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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
other information
Study period:
The study was conducted between 4 October 2011 and 2 November 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was 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)
Version / remarks:
referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 3 October 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
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 2.5 g/l prior to use.

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

Duration of test (contact time):
28 d
Initial conc.:
13.6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Culture medium
The culture medium used in this study (see Appendix 2) was that recommended in the OECD Guidelines.

Appendix 2 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

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


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 21ºC, 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 36.0 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 30 to 100 ml/min per vessel and stirred continuously by magnetic stirrer. The flow rates were adjusted to 40 ml/minute where necessary.
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 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 reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis
Samples (30 ml) were removed from the test item and toxicity control vessels on Day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (approximately 5 ml discarded) prior to DOC analysis. DOC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.

On Days 0 and 28 samples (30 ml) were removed from the control and reference item vessels 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 and a Shimadzu TOC-LCSH TOC Analyser. Samples (50 or 800 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680ºC 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 pH/Oxi 340I pH and dissolved oxygen meter.

Evalaution of data:
Determination of carbon content
The test item contains 73.47% carbon (data supplied by the Sponsor) and so for a concentration of 10 mg C/l (a total of 40.8 mg of test item in 3 litres) the total organic carbon present was 30 mg C.
The theoretical amount of the carbon present in the reference item was calculated using the equation: number of carbon atoms x molecular wt of carbon divided by molecular wt of the reference item multiplied by 100%. Thus for a 10 mg C/l test concentration (a total of 51.4 mg of sodium benzoate in 3 litres) the total organic carbon present for sodium benzoate was 30 mg C.


Percentage degradation:
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, in the following equation:
The values of Replicates R1 and R2 are meaned for the control, test and reference items before substitution in the equation.
%ThCo2(=%degradation)=mgICin test flask-mgICin control divided by mg TOC as test chemical multiplied by 100%.
The percentage degradation from the results of the DOC analysis, see Table 4, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation

Equation: % degradation=[1-mg DOC in test flask on day 28/mg DOC in test flask on day 0] x 100%.

The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation.

Total CO2 evolution = mgIC in control x 100/%C of CO2 x 1/test volume.

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.

All tables are under other informations on results including tables section.

Reference substance:
other: Sodium benzoate
Test performance:
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 attached 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. Observations made on the contents of the test vessels are given in Table 6. The total CO2 evolution in the control vessels on Day 28 was 26.75 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) 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.

All tables under results incl. tables section.
Parameter:
% degradation (CO2 evolution)
Value:
17
Sampling time:
28 d
Details on results:
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 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 item attained 17% 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 45% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Results with reference substance:
Sodium benzoate attained 77% degradation after 14 days and 91% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Analysis of the test media taken from the reference item culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), (see Table 4), gave percentage degradation values of 99% and 100% respectively for Replicates R1 and R2. 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.

For tables, see under results incl. tables section.

Table 1              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

1.17

1.75

1.05

1.05

1.40

1.17

1.75

1.17

1.05

1.40

1.28

1.75

1.28

1.17

2

5.22

-

8.35

-

22.27

-

19.03

-

5.68

-

9.40

-

24.36

-

6

11.65

-

16.72

-

38.75

-

35.41

-

15.22

-

17.19

-

40.37

-

8

12.96

-

20.87

-

43.34

-

39.33

-

18.58

-

19.84

-

43.23

-

10

13.68

-

18.70

-

43.43

-

40.59

-

19.38

-

19.72

-

44.80

-

14

18.25

-

21.87

-

45.67

-

40.46

-

22.10

-

21.99

-

46.92

-

21

20.96

-

20.73*

-

44.95

-

42.02*

-

25.01

-

22.31

-

-

-

28

21.39

-

22.40

-

52.75

-

44.46

-

28.22

-

23.97

-

-

-

29

23.16

2.90

24.16

3.25

54.11

3.37

47.09

3.48

30.06

3.48

26.39

3.48

-

-


R1– R2= Replicates 1 and 2

Abs= CO2absorber vessels

- = No value determined

*Duplicate sample analysed as the original sample result was deemed anomalous

Table 2              Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Item

% Degradation

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

46

3

29

6

76

7

44

8

81

8

44

10

86

11

48

14

77

7

45

21

75

9

-

28

89

14

-

29*

91

17

-


-= 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

Table 3              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

10.10

0.38

4

Sodium Benzoate

10 mg C/l R2

9.99

-0.13

0

Test Item

10 mg C/l R1

9.65**

-0.34

0

Test Item

10 mg C/l R2

9.85**

-0.68

0

Test Item plus Sodium Benzoate Toxicity Control

20 mg C/l

20.84**

0.81

4


R1– R2= Replicates 1 and 2

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

**Total carbon value given is the sumof the TC value obtained from analysis and the nominal TC contribution of the test item and sodium benzoate where applicable

Table 4              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

9.71

97

0.05

1

99

Sodium Benzoate

10 mg C/l R2

10.12

101

      <control

0

100


R1– R2= Replicates 1 and 2

*Corrected for control values.

Table 5              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

7.6

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

Table 6              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 cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

 

R2

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Reference Item

R1

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

 

R2

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Test Item

R1

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

 

R2

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Toxicity Control

 

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved 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: Cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Conclusions:
The test item attained 17% 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 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; 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.

Results and Conclusion The test item attained 17% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
The study was conducted between 4 October 2011 and 2 November 2011.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was 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.
Justification for type of information:
PU TDI-1 substances all contain the same core structure, with predominantly linear alkyl chains (C8 – C18) attached. The same substance can contain structures with different alkyl chain lengths, and some substances may contain small amounts of structures with cyclic groups. PU TDI-1 structures are therefore similar between all category members, and organisms will be exposed to very similar compounds. Organisms would be exposed to common structures, only differing by the length of the alkyl chain or whether cyclic groups are present. In the body, there may be metabolism of the PU TDI-1 structures, however due to the structural similarity of the parent compounds any metabolites are also likely to be similar.

Further information is available in the read-across justification document in Section 13 of IUCLID.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 3 October 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
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 2.5 g/l prior to use.

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

Duration of test (contact time):
28 d
Initial conc.:
13.6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Culture medium
The culture medium used in this study (see Appendix 2) was that recommended in the OECD Guidelines.

Appendix 2 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

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


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 21ºC, 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 36.0 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 30 to 100 ml/min per vessel and stirred continuously by magnetic stirrer. The flow rates were adjusted to 40 ml/minute where necessary.
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 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 reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis
Samples (30 ml) were removed from the test item and toxicity control vessels on Day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (approximately 5 ml discarded) prior to DOC analysis. DOC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.

On Days 0 and 28 samples (30 ml) were removed from the control and reference item vessels 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 and a Shimadzu TOC-LCSH TOC Analyser. Samples (50 or 800 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680ºC 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 pH/Oxi 340I pH and dissolved oxygen meter.

Evalaution of data:
Determination of carbon content
The test item contains 73.47% carbon (data supplied by the Sponsor) and so for a concentration of 10 mg C/l (a total of 40.8 mg of test item in 3 litres) the total organic carbon present was 30 mg C.
The theoretical amount of the carbon present in the reference item was calculated using the equation: number of carbon atoms x molecular wt of carbon divided by molecular wt of the reference item multiplied by 100%. Thus for a 10 mg C/l test concentration (a total of 51.4 mg of sodium benzoate in 3 litres) the total organic carbon present for sodium benzoate was 30 mg C.


Percentage degradation:
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, in the following equation:
The values of Replicates R1 and R2 are meaned for the control, test and reference items before substitution in the equation.
%ThCo2(=%degradation)=mgICin test flask-mgICin control divided by mg TOC as test chemical multiplied by 100%.
The percentage degradation from the results of the DOC analysis, see Table 4, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation

Equation: % degradation=[1-mg DOC in test flask on day 28/mg DOC in test flask on day 0] x 100%.

The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation.

Total CO2 evolution = mgIC in control x 100/%C of CO2 x 1/test volume.

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.

All tables are under other informations on results including tables section.

Reference substance:
other: Sodium benzoate
Test performance:
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 attached 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. Observations made on the contents of the test vessels are given in Table 6. The total CO2 evolution in the control vessels on Day 28 was 26.75 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) 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.

All tables under results incl. tables section.
Parameter:
% degradation (CO2 evolution)
Value:
17
Sampling time:
28 d
Details on results:
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 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 item attained 17% 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 45% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Results with reference substance:
Sodium benzoate attained 77% degradation after 14 days and 91% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Analysis of the test media taken from the reference item culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), (see Table 4), gave percentage degradation values of 99% and 100% respectively for Replicates R1 and R2. 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.

For tables, see under results incl. tables section.

Table 1              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

1.17

1.75

1.05

1.05

1.40

1.17

1.75

1.17

1.05

1.40

1.28

1.75

1.28

1.17

2

5.22

-

8.35

-

22.27

-

19.03

-

5.68

-

9.40

-

24.36

-

6

11.65

-

16.72

-

38.75

-

35.41

-

15.22

-

17.19

-

40.37

-

8

12.96

-

20.87

-

43.34

-

39.33

-

18.58

-

19.84

-

43.23

-

10

13.68

-

18.70

-

43.43

-

40.59

-

19.38

-

19.72

-

44.80

-

14

18.25

-

21.87

-

45.67

-

40.46

-

22.10

-

21.99

-

46.92

-

21

20.96

-

20.73*

-

44.95

-

42.02*

-

25.01

-

22.31

-

-

-

28

21.39

-

22.40

-

52.75

-

44.46

-

28.22

-

23.97

-

-

-

29

23.16

2.90

24.16

3.25

54.11

3.37

47.09

3.48

30.06

3.48

26.39

3.48

-

-


R1– R2= Replicates 1 and 2

Abs= CO2absorber vessels

- = No value determined

*Duplicate sample analysed as the original sample result was deemed anomalous

Table 2              Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Item

% Degradation

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

46

3

29

6

76

7

44

8

81

8

44

10

86

11

48

14

77

7

45

21

75

9

-

28

89

14

-

29*

91

17

-


-= 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

Table 3              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

10.10

0.38

4

Sodium Benzoate

10 mg C/l R2

9.99

-0.13

0

Test Item

10 mg C/l R1

9.65**

-0.34

0

Test Item

10 mg C/l R2

9.85**

-0.68

0

Test Item plus Sodium Benzoate Toxicity Control

20 mg C/l

20.84**

0.81

4


R1– R2= Replicates 1 and 2

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

**Total carbon value given is the sumof the TC value obtained from analysis and the nominal TC contribution of the test item and sodium benzoate where applicable

Table 4              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

9.71

97

0.05

1

99

Sodium Benzoate

10 mg C/l R2

10.12

101

      <control

0

100


R1– R2= Replicates 1 and 2

*Corrected for control values.

Table 5              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

7.6

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

Table 6              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 cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

 

R2

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Reference Item

R1

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

 

R2

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Test Item

R1

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

 

R2

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Toxicity Control

 

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved 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: Cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Conclusions:
The test item attained 17% 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 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; 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.

Results and Conclusion The test item attained 17% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 4 October 2011 and 2 November 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The 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)
Version / remarks:
referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 3 October 2011 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Procedure
Preparation of inoculum
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 2.5 g/l prior to use.

* Rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven
Duration of test (contact time):
28 d
Initial conc.:
14 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
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 36.0 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 30 to 100 ml/min per vessel and stirred continuously by magnetic stirrer. The flow rates were adjusted to 40 ml/minute where necessary.
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 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 reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved Organic carbon (DOC) analysis:
Samples (30 ml) were removed from the test item and toxicity control vessels on Day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (approximately 5 ml discarded) prior to DOC analysis.
DOC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.
On Days 0 and 28 samples (30 ml) were removed from the control and reference item vessels 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-VCPN TOC analyser and a Shimadzu TOC-LCSH TOC Analyser. Samples (50 or 800 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 reference 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/Oxi 340I pH and dissolved oxygen meter.


Evalaution of data:
The test item contains 71.41% carbon (data supplied by the Sponsor) and so for a concentration of 10 mg C/l (a total of 42.0 mg of test item in 3 litres) the total organic carbon present was 30 mg C.
The theoretical amount of the carbon present in the reference item was calculated using the equation: number of carbon atoms x molecular wt of carbon divided by molecular wt of the reference item multiplied by 100%. Thus for a 10 mg C/l test concentration (a total of 51.4 mg of sodium benzoate in 3 litres) the total organic carbon present for sodium benzoate was 30 mg C.


Percentage degradation:
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, in the following equation:

The values of Replicates R1 and R2 are meaned for the control, test and reference items before substitution in the equation.

%ThCo2(=%degradation)=mgICin test flask-mgICin control divided by mg TOC as test chemical multiplied by 100%.

The percentage degradation from the results of the DOC analysis, see Table 4, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.

Equation: % degradation=[1-mg DOC in test flask on day 28/mg DOC in test flask on day 0] x 100%.

The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation.

Total CO2 evolution = mgIC in control x 100/%C of CO2 x 1/test volume.

Validation criteria:
The results of the degradation test are considered valid if in the same test the reference item yields equal to or greater than 60% degradation by Day 14. The test item may be considered to be readily biodegradable if equal 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.

For tables, see under results incl. tables section.


Reference substance:
other: Sodium benzoate
Test performance:
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 attached 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. Observations made on the contents of the test vessels are given in Table 6.
The total CO2 evolution in the control vessels on Day 28 was 26.75 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) 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.

For tables, see under results inlc. tables section.
Parameter:
% degradation (CO2 evolution)
Value:
26
Sampling time:
28 d
Details on results:
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 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 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 toxicity control attained 34% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.
Results with reference substance:
Sodium benzoate attained 77% degradation after 14 days and 91% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Analysis of the test media taken from the reference item culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), (see Table 4), gave percentage degradation values of 99% and 100% respectively for Replicates R1 and R2. 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.

For tables, see under results incl. tables section.

Table 1              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

1.17

1.75

1.05

1.05

1.40

1.17

1.75

1.17

1.63

1.28

1.28

1.87

1.05

1.28

2

5.22

-

8.35

-

22.27

-

19.03

-

8.70

-

8.00

-

23.43

-

6

11.65

-

16.72

-

38.75

-

35.41

-

17.53

-

15.92

-

35.18

-

8

12.96

-

20.87

-

43.34

-

39.33

-

20.07

-

18.69

-

39.22

-

10

13.68

-

18.70

-

43.43

-

40.59

-

23.71

-

20.75

-

38.99

-

14

18.25

-

21.87

-

45.67

-

40.46

-

23.12

-

24.03

-

40.57

-

21

20.96

-

20.73*

-

44.95

-

42.02*

-

25.80

-

27.72

-

-

-

28

21.39

-

22.40

-

52.75

-

44.46

-

28.45

-

31.02

-

-

-

29

23.16

2.90

24.16

3.25

54.11

3.37

47.09

3.48

29.62

3.13

32.73

3.48

-

-


R1– R2= Replicates 1 and 2

Abs= CO2absorber vessels

*Duplicate sample analysed as the original sample result was deemed anomalous

Table 2              Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Item

% Degradation

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

46

5

28

6

76

8

35

8

81

8

37

10

86

20

38

14

77

12

34

21

75

20

-

28

89

26

-

29*

91

26

-


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

*Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Table 3              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

10.10

0.38

4

Sodium Benzoate

10 mg C/l R2

9.99

-0.13

0

Test Item

10 mg C/l R1

10.21**

-0.35

0

Test Item

10 mg C/l R2

10.34**

0.12

1

Test Item plus Sodium Benzoate Toxicity Control

20 mg C/l

20.20**

0.16

1


R1– R2= Replicates 1 and 2

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

**Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item and sodium benzoate where applicable

Table 4              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

9.71

97

0.05

1

99

Sodium Benzoate

10 mg C/l R2

10.12

101

     <control

0

100


R1– R2= Replicates 1 and 2

*Corrected for control values.

Table 5              pH Values of the Test Preparations on Day 28

Test Vessel

pH

Control R1

7.5

Control R2

7.5

Sodium Benzoate

10 mg C/l R1

7.6

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

Table 6              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 cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

 

R2

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Reference Item

R1

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

 

R2

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Test Item

R1

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

 

R2

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Toxicity Control

 

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved 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: Cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
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:

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 301B, "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.

Results and Conclusion

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.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
The study was conducted between 4 October 2011 and 2 November 2011.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The 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.
Justification for type of information:
PU TDI-1 substances all contain the same core structure, with predominantly linear alkyl chains (C8 – C18) attached. The same substance can contain structures with different alkyl chain lengths, and some substances may contain small amounts of structures with cyclic groups. PU TDI-1 structures are therefore similar between all category members, and organisms will be exposed to very similar compounds. Organisms would be exposed to common structures, only differing by the length of the alkyl chain or whether cyclic groups are present. In the body, there may be metabolism of the PU TDI-1 structures, however due to the structural similarity of the parent compounds any metabolites are also likely to be similar.

Further information is available in the read-across justification document in Section 13 of IUCLID.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 3 October 2011 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Procedure
Preparation of inoculum
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 2.5 g/l prior to use.

* Rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven
Duration of test (contact time):
28 d
Initial conc.:
14 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
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 36.0 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 30 to 100 ml/min per vessel and stirred continuously by magnetic stirrer. The flow rates were adjusted to 40 ml/minute where necessary.
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 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 reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved Organic carbon (DOC) analysis:
Samples (30 ml) were removed from the test item and toxicity control vessels on Day 0 prior to the addition of the test item in order to calculate the Inorganic Carbon content in the test media. The samples were filtered through 0.45 µm Gelman AcroCap filters (approximately 5 ml discarded) prior to DOC analysis.
DOC analysis of the test item dispersions after dosing was not possible due to the insoluble nature of the test item in water.
On Days 0 and 28 samples (30 ml) were removed from the control and reference item vessels 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-VCPN TOC analyser and a Shimadzu TOC-LCSH TOC Analyser. Samples (50 or 800 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 reference 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/Oxi 340I pH and dissolved oxygen meter.


Evalaution of data:
The test item contains 71.41% carbon (data supplied by the Sponsor) and so for a concentration of 10 mg C/l (a total of 42.0 mg of test item in 3 litres) the total organic carbon present was 30 mg C.
The theoretical amount of the carbon present in the reference item was calculated using the equation: number of carbon atoms x molecular wt of carbon divided by molecular wt of the reference item multiplied by 100%. Thus for a 10 mg C/l test concentration (a total of 51.4 mg of sodium benzoate in 3 litres) the total organic carbon present for sodium benzoate was 30 mg C.


Percentage degradation:
The percentage degradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, in the following equation:

The values of Replicates R1 and R2 are meaned for the control, test and reference items before substitution in the equation.

%ThCo2(=%degradation)=mgICin test flask-mgICin control divided by mg TOC as test chemical multiplied by 100%.

The percentage degradation from the results of the DOC analysis, see Table 4, is calculated from the equation below. Replicate values are corrected for the mean control value prior to calculation of percentage degradation.

Equation: % degradation=[1-mg DOC in test flask on day 28/mg DOC in test flask on day 0] x 100%.

The total CO2 evolution in the control vessels at the end of the test is calculated from the equation below. The inorganic carbon values for Replicates R1 and R2 on Day 28 are meaned before substitution into the equation.

Total CO2 evolution = mgIC in control x 100/%C of CO2 x 1/test volume.

Validation criteria:
The results of the degradation test are considered valid if in the same test the reference item yields equal to or greater than 60% degradation by Day 14. The test item may be considered to be readily biodegradable if equal 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.

For tables, see under results incl. tables section.


Reference substance:
other: Sodium benzoate
Test performance:
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 attached 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. Observations made on the contents of the test vessels are given in Table 6.
The total CO2 evolution in the control vessels on Day 28 was 26.75 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) 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.

For tables, see under results inlc. tables section.
Parameter:
% degradation (CO2 evolution)
Value:
26
Sampling time:
28 d
Details on results:
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 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 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 toxicity control attained 34% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.
Results with reference substance:
Sodium benzoate attained 77% degradation after 14 days and 91% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Analysis of the test media taken from the reference item culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), (see Table 4), gave percentage degradation values of 99% and 100% respectively for Replicates R1 and R2. 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.

For tables, see under results incl. tables section.

Table 1              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

1.17

1.75

1.05

1.05

1.40

1.17

1.75

1.17

1.63

1.28

1.28

1.87

1.05

1.28

2

5.22

-

8.35

-

22.27

-

19.03

-

8.70

-

8.00

-

23.43

-

6

11.65

-

16.72

-

38.75

-

35.41

-

17.53

-

15.92

-

35.18

-

8

12.96

-

20.87

-

43.34

-

39.33

-

20.07

-

18.69

-

39.22

-

10

13.68

-

18.70

-

43.43

-

40.59

-

23.71

-

20.75

-

38.99

-

14

18.25

-

21.87

-

45.67

-

40.46

-

23.12

-

24.03

-

40.57

-

21

20.96

-

20.73*

-

44.95

-

42.02*

-

25.80

-

27.72

-

-

-

28

21.39

-

22.40

-

52.75

-

44.46

-

28.45

-

31.02

-

-

-

29

23.16

2.90

24.16

3.25

54.11

3.37

47.09

3.48

29.62

3.13

32.73

3.48

-

-


R1– R2= Replicates 1 and 2

Abs= CO2absorber vessels

*Duplicate sample analysed as the original sample result was deemed anomalous

Table 2              Percentage Biodegradation Values

Day

% Degradation

Sodium Benzoate

% Degradation

Test Item

% Degradation

Test Item plus Sodium Benzoate Toxicity Control

0

0

0

0

2

46

5

28

6

76

8

35

8

81

8

37

10

86

20

38

14

77

12

34

21

75

20

-

28

89

26

-

29*

91

26

-


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

*Day 29 values corrected to include any carry-over of CO2 detected in Absorber 2

Table 3              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

10.10

0.38

4

Sodium Benzoate

10 mg C/l R2

9.99

-0.13

0

Test Item

10 mg C/l R1

10.21**

-0.35

0

Test Item

10 mg C/l R2

10.34**

0.12

1

Test Item plus Sodium Benzoate Toxicity Control

20 mg C/l

20.20**

0.16

1


R1– R2= Replicates 1 and 2

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

**Total carbon value given is the sum of the TC value obtained from analysis and the nominal TC contribution of the test item and sodium benzoate where applicable

Table 4              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

9.71

97

0.05

1

99

Sodium Benzoate

10 mg C/l R2

10.12

101

     <control

0

100


R1– R2= Replicates 1 and 2

*Corrected for control values.

Table 5              pH Values of the Test Preparations on Day 28

Test Vessel

pH

Control R1

7.5

Control R2

7.5

Sodium Benzoate

10 mg C/l R1

7.6

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

Table 6              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 cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

 

R2

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Light brown cloudy dispersion

Reference Item

R1

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

 

R2

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Light brown cloudy dispersion, no undissolved reference item visible

Test Item

R1

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

 

R2

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout

Toxicity Control

 

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small particles of test item visible floating on surface and dispersed throughout. No undissolved 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: Cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
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:

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 301B, "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.

Results and Conclusion

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.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 September 2009 - 30 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Version / remarks:
Adopted 7 July 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
EC Publication Number L142/496, May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Sewage plant of Rossdorf, Germany
- Preparation of inoculum for exposure: The aerobic activated sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and centrifuged again; and the procedure was repeated three times. The sediment was re-suspended in test water and aerated overnight. An aliquot of the final sludge suspension was weighed, and dried and the ration of wet sludge to dry weight was determined.
- Concentration of sludge: 1.5 g dry material per litre were mixed with test water
- Initial concentration: 5 mL activated sludge added to test vessel with a final volume including test water and treatment of 244 mL
- Water filtered: no
- Type and size of filter used, if any: Not applicable
Duration of test (contact time):
28 d
Initial conc.:
103 mg/L
Based on:
test mat.
Remarks:
Loading rate
Initial conc.:
281 mg/L
Based on:
ThOD
Remarks:
ThOD Nh4
Initial conc.:
322 mg/L
Based on:
ThOD
Remarks:
ThOD NO3
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
BOD demand calculated
Details on study design:
TEST CONDITIONS
- Composition of medium: 5 mL of the prepared activated sludge was applied in each test along with reconstituted test water. This was prepared by adding analytical grade salts to deionised water as follows:
a) 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 x 2 H20, 0.5 g NH4CL filled up with deionised water to 1000 mL
b) 22.5 g MgSO4 x 7 H20 filled up with deionised water to 1000 mL
c) 36.4 g CaCl2 x 2 H20 filled up with deionised water to 1000 mL
d) 0.25 g FeCl3 x 6 H20 filled up with deionised water to 1000 mL;
In order to avoid precipitation of iron hydroxide after storage and before use, one drop of concentrated HCl per litre was added.
10 mL of stock solution a) and 1 mL of stock solutions b) to d) were combined and filled up to a final volume of 1000 mL with deionised water.
- Solubilising agent (type and concentration if used): None
- Test temperature: 22 °C
- pH: 7.6 at the start of the test; 6.6 - 7.6 at the end of the test
- pH adjusted: no
- CEC (meq/100 g): Not reported
- Aeration of dilution water: None reported
- Suspended solids concentration: Not reported
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Manometric Test System with test flasks containing a volume of approximately 500 mL
- Number of culture flasks/concentration: 2 for test item and inoculum control; 1 for the procedure control, abiotic control and toxicity control.
- Method used to create aerobic conditions: Flasks were incubated under continuous stirring
- Method used to create anaerobic conditions: Not applicable
- Measuring equipment: BSB/BOD-Sensor-System
- Test performed in closed vessels due to significant volatility of test substance: Yes; test performed in closed flask to capture evolved carbon dioxide and to measure the change in pressure.
- Test performed in open system: No
- Details of trap for CO2 and volatile organics if used: Evolved CO2 was absorbed in an aqueous solution (45 %) of potassium hydroxide
- Other: pH measured in a separately prepared test flask with test item at test start, and in all flasks at the end of the test using a pH electrode.

SAMPLING
- Sampling frequency: Daily
- Sampling method: The change in pressure was measured by manometric method.
- Sterility check if applicable: not applicable
- Sample storage before analysis: Not applicable

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control: Yes; poisoned with HgCl2 (stock solution 48.72 mg/mL)
- Reference item: Yes, sodium Benzoate
- Toxicity control: Yes, sodium Benzoate and test item

STATISTICAL METHODS: The biodegradability (% BOD = mg O2 per mg test item) exerted after each period was calculated as:
BOD = (mg O2 uptake of test item – mg O2 uptake of inoculum control) / mg test item in flask
For the toxicity control, the BOD of the treatment is the sum of the test item and reference item and is calculated as the sum of test item and reference item concentration in the flask. The percentage biodegradation of the test item and of the reference item was calculated as:
% biodegradation = (BOD/ThODNH4) * 100
For nitrification it was calculated as:
% biodegradation = (BOD/ThODNO3) * 100
The ThODNH4 was calculated to be 2.728 mg O2/mg test item, and ThODNO3 was calculated to 3.130 mg O2/mg test item
Reference substance:
benzoic acid, sodium salt
Test performance:
Inoculum control: The oxygen demand of the inoculum control (medium and inoculum) was 20 mg O2/L; and thus not greater than 60 mg O2/L within 28 days as required by the test guideline.

pH-value: The pH-value of the test item flasks at end of the was within the range of pH 6.0 to 8.5 (observer 7.5) as required by the test guideline

Reference item: The percentage degradation of the regerence should reach the level of ready biodegradability (>60 %) within 14 days as required by the test guideline (97 % observed)
Key result
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
ThODNH4; The occurrence of nitrification was considered but not experimentally confirmed. The mean percentage biodegradation at the end of the 28-day exposure period was 0 % (ThODNH4). No 10-day window was passed.

ThODNH3: The mean percentage biodegradation at the end of the 28-day exposure period was also 0 % (ThODNH3). No 10-day window was passed.

The degradation rate of the test item did not reach 60 % after 28 days of incubation and it is therefore not considered to be readily biodegradable.
Parameter:
ThOD
Value:
2.728 mg O2/g test mat.
Results with reference substance:
The reference item sodium benzoate was sufficiently degraded to 85 % after 14 days and to 97 % after 29 days of incubation. If nitrification occurs, sodium benzoate will be degraded by 85 % after 14 days and 97 % at the end of the test. The percentage degradation of the reference item confirms the suitability of the used aerobic activated sludge inoculum.

In the toxicity control, 31 % biodegradation was noted within 14 days and 32 % biodegradation after 28 days of incubation based on ThODNH4. If nitrification occurs the biodegradation was 28 % and 29 % after 14 and 28 days, respectively, based on ThODNH3. The test item can be assumed to be not inhibitory to the aerobic activated sludge microorganisms.

In the abiotic control, the oxygen demand was zero; therefore, no correction of the test item degradation rates were required.

Table 2: Cumulative Biological Oxygen Demand (mg O2/L) in Test Flasks During the test period

 Time (days)  Flask No.
 1  2  3  4  5  6  7
 1  0  0  0  0  55  0  50
 2  0  0  0  0  85  0  80
 3  5  0  0  5  115  0  105
 4  5  5  5  5  120  0  110
 5  5  5  5  10  125  0  120
 6  5  5  5  10  130  0  125
 7  5  10  10  10  135  0  130
 8  10  10  10  10  140  0  135
 9  10  10  10  10  145  0  140
 10  10  10  10  10  145  0  140
 11  10  10  10  10  150  0  145
 12  10  10  10  10  150  0  145
 13  10  10  10  10  155  0  145
 14  10  10  10  10  155  0  150
 15  15  10  15  15  160  0  150
 16  15  15  15  15  165  0  155
 17  15  15  15  15  165  0  155
 18  15  15  15  15  170  0  160
 19  15  15  15  15  175  0  160
 20  15  15  15  15  175  0  160
 21  15  15  15  15  175  0  160
 22  15  15  15  15  180  0  160
 23  15  15  15  15  180  0  160
 24  15  15  15  15  180  0  160
 25  15  15  15  20  180  0  160
 26  15  20  15  20  180  0  160
 27  20  20  20  20  185  0  165
 28  20  20  20  20  185  0  165

Flasks 1 and 2: Test item

Flasks 3 and 4: Inoculum control

Flask 5: reference (procedure control)

Flask 6: abiotic control

Flask 7: toxicity control

Table 3: Percentage Biodegradation of Test Item, Sodium Benzoate and of the Toxicity Control based on ThODNH4

Time (days)  Percentage Biodegradation1
 Test Item1  Sodium Benzoate2  Toxicity Control1,2
 Flask 1 (%)  Flask 2 (%)  Flask 5 (%)  Flask 7 (%)
 1  0  0  32  11
 2  0  0  50  18
 3  1  -1  66  23
 4  0  0  68  23
 5  -1  -1  69  25
 6  -1  -1  72  26
 7  -2  0  74  27
 8  0  0  76  28
 9  0  0  79  29
 10  0  0  79  29
 11  0  0  82  30
 12  0  0  82  30
 13  0  0  85  30
 14  0  0  85  31
 15  0  -2  85  30
 16  0  0  88  31
 17  0  0  88  31
 18  0  0  91  32
 19  0  0  94  32
 20  0  0  94  32
 21  0  0  94  32
 22  0  0  97  32
 23  0  0  97  32
 24  0  0  97  32
 25  -1  -1  96  32
 26  -1  1  96  32
 27  0  0  97  32
 28  0  0  97  32

1ThODNH4 of test: 2.728 mg/mg

2ThODNH4 of sodium benzoate: 1.666 mg/mg

Table 4: Percentage Biodegradation of Test Item, Sodium Benzoate and of the Toxicity Control based on ThODNO3

Time (Days)   Percentage Biodegradation
 Test item  Sodium Benzoate  Toxicity Control
 Flask 1 (%)  Flask 2 (%)  Flask 5 (%)  Flask 7 (%)
 1  0  0  32  10
 2  0  0  50  16
 3  1  -1  66  21
 4  0  0  68  21
 5  -1  -1  69  23
 6  -1  -1  72  24
 7  -2  0  74  24
 8  0  0  76  25
 9  0  0  79  26
 10  0  0  79  26
 11  0  0  82  27
 12  0  0  82

 27

 13  0  0  85  27
 14  0  0  85  28
 15  0  -2  85  27
 16  0  0  88  28
 17  0  0  88  28
 18  0  0  91  29
 19  0  0  94  29
 20  0  0  94  29
 21  0 0  94  29
 22  0  0  97  29
 23  0  0  97  29
 24  0  0  97  29
 25  -1  -1  96  29
 26  -1  1  96  29
 27  0  0  97  29
 28  0  0  97  29

1ThODNO3of test: 3.130 mg/mg

2ThODNH4/ThODNO3 of sodium benzoate: 1.666 mg/mg

Table 5: pH-Values at the End of the Test

 Flask No.  Treatment  pH-value
 1  Test item  7.5
 2  Test item  7.5
 3  Inoculum control  7.5
 4  Inoculum control  7.5
 5  Procedure control  7.5
 6  Abiotic control  6.6
 7  Toxicity control  7.6
Validity criteria fulfilled:
yes
Remarks:
The oxygen demand of the inoculum control did not met the guideline requirement, but the test was deemed acceptable.
Interpretation of results:
not readily biodegradable
Conclusions:
The biodegradation of the test item was investigated using activated sludge under aerobic conditions, and 0 % degradation was observed during the 28 days of the study. The test item is therefore deemed not readily biodegradable.
Executive summary:

The ready biodegradability of the test item was investigated in an OECD 301-F and EC C.4-D guideline study using aerobic activated sludge. Due to the test item containing nitrogen, the evaluation of biodegradation was based on ThODNH4and ThODNO3. The study was conducted at a test item concentration of 103 mg/L corresponding to an oxygen demand of about 281 mg/L and 322 mg/L ThODNH4and ThODNO3, respectively. Alongside the test item, a reference item (sodium benzoate), a toxicity control (sodium benzoate and test item), an inoculum control and an abiotic control were also tested. The test item and inoculum control were performed in duplicate and the procedure, abiotic and toxicity controls consisted of one replicate. The change of pressure in the test flasks was measured by a manometric method, and the pH values were measured at the start and end of the test. All validity criteria for the study were met.

 

The mean percentage biodegradation based on ThODNH4at the end of the 28-day exposure period was 0 %. The mean percentage biodegradation based on ThODNH3at the end of the 28-day exposure period was also 0 %. The degradation rate of the test item did not reach 60 % after 28 days of incubation and it is therefore not considered to be readily biodegradable.

The study is a GLP compliant guideline experimental study and is fully adequate for assessment of this endpoint.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
The ready biodegradability of the test item was determined using the regulatory validated model Biowin v4.10 as supporting evidence for the read-across of the available experimental data. Further information on the predictions and model performance are available in the attached QMRF and QPRF.
Principles of method if other than guideline:
Ready biodegradability was determined using the model Biowin 4.10 in EPISuite v4.11 from the SMILES codes. Further information is available in the attached QPRF and QMRF.
GLP compliance:
no
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

Table 1: Ready biodegradation predictions

 Model  Sub-Model  SMILES  Prediction
 EPISuite v4.11  Biowin v4.10

 CCCCCCCCCCCCCCCCNC(=O)NC1=CC=C(C)C(NC(NCCCC

(=O)CC2=C(C)C=CC(NC(=O)NCCCCCCCC/C=C/CCCCCC)=C2)=O)=C1

Biowin1 (Linear Model Prediction): Biodegrades Fast

  Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Recalcitrant

  Biowin4 (Primary Biodegradation Timeframe): Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO 
  EPISuite v4.11    Biowin v4.10  

C(CC)CCCCCCCCCCCCCCCNC(=O)NC1=CC=C(C)C(NC(NCCCC(=O)CC2

=C(C)C=CC(NC(=O)NCCCCCCCC/C=C/CCCCCCCC)=C2)=O)=C1

 

Biowin1 (Linear Model Prediction): Biodegrades Fast

  Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Recalcitrant

  Biowin4 (Primary Biodegradation Timeframe): Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO

 

Validity criteria fulfilled:
yes
Remarks:
See QPRF for further information
Interpretation of results:
not readily biodegradable
Conclusions:
The test item was determined not to be readily biodegradable using the QSAR Biowin v4.10.
Executive summary:

The ready biodegradability of the test item was determined using Biowin v4.10 in the EPISuite v4.11 model based on the SMILES codes of representative constituents, to support the read-across of the available experimental data. All constituents within the test item are not predicted to readily biodegrade, supporting the read-across of the available data.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
The ready biodegradability of the test item was determined using the regulatory validated model Biowin v4.10 as supporting evidence for the read-across of the available experimental data. Further information on the predictions and model performance are available in the attached QMRF and QPRF.
Principles of method if other than guideline:
Ready biodegradability was determined using the model Biowin 4.10 in EPISuite v4.11 from the SMILES codes. Further information is available in the attached QPRF and QMRF.
GLP compliance:
no
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

Table 1: Ready biodegradation predictions

 Model  Sub-Model  SMILES  Prediction
 EPISuite v4.11  Biowin v4.10

CCCCCCCCCCCCCCCCNC(=O)NC1=CC=C(C)C(NC(=O)(Nc2cc

(CNC(=O)Nc3c(C)ccc(NC(=O)NCCCCCCCCC=CCCCCCC)c3)ccc2))C1

Biowin1 (Linear Model Prediction): Biodegrades Fast

  Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Recalcitrant

  Biowin4 (Primary Biodegradation Timeframe): Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO
  EPISuite v4.11    Biowin v4.10  

CCCCCCCCC=CCCCCCCCCNC(=O)Nc3ccc(C)c(NC(=O)NCc2cccc

(NC(=O)Nc1cc(NC(=O)NCCCCCCCCCCCCCCCC)ccc1C)c2)c3

Biowin1 (Linear Model Prediction): Biodegrades Fast

 Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Recalcitrant

  Biowin4 (Primary Biodegradation Timeframe): Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO

 

Validity criteria fulfilled:
yes
Remarks:
See QPRF for further information
Interpretation of results:
not readily biodegradable
Conclusions:
The test item was determined not to be readily biodegradable using the QSAR BioWin v4.10.
Executive summary:

The ready biodegradability of the test item was determined using Biowin v4.10 in the EPISuite v4.11 model based on the SMILES codes of representative constituents, to support the read-across of the available experimental data. All constituents within the test item are not predicted to readily biodegrade, supporting the read-across of the available data.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
The ready biodegradability of the test item was determined using the regulatory validated model Biowin v4.10 as supporting evidence for the read-across of the available experimental data. Further information on the predictions and model performance are available in the attached QMRF and QPRF.
Principles of method if other than guideline:
Ready biodegradability was determined using the model Biowin 4.10 in EPISuite v4.11 from the SMILES codes. Further information is available in the attached QPRF and QMRF.
GLP compliance:
no
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

Table 1: Ready biodegradation predictions

 Model  Sub-Model  SMILES  Prediction
 EPISuite v4.11  Biowin v4.10

 CCCCCCCCNC(=O)Nc2ccc(C)c(NC(=O)Nc1ccc(C)cc1)c2

Biowin1 (Linear Model Prediction): Biodegrades Fast

  Biowin2 (Non-Linear Model Prediction): Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Weeks - Months

  Biowin4 (Primary Biodegradation Timeframe): Days - Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO
  EPISuite v4.11    Biowin v4.10

Cc3ccc(NC(=O)Nc2ccc(C)c(NC(=O)Nc1ccc(C)cc1)c2)cc3

Biowin1 (Linear Model Prediction): Biodegrades Fast

  Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Months

  Biowin4 (Primary Biodegradation Timeframe): Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO

   EPISuite v4.11

  Biowin v4.10

 CCCCCCCCNC(=O)Nc1ccc(C)c(NC(=O)NCCCCCCCC)c1

 

Biowin1 (Linear Model Prediction): Biodegrades Fast

  Biowin2 (Non-Linear Model Prediction): Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Weeks

  Biowin4 (Primary Biodegradation Timeframe): Days - Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO

 

Validity criteria fulfilled:
yes
Remarks:
See QPRF for further information
Interpretation of results:
not readily biodegradable
Conclusions:
The test item was determined not to be readily biodegradable using the QSAR BioWin v4.10.
Executive summary:

The ready biodegradability of the test item was determined using Biowin v4.10 in the EPISuite v4.11 model based on the SMILES codes of representative constituents, to support the read-across of the available experimental data. All constituents within the test item are not predicted to readily biodegrade, supporting the read-across of the available data.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
The ready biodegradability of the test item was determined using the regulatory validated model Biowin v4.10 as supporting evidence for the read-across of the available experimental data. Further information on the predictions and model performance are available in the attached QMRF and QPRF.
Principles of method if other than guideline:
Ready biodegradability was determined using the model Biowin 4.10 in EPISuite v4.11 from the SMILES codes. Further information is available in the attached QPRF and QMRF.
GLP compliance:
no
Parameter:
probability of ready biodegradability (QSAR/QSPR)
Remarks on result:
not readily biodegradable based on QSAR/QSPR prediction

Table 1: Ready biodegradation predictions

 Model  Sub-Model  SMILES  Prediction
EPISuite v4.11 Biowin v4.10

CCCCCCCCCCCCCCCCNC(=O)NC1=CC(=CC=C1C)NC(=O)NCCCCCC

NC(=O)NC2=CC=C(C)C(=C2)NC(=O)NCCCCCCCCCCCCCCCC 

Biowin1 (Linear Model Prediction): Biodegrades Fast

  Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Recalcitrant

  Biowin4 (Primary Biodegradation Timeframe): Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO

EPISuite v4.11

Biowin v4.10

CCCCCCCCCCCCCCCCCCNC(=O)NC1=CC(=CC=C1C)NC(=O)N

CCCCCCNC(=O)NC2=CC=C(C)C(=C2)NC(=O)NCCCCCCCCCCCCCCCC

Biowin1 (Linear Model Prediction): Biodegrades Fast

  Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Recalcitrant

  Biowin4 (Primary Biodegradation Timeframe): Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO

EPISuite v4.11

Biowin v4.10

CCCCCCCCCCCCCCCCCCNC(=O)NC1=CC(=CC=C1C)NC(=O)NCCCCC

CNC(=O)NC2=CC=C(C)C(=C2)NC(=O)NCCCCCCCCCCCCCCCCCC

Biowin1 (Linear Model Prediction): Biodegrades Fast

  Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Recalcitrant

  Biowin4 (Primary Biodegradation Timeframe): Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO
EPISuite v4.11

Biowin v4.10

 CCCCCCCCC\C=C\CCCCCCCNC(=O)NC1=CC(=CC=C1C)NC(=O)

NCCCCCCNC(=O)NC2=CC=C(C)C(=C2)NC(=O)NCCCCCCCC\C=C\CCCCCCCC

Biowin1 (Linear Model Prediction): Biodegrades Fast

  Biowin2 (Non-Linear Model Prediction): Does Not Biodegrade Fast

  Biowin3 (Ultimate Biodegradation Timeframe): Recalcitrant

  Biowin4 (Primary Biodegradation Timeframe): Weeks

  Biowin5 (MITI Linear Model Prediction): Not Readily Degradable

  Biowin6 (MITI Non-Linear Model Prediction): Not Readily Degradable

  Biowin7 (Anaerobic Model Prediction): Does Not Biodegrade Fast

  Ready Biodegradability Prediction: NO 

 

Validity criteria fulfilled:
yes
Remarks:
See QPRF for further information
Interpretation of results:
not readily biodegradable
Conclusions:
The test item was determined not to be readily biodegradable using the QSAR BioWin v4.10.
Executive summary:

The ready biodegradability of the test item was determined using Biowin v4.10 in the EPISuite v4.11 model based on the SMILES codes of representative constituents, to support the read-across of the available experimental data. All constituents within the test item are not predicted to readily biodegrade, supporting the read-across of the available data.

Description of key information

On the basis of experimental data for three category members, and supporting QSAR predictions, TDI-category I substances are not considered readily biodegradable. The limited degradation may be a result of the low predicted water solubility of category members reducing the concentration and bioavailable fraction for degradation by micro-organisms. The category members only occur in a grease base and, in realistic use scenarios, the formulated greases are specifically designed to minimise leaching of the thickener. As such, exposure to the isolated thickener is not expected to occur and biodegradation processes in water, sediment or soil are not considered to be a relevant environmental fate process.

Key value for chemical safety assessment

Additional information

Biodegradation studies have been conducted on three substance that would fall within the category definition for the TDI-I category: Diurea 8 (Clarke 2011a), Tetraurea 2 (Clarke 2011b) and Reaction product of m-tolylidene diisocyanate and cyclohexylamine and cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine (Fiel 2009). Studies with Diurea 8 and Tetraurea 2 were conducted according to the OECD 301B guideline and demonstrated 17 % and 26 % biodegradation, respectively, after 28 days. The study Fiel (2009) was performed following the OECD 301F guideline, and the test item demonstated 0 % biodegradation during the test. Therefore, the experimental data on the substances indicate they are not ready biodegradable.

 

QSAR modelling using Biowin v4.10 in EPISuite v4.11 was performed for each category member where data are not available, to support read across of the experimental data. The QSAR modelling indicated that none of the category members are expected to be ready biodegradable, therefore the experimental data is deemed suitable for read across to the other category members. 

A conclusion of not readily biodegradable is considered appropriate for all category members.