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Environmental fate & pathways

Biodegradation in water: screening tests

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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 29 November 2013 and 21 February 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
EC Number:
294-430-8
EC Name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
Cas Number:
91722-28-0
IUPAC Name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
Test material form:
liquid
Details on test material:
Identification: Internal Olefin Sulfonate
Description: pale yellow liquid
Storage conditions: approximately 4°C in the dark
The test item was heated to approximately 30°C and homogenized prior to use.
Specific details on test material used for the study:
Expiry date: 01 May 2013

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
-Test Species:

A mixed population of activated sewage sludge micro-organisms was obtained on 21 January 2013 from the aeration stage of the Severn Trent Water PIc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

-Preparation of Inoculum:
The activated sewage sludge sample was washed two times by settlement and resuspension in mineral 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 21°C 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 preweighed 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 deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105°C 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 3.0 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 test substance concentration
Initial conc.:
6.63 mg/L
Based on:
DOC
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
-Mineral Medium:
The mineral medium used in this study was that recommended in the OECD Test Guidelines.

-Preliminary Investigational Work:
In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge, samples were taken for Dissolved Organic Carbon (DOC) analysis and as part of the sample preparation the samples were either filtered or centrifuged to remove the sewage sludge solids. Thus the following work was conducted and samples analyzed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-LCSH TOC analyzer.

An amount of test item (500 mg) was dissolved in mineral medium (1 liter) to give a 500 mg/L stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter). A further amount of test item (500 mg) was dissolved in mineral medium and inoculated at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1 liter. Two samples were taken for DOC analysis; one after filtration through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and the other after centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 30 mg ss/L and then filtering or centrifuging as per the test item samples.


-Experimental Preparation:
For the purpose of the test, the test item was dissolved directly in mineral medium.

An amount of test item (1000 mg) was dissolved in mineral medium with the aid of ultrasonication (5 minutes) and the volume adjusted to 1 liter to give a 1000 mg/L stock solution. An aliquot (143.1 mL) of this stock solution was dispersed in inoculated mineral medium and the volume adjusted to 3 liters to give a final nominal concentration of 47.7 mg/L. The volumetric flask containing the test item stock solution was inverted several times to ensure homogeneity of the solution.

The test item contains 20.97% carbon (data supplied by the Sponsor) and so for a nominal concentration of 47.7 mg/L would give a test concentration of 10 mg carbon/L as recommended by the Test Guidelines. Dissolved Organic Carbon (DOC) analysis of the test preparations on Day 0 showed a measured concentration of 6.63 mg C/L and hence this value was used as the carbon content for the calculation of biodegradation values.


-Reference Item:
For the purposes of the test, a reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 5 minutes. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item stock solution was inverted several times to ensure homogeneity of the solution.


-Toxicity Control:
For the purposes of the test, a toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge microorganisms used in the test.

An aliquot (143.1 mL) of the test item stock solution was dispersed in inoculated mineral medium along with an aliquot (51.4 mL) of the sodium benzoate stock solution. The volume was adjusted to 3 liters to give a final concentration of 47.7 mg test item/L plus 17.1 mg sodium benzoate/L. Based on the measured DOC values in the test item vessels and the nominal carbon content of sodium benzoate this gave an equivalent test concentration of 16.63 mg carbon/L.


-Preparation of Test System:
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:

a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 6.63 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 16.63 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 22 ± 2°C in darkness.

Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 30 mL of inoculum and aerated overnight. On Day 0 the test and reference items were added and the pH of all vessels measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. The pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.

The test 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 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 first CO2 absorber vessels on Days 0, 3, 6, 9, 12, 14, 17, 22, 28 and 29. The second absorber vessels were all sampled on Days 0 and 29.

The samples taken on Days 0, 3, 6, 9, 12, 14, 17, 22, 28 and 29 were analyzed 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 analyzed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyzer or a Shimadzu TOC-VCSH TOC analyzer. Samples (300 or 50 µL) were injected into the IC (Inorganic Carbon) channel of the TOC analyzer. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M hydrochloric 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.


-Inorganic Carbon/Total Carbon Analysis:
Samples (30 mL) were removed from the inoculum control and test item vessels on Day 0 and filtered through 0.45 µm Gelman AcroCap filters (first approximate 5 mL discarded in order to pre-condition the filter) prior to DOC analysis.

The samples were analyzed for IC and TC using a Shimadzu TOC-VCPH TOC analyzer. Samples (50 µL) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyzer. 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 deionized water. Each analysis was carried out in triplicate.


-pH Measurements:
The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a WTW pH/Oxi 340I pH and dissolved oxygen meter.


-Evaluation of Data:
Calculation of Carbon Content:
The test item contains 20.97% carbon (data supplied by the Sponsor) and so for a nominal concentration of 47.7 mg/L the total organic carbon present would be 10 mg C/L, however DOC analysis of the test preparations on Day 0 showed measured organic carbon values of 6.63 mg C/L which were used for the calculation of biodegradation values.

The theoretical amount of carbon present in the reference item, sodium benzoate (C6H5COONa) was calculated as follows:

(No. of C atoms x mol wt of C / mol wt of sodium benzoate) x 100

(7 x 12.011/144.11) x 100 = 58.34%

Thus for a 10 mg C/L test concentration (a total of 51.4 mg of sodium benzoate in 3 liters) 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 provided, into the following equation. The values of Replicates R1 and R2 are averaged for the inoculum control, test and reference items before substitution into the following equation:

%ThCO2(= %degradation*) =
(mg IC in test flask - mg IC in control flask/ mg TOC added as test chemicaI) x 100

*the conversion factor for carbon to carbon dioxide is 3.67

The total CO2 evolution in the inoculum 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 (mg C/L) =
= (mg IC in controI) x (100/ %C of CO2) x (1/test volume)
= (mg IC in control) x (100 / 27.29) x (1/3)


-Validation Criteria:
The results of the degradation test are considered valid if in the same test the reference 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 time the plateau is reached, at the end of the test or at the end of the 10-Day window, as appropriate, is less than 20%.

The total CO2 evolution in the inoculum control vessels at the end of the test should not normally exceed 40 mg/L medium (= 120 mg/3 liters, corresponding to 33 mg C per flask), however values up to 70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are obtained should be critically examined.

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:
Preliminary Investigational Work:
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. 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.

The results did show that it was not possible to obtain DOC values that were near nominal based on the percentage carbon content of the test item. A reason could not be found for this, however similar results were obtained from analysis of the Day 0 test item samples and hence it was considered that this was a true effect. Therefore, after consultation with the Sponsor, measured DOC values on Day 0 of the test were used for the calculation of biodegradation values.
Test performance:
Test satisfied the validation criterion given in the OECD Test Guideline 301B.
% Degradation
Key result
Parameter:
% degradation (CO2 evolution)
Value:
105
Sampling time:
28 d
Details on results:
Definitive Test
An initial test started was on the 8 January 2013 however due to low results of percentage carbon this test was terminated and re-started on the 22 January 2013.

Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are provided. Percentage biodegradation values of the test and reference items and the toxicity control are provided and the biodegradation curves were plotted. Total and Inorganic Carbon values in the culture vessels on Day 0 are provided. The pH values of the test preparations on Days 0 and 28 are provided. Observations made on the contents of the test vessels are provided.

The total CO2 evolution in the inoculum control vessels on Day 28 was 33.39 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines 301B.

The IC content of the test item suspension in the mineral medium for Test Vessel R1 at the start of the test, 5.04% slightly exceeded the recommended level of 5% of the TC content given in the OECD Test Guidelines 301B. This was considered to be due to the relatively low TC concentration in the mineral medium and hence the IC contribution was relatively high. This was considered to have had no adverse effect on the study given that the CO2 evolution in the control vessels 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 Inoculum Control R1 and R2. This decrease was considered to be due to sampling/analytical variation. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The test item attained 105% 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.

The toxicity control attained 82% degradation after 14 days and 85% degradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 85% degradation after 14 days and 102% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions. Degradation values in excess of 100% were considered to be due to sampling/analytical variation.

Any other information on results incl. tables

Inorganic Carbon Values on Each Analysis Occasion

 

Day

Inoculum Control

(mg IC)

Sodium Benzoate

Procedure Control

(mg IC)

Test Item

(mg IC)

Test Item Plus Sodium Benzoate Toxicity Control

(mg IC)

R1

R2

R1

R2

R1

R2

R1

Abs 1

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

3.15

1.52

1.87

1.40

1.63

3.15

1.63

1.52

1.63

1.63

1.98

1.87

1.75

1.63

3

6.96

-

9.74

-

26.10

-

25.52

-

11.95

-

8.70

-

29.00

-

6

11.30

-

12.11

-

35.29

-

35.18

-

22.49

-

16.61

-

45.67

-

9

14.79

-

18.92

-

40.59

-

39.10

-

29.35

-

23.74

-

54.24

-

12

16.65

-

20.52

-

45.72

-

41.72

-

34.43

-

28.39

-

58.37

-

14

17.91

-

20.63

-

47.60

-

41.82

-

38.19

-

30.83

-

60.29

-

17

20.51

-

24.22

-

55.66

-

49.80

-

44.05

-

36.84

-

68.05

-

22

23.52

-

29.23

-

61.49

-

52.75

-

48.72

-

40.99

-

70.56

-

28

25.61

-

29.06

-

61.35

-

49.43

-

51.55

-

41.97

-

68.47

-

29

24.35

3.02

27.89

2.78

62.08

2.90

51.35

2.55

51.57

2.20

42.50

2.32

68.72

2.90

 

R1-R2 = Replicates 1 and 2

Abs = CO2 absorber vessels

 

Percentage Biodegradation Values

 

Day

% Degradation Sodium Benzoate Procedure Control

% Degradation Test Item

% Degradation Test Item Plus Sodium Benzoate Toxicity Control

0

0

0

0

3

58

10

41

6

78

39

68

9

77

49

75

12

84

65

80

14

85

77

82

17

101

91

92

22

102

93

89

28

94

98

82

29*

102

105

85

 

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

 

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)

Test Item

10 mg C/L R1

6.94

0.35

5

Test Item

10 mg C/L R2

6.33

-0.32

0

 

R1 -R2= Replicates 1 and 2

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

pH Values Of The Test Preparations On Days 0 And 28

 

Test Vessel

pH Prior To Adjustment On Day 0

pH After Adjustment On Day 0

pH On Day 28

Inoculum Control R1

 

7.8

7.5

7.5

Inoculum Control R2

 

7.7

7.4

7.5

Procedure Control

10 mgC/L R1

7.7

7.4

7.7

Procedure Control

10 mgC/L R2

7.6

-

7.6

Test Item

10 mg C/L R1

7.7

7.5

7.5

Test Item

10 mg C/L R2

7.7

7.4

7.5

Toxicity Control

20 mg C/L

7.6

-

7.7

 

R1 – R2 = Replicates 1 and 2

-         = No adjustment necessary

Observations On The Test Preparations Throughout The Test Period

 

Test Vessel

Observations On Test Preparation

Day 0

Day 6

Day 13

Day 20

Day 28

Inoculum Control  R1

               

               R2

 

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Light brown dispersion

Procedure Control     R1

 

  

            

               R2

Light brown dispersion no undissolved reference item visible

Light brown dispersion no undissolved reference item visible

Light brown dispersion no undissolved reference item visible

Light brown dispersion no undissolved reference item visible

Light brown dispersion no undissolved reference item visible

Light brown dispersion no undissolved reference item visible

Light brown dispersion no undissolved reference item visible

Light brown dispersion no undissolved reference item visible

Light brown dispersion no undissolved reference item visible

Light brown dispersion no undissolved reference item visible

Test Item                

                R1

 

 

 

 

                 

                R2

Light brown dispersion with foam on the surface, 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

Light brown dispersion with foam on the surface, 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 with foam on the surface, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

Light brown dispersion, no undissolved test or reference item visible

R1 -R2= Replicates 1 and 2

Dissolved Organic Carbon (DOC) Values From The Preliminary Investigational Work

 

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

 

Sample

DOC Concentration

% of Nominal Carbon Content

mg C/L

mg C/L Corrected For Appropriate Control

Mineral Medium

<LOQ

-

-

Control, Inoculated at 30 mg ss/L, Filtered

<LOQ

-

-

Control, Inoculated at 30 mg ss/L, Centrifuged

<LOQ

-

-

500 mg/L Untreated

72.93

72.93

70

500 mg/L Filtered

71.75

71.75

68

500 mg/L, Inoculated at 30 mg ss/L, Filtered

71.19

71.19

68

500 mg/L, Inoculated at 30 mg ss/L, Centrifuged

73.15

73.15

70

 

LOQ = Limit of Quantification (assessed as 1.0 mg C/L).

 

 

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

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item attained 105% 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 a biodegradation test. 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 an initial measured concentration of 6.63 mg Carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at 22 ± 2 °C for 28 days.


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


 


 


Results


The test item attained 105% 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.