Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 7 January 2010 and 12 February 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted to current accepted guidelines and to GLP with one exception (No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP.), which was considered not to affect the integrity of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15/09/09 Date of Signature: 26/11/09

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Sponsor's identification : Potassium 2-Phenyl Acetate
Description : white powder
Batch number : POK(S)-01
Date received : 14 December 2009
Expiry date : 24 November 2010
Storage conditions : room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge: Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK
- Laboratory culture: not reported
- Method of cultivation: not reported
- Storage conditions: not reported
- Storage length: not reported
- Preparation of inoculum for exposure: The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.8 g/l prior to use.
- Pretreatment: not reported
- Concentration of sludge: not reported
- Initial cell/biomass concentration: not reported
- Water filtered: yes
- Type and size of filter used, if any: GF/A filter paper was used inside a Buchner funnel.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
10 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST SPECIES:
A mixed population of activated sewage sludge micro-organisms was obtained on 12 January 2010 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

EXPERIMENTAL PREPARATION:
For the purpose of the test, the test item was dissolved directly in culture medium.
An amount of test item (300 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 300 mg/l stock solution. An aliquot (181 ml) of this stock solution was dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 18.1 mg/l, equivalent to 10 mg carbon/l. The volumetric flask containing the test item was inverted several times to ensure homogeneity of the solution.
A test concentration of 10 mg carbon/l was employed in the test following the recommendations of the Test Guideline.
Data from the control vessels was shared with similar concurrent studies.

TEST SYSTEM:
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard 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 standard 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 21°C, in darkness.
Approximately 24 hours prior to addition of the test and standard items the vessels were filled with 2400 ml of culture medium and 23.7 ml of inoculum and aerated overnight. On Day 0 the test and standard items were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.

The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute and stirred continuously by magnetic stirrer.

The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 ml Dreschel bottles containing 350 ml of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified de-gassed water.

SAMPLING:
CO2 analysis:
Samples (2 ml) were taken from the control, standard 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, standard and test item and on Day 0 for the toxicity control. All samples 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 standard solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.

Dissolved organic carbon (DOC) analysis:
Samples (20 ml) were removed from all culture vessels on Day 0 and from the control, standard and test item culture vessels on Day 28 and filtered through Gelman 0.45 µm AcroCap filters (approximately 5 ml discarded) prior to DOC analysis.
The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (C8H5KO4) and sodium carbonate (Na2CO3) in deionised water. Each analysis was carried out in triplicate.

pH 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.


CONTROL AND BLANK SYSTEM:
Toxicity Control
For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An aliquot (181 ml) of the test item stock solution was dispersed in inoculated culture medium along with an aliquot (51.4 ml) of the sodium benzoate stock solution. The volume was adjusted to 3 litres to give a final concentration of 18.1 mg test item/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.

Validation criteria
The results of the degradation test are considered valid if in the same test the standard item yields > 60% degradation by Day 14.
The test item may be considered to be readily biodegradable if > 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  25% degradation by Day 14 for the test item to be considered as non-inhibitory.
The test is considered valid if the difference of the extremes of replicate values of production of CO2 at the end of the test is less than 20%.
The total CO2 evolution in the control vessels at the end of the test should not normally exceed 40 mg/l medium.
The IC content of the test item suspension in the mineral medium at the beginning of the test should be < 5% of the TC.

Reference substance
Reference substance:
other: sodium benzoate

Results and discussion

Preliminary study:
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge (see attachment 1 - Appendix 2). Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.
Test performance:
The test item attained 103% degradation after 28 days. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
103
Sampling time:
28 d
Details on results:
Inorganic carbon values for the test item, standard item, toxicity control and control vessels at each analysis occasion are given in Table 1 (all tables are shown in the any other information on results section). Percentage biodegradation values of the test and standard items and the toxicity control are given in Table 2

Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3, and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4. The pH values of the test preparations on Day 28 are given in Table 5.
The total CO2 evolution in the control vessels on Day 28 was 43.57 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines. Although the CO2 evolution was in excess of 40 mg/l at the end of the test, this was considered not to affect the integrity of the study given that the upper level of 70 mg CO2/l given in the OECD Test Guidelines was not exceeded, and that all other validation criteria were satisfied.
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.

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 with the exception of control replicates R1 and R2 and standard item replicate R2. 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 103% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.

BOD5 / COD results

Results with reference substance:
The toxicity control attained 51% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Sodium benzoate attained 63% degradation after 14 days and 99% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Analysis of the test media from the test item culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4, gave percentage degradation values of 100% for both test item Replicates R1 and R2. Sodium benzoate attained 100% degradation for both Replicates R1 and R2 calculated from the results of the DOC analyses.
Observations made throughout the test period (see Table 6) showed the contents of the control vessels to be light brown dispersions and the contents of the standard item vessels to be light brown dispersions with no undissolved standard item visible. The contents of the test item vessels were light brown dispersions with no undissolved test item visible and the contents of the toxicity control vessel was a light brown dispersion with no undissolved test or standard item visible.

Any other information on results incl. tables

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

1.63

1.87

1.63

1.75

1.98

1.75

1.75

1.63

1.98

2.10

1.63

1.75

1.52

1.63

2

7.54

-

6.38

-

21.58

-

16.94

-

14.73

-

18.79

-

26.10

-

6

15.92

-

15.80

-

39.79

-

33.91

-

33.79

-

34.02

-

42.56

-

8

25.34

-

23.85

-

46.33

-

45.29

-

44.38

-

43.57

-

51.14

-

10

20.07

-

19.49

-

40.59

-

37.51

-

40.81

-

39.67

-

59.62

-

14

25.84

-

23.23

-

43.52

-

43.29

-

43.52

-

43.75

-

55.31

-

21

37.63

-

37.29

-

66.92

-

62.53

-

62.64

-

57.69

-

-

-

28

35.95

-

35.39

-

64.40

-

60.71

-

64.96

-

61.15

-

-

-

29

31.62

1.63

34.74

1.74

65.13

1.63

60.68

1.63

65.80

1.63

61.35

2.44

-

-



R1– R2= Replicates 1 and 2

Abs= CO2absorber vessels

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

41

33

32

6

70

60

45

8

71

65

44

10

64

68

66

14

63

64

51

21

91

76

-

28

90

91

-

29*

99

103

-


-= 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

10.73

0.07

1

Sodium Benzoate

10 mg C/l R2

10.46

-0.38

0

Test Item

10 mg C/l R1

10.55

0.20

2

Test Item

10 mg C/l R2

9.46

-0.18

0

Test Item plus Sodium Benzoate Toxicity Control

20 mg C/l

20.85

-0.14

0

 


R1– R2= Replicates 1 and 2

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

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

10.65

107

<control

0

100

Sodium Benzoate

10 mg C/l R2

10.83

108

<control

0

100

Test Item

10 mg C/l R1

10.34

103

<control

0

100

Test Item

10 mg C/l R2

9.62

96

<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

7.7

Control R2

7.7

Sodium Benzoate

10 mg C/l R1

7.8

Sodium Benzoate

10 mg C/l R2

7.7

Test Item

10 mg C/l R1

7.8

Test Item

10 mg C/l R2

7.7


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 5

Day 12

Day 19

Day 26

Control

R1

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

 

R2

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Standard item

R1

Light brown dispersion, no undissolved standard item visible

Light brown dispersion, no undissolved standard item visible

Light brown dispersion, no undissolved standard item visible

Light brown dispersion, no undissolved standard item visible

Light brown dispersion, no undissolved standard item visible

 

R2

Light brown dispersion, no undissolved standard item visible

Light brown dispersion, no undissolved standard item visible

Light brown dispersion, no undissolved standard item visible

Light brown dispersion, no undissolved standard item visible

Light brown dispersion, no undissolved standard item visible

Test Item

R1

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

 

R2

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Light brown dispersion, no undissolved test item visible

Toxicity Control

 

Light brown dispersion, no undissolved test or standard item visible

Light brown dispersion, no undissolved test or standard item visible

Light brown dispersion, no undissolved test or standard item visible

-

-



R1– R2= Replicates 1 and 2

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

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Please refer to section overall remarks
Interpretation of results:
readily biodegradable
Conclusions:
The test item attained 103% degradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.
Executive summary:

Introduction:

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

Methods:

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

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

Results:

The test item attained 103% degradation after 28 days and satisfied the10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.