodium Benzoate

10 mg C/l R2

9.61

-0.31

0

Test Material

10 mg C/l R1

9.32

-0.36

0

Test Material

10 mg C/l R2

9.45

-0.33

0

Test Material plus Sodium Benzoate Toxicity Control

20 mg C/l

19.79

0.35

2

 


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/l R1

10.89

109

0.38

3

97

Sodium Benzoate

10 mg C/l R2

9.92

99

0.27

3

97

Test Material

10 mg C/l R1

9.68

97

0.79

8

92

Test Material

10 mg C/l R2

9.78

98

0.20

2

98

Test Material plus

Sodium Benzoate Toxicity Control

20 mg C/l

19.44

97

0.61

3

97

 


Table5               pH Values of the Test Preparations on Day 28

Test Vessel

pH

ControlR1

8.1

Control R2

8.1

Sodium Benzoate

10 mg C/l R1

8.0

Sodium Benzoate

10 mg C/l R2

8.0

Test Material

10 mg C/l R1

7.9

Test Material

10 mg C/l R2

8.0

Test Material

plus Sodium Benzoate Toxicity Control 20 mg C/l

7.9

 


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

Light brown dispersion, no undissolved test material visible

Light brown dispersion, no undissolved test material visible

Light brown dispersion, no undissolved test material visible

Light brown dispersion, no undissolved test material visible

Light brown dispersion, no undissolved test material visible

 

R2

Light brown dispersion, no undissolved test material visible

Light brown dispersion, no undissolved test material visible

Light brown dispersion, no undissolved test material visible

Light brown dispersion, no undissolved test material visible

Light brown dispersion, no undissolved test material visible

Toxicity Control

 

Light brown dispersion, no undissolved test or standard material visible

Light brown dispersion, no undissolved test or standard material visible

Light brown dispersion, no undissolved test or standard material visible

Light brown dispersion, no undissolved test or standard material visible

Light brown dispersion, no undissolved test or standard material visible


R1– R2= Replicates 1 and 2

Abs= CO2absorber vessels

*Day 29 values corrected to include any carry-over of CO2detected in Absorber 2

R1– R2= Replicates 1 and 2

*Corrected for control values. Negative values are due to measured concentrations being less than control values

 

R1– R2= Replicates 1 and 2

*Corrected for control values. Negative values are due to measured concentrations being less than control values

R1– R2= Replicates 1 and 2

R1– R2= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
not inherently biodegradable
Conclusions:
The test material attained 55% 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 55% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.