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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:
The study was conducted between 10 June 2011 and 20 July 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge:
Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on
20 June 2011 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 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 filter 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.

* Rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven

- Concentration of sludge: 30 mg ss/l

Duration of test (contact time):
28 d
Initial conc.:
10 other: mg carbon/l
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preliminary Investigational Work
In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge, samples were taken for Dissolved Organic Carbon (DOC) analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge solids. Thus the following work was conducted and samples analysed for Dissolved Organic Carbon (DOC) using a Shimadzu
TOC-VCPH TOC analyser (see Appendix 2 in preliminary study section in results and discussion).

An amount of test item (100 mg) was dissolved in culture medium (1000 ml) to give a 100 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 (100 mg) was dissolved in culture medium and inoculated at a concentration of 30 mg suspended solids (ss)/l prior to adjusting to a final volume of 1000 ml. 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 3500 rpm for 15 minutes. Control samples were prepared by inoculating culture medium (1000 ml) at a suspended solids level of 30 mg ss/l and then filtering or centrifuging as per the test item samples.

A mixed population of activated sewage sludge micro-organisms was obtained on
10 June 2011 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
For the purpose of the test, the test item was dissolved directly in culture medium.

Experimental Preparation
An amount of test item (1000 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give a 1000 mg/l stock solution. An aliquot (150 ml) of this stock solution was dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 50 mg/l, equivalent to 10 mg carbon/l. The volumetric flask containing the test item was inverted several times to ensure homogeneity of the solution.
A test concentration of 10 mg carbon/l was employed in the test following the recommendations of the Test Guideline.

Reference Item
For the purposes of the test, a reference item, sodium benzoate (C6H5COONa) (Sigma Aldrich Lot No MKBC1469), was used. An initial stock solution of 1000 mg/l was prepared by dissolving the reference item directly in culture medium with the aid of ultrasonication for approximately 5 minutes. An aliquot (51.4 ml) of this stock solution was added to the test vessel containing inoculated culture medium and the volume adjusted to 3 litres to give a final test concentration of 17.1 mg/l, equivalent to 10 mg carbon/l. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Toxicity Control
For the purposes 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 (150 ml) of the test item 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 50 mg test item/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.

Culture medium
The culture medium used in this study (in section any other information on results incl tables) was that recommended in the OECD Guidelines

Preparation of 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 reference item (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test item, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
d) The test item plus the reference item 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 21DegC, in darkness.
Approximately 24 hours prior to addition of the test and reference items 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 reference items 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 and analysis
CO2 analysis
Samples (2 ml) were taken from the control, reference and test item first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 and from the toxicity control first CO2 absorber vessel on Days 0, 2, 6, 8, 10 and 14. The second absorber vessel was sampled on Days 0 and 29 for the control, reference and test item and on Day 0 for the toxicity control.
The samples taken on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29 were analysed for CO2 immediately.
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. Samples (300 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 reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis
Samples (30 ml) were removed from all culture vessels on Day 0 and from the control, reference and test item culture vessels on Day 28 and filtered through 0.45 µm Gelman AcroCap filters (approximately 5 ml discarded) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-VCPH TOC Analyser. Samples (50 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680DegC 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 reference solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

pH measurements
The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH320 pH meter.

Evaluation of Data
Please see attached evaluation of data attached in background material section.


Validation Criteria
The results of the degradation test are considered valid if in the same test the reference item yields equalt to or greater than 60% degradation by Day 14.
The test item may be considered to be readily biodegradable if equalt to or greater than 60% degradation is attained within 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.
The toxicity control (test item and sodium benzoate) should attain equal to or greater than 25% degradation by Day 14 for the test item to be considered as non-inhibitory.
The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the end of the test is less than 20%.
The total CO2 evolution in the control vessels at the end of the test should not normally exceed 40 mg/l medium.
The IC content of the test item suspension in the mineral medium at the beginning of the test should be <5% of the TC.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
Appendix 2 Dissolved Organic Carbon (DOC) Values from the Preliminary Investigational Work

In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge the following samples were analysed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-VCPH TOC analyser.

Sample DOC Concentration % of Nominal Carbon Content
mg C/l mg C/l corrected for appropriate control
Culture medium Control, inoculated at 30 mg ss/l, Filtered Control, inoculated at 30 mg ss/l, Centrifuged 100 mg/l Untreated 22.11 22.11 111
100 mg/l Filtered 21.96 21.96 110
100 mg/l, inoculated at 30 mg ss/l, Filtered 24.17 24.17 121
100 mg/l, inoculated at 30 mg ss/l, Centrifuged 23.91 23.91 120

These results indicated that the test item did not adsorb to filter matrices or activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without causing a loss of any test item.

Key result
Parameter:
% degradation (inorg. C analysis)
Value:
26
Sampling time:
28 d
Remarks on result:
other: No St dev.
Parameter:
% degradation (DOC removal)
Value:
4
Sampling time:
28 d
Remarks on result:
other: No St dev.
Details on results:
Inorganic carbon values for the test item, reference item, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1 (attached in background material section). 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. Observations made on the contents of the test vessels are given in Table 6. All tables are in any other infromation on material and methods including tables section
The total CO2 evolution in the control vessels on Day 28 was 27.85 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test (see Table 3 in any other infromation on material and methods including tables section ) 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 item.
The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed a decrease in all replicate vessels. This decrease was considered to be due to sampling/analytical variation. 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.
Results with reference substance:
Sodium benzoate attained 106% degradation after 14 days and 103% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. This slight decrease in degradation between days 14 and 28 was considered to be due to sampling/analytical variation. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.

Table1              Inorganic Carbon Values on Each Analysis Occasion

Day

Control (mg IC)

Sodium Benzoate
(mg IC)

Test Item (mg IC)

Test Item
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

0.82

0.70

0.82

0.58

1.40

0.70

0.70

1.40

0.70

0.70

1.05

0.70

0.70

0.82

2

5.22

-

2.44

-

2.55

-

18.10

-

7.42

-

11.14

-

21.11

-

6

10.84

-

10.38

-

31.60

-

36.22

-

13.84

-

15.80

-

38.75

-

8

12.73

-

12.73

-

39.10

-

38.76

-

16.63

-

18.12

-

39.90

-

10

13.68

-

14.02

-

34.54

-

41.61

-

16.87

-

20.75

-

43.66

-

14

16.43

-

15.30

-

48.96

-

46.13

-

22.78

-

25.27

-

48.73

-

21

18.59

-

18.70

-

51.83

-

54.53

-

26.25

-

28.73

-

-

-

28

22.85

-

22.74

-

53.09

-

55.10

-

29.90

-

29.57

-

-

-

29

20.82

2.44

20.15

3.02

50.10

3.13

51.44

3.37

27.39

2.78

28.95

2.55

-

-


R1– R2= Replicates 1 and 2

Abs= CO2absorber vessels

- = No value dtermined

Table2              Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Item

% Degradation

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

22

18

29

6

78

14

47

8

87

16

45

10

81

17

50

14

106

27

55

21

115

29

-

28

104

23

-

29*

103

26

-


-= No degradation result obtained due to toxicity control being terminated after 14 days.

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

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

10.04

-0.02

0

Sodium Benzoate

10 mg C/l R2

9.40

-0.39

0

Test Item

10 mg C/l R1

10.68

-0.16

0

Test Item

10 mg C/l R2

11.40

0.36

3

Test Item plus Sodium Benzoate Toxicity Control

20 mg C/l

20.83

-0.02

0


R1– R2= Replicates 1 and 2

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

 

 

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

10.05

101

0.15

1

99

Sodium Benzoate

10 mg C/l R2

9.78

98

0.05

1

99

Test Item

10 mg C/l R1

10.84

108

10.62

98

2

Test Item

10 mg C/l R2

11.03

110

10.40

94

6


R1– R2= Replicates 1 and 2

*Corrected for control values.

Table5              pH Values of the Test Preparations on Day 28

Test Vessel

pH

ControlR1

7.7

Control R2

7.7

Sodium Benzoate

10 mg C/l R1

7.8

Sodium Benzoate

10 mg C/l R2

7.6

Test Item

10 mg C/l R1

7.5

Test Item

10 mg C/l R2

7.5


R1– R2= Replicates 1 and 2

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

Reference Item

R1

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

 

R2

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Light brown dispersion, no undissolved reference item visible

Test Item

R1

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

 

R2

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Toxicity Control

 

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

-

-


R1– R2= Replicates 1 and 2

-= No observations made due to toxicity control being terminated after 14 days


Validity criteria fulfilled:
yes
Interpretation of results:
other: The test item attained 26% degradation after 28 days and therefore cannot be considered to be readily biodegradable
Conclusions:
The test item attained 26% 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:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; CO2Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).

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

The test item attained 26% 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 test item attained 26% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Description of key information

A single key study was performed to assess the ready biodegradability of the test item in accordance with OECD 301B (CO2 evolution). The test item attained 26% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Introduction.

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301 B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (M)).

Methods.

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

Results.

The test item attained 26% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301 B.