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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:
Experimental Starting Date: 12 May 2014 Experimental Completion Date: 26 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

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
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed. An approach endorsed by the International Standards Organisation (ISO, 1995) is to dissolve the test item in an auxiliary solvent prior to adsorption onto filter paper. High shear mixing was also applied to break up the filter paper containing the test item. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: BMS-587172-01
- Substance type: Chemical intermediate
- Physical state: White powder
- Analytical purity: 99.9%
- Lot/batch No.: 2G71626N
- Expiration date of the lot/batch: 27 August 2014
- Storage condition of test material:Room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 27 May 2014 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 twice 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 ºC 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 deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC 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 3.3 g/L prior to use.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
30 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
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 plus a filter paper*.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium plus a filter paper* to give a final concentration of 10 mg carbon/L.
c) The test item on a filter paper*, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item on a filter paper* plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).

TEST CONDITIONS:
Mineral medium: The mineral medium used in this study was that recommended in the OECD Guidelines.

A filter paper with acetone evaporated to dryness was added to the inoculum control and procedure control vessels in order to maintain consistency between these vessels and the test item vessels.

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 reference items, the vessels were filled with 2400 mL of mineral medium and 27.3 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 Hach HQ40d Flexi handheld meter. If necessary 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.

The pH of the test preparations was determined on Day 0 and on Day 28 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter

EXPERIMENTAL PREPARATION:
TEST ITEM:
Following the recommendations of the International Standards Organisation (ISO, 1995) and in the published literature (Handley et al, 2002) the test item was dissolved in an auxiliary solvent prior to adsorption onto filter paper*. High shear mixing was also applied to break up the filter paper containing the test item. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

An amount of test item (1031 mg) was dissolved in 10 mL of acetone to give a 1031 mg/10 mL solvent stock solution. An aliquot (483 µL) of this solvent stock solution was dispensed onto a filter paper and the solvent allowed to evaporate to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to inoculated mineral medium. The volume was then adjusted to 3 liters to give a final concentration of 16.6 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the solution.

Preparation of the test item dispersion was carried out under laboratory safety lighting.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.


CONTROL AND BLANK SYSTEM:
- Reference item:
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 was inverted several times to ensure homogeneity of the solution.

A filter paper* was added to each vessel in order to maintain consistency between the test and procedure control vessels. Acetone (483 µL) was dispensed onto each filter paper and evaporated to dryness for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition of each vessel.


- Toxicity control:
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 (483 µL) of the test item solvent stock solution was dispensed onto a filter paper* and the solvent allowed to evaporate for approximately 15 minutes. The filter paper was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes) prior to addition to the test vessel containing inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume was adjusted to 3 liters to give a final concentration of 16.6 mg test item/L plus 17.1 mg sodium benzoate/L equivalent to a total of 20 mg carbon/L.


DATA EVALUATION:
Observations: The appearance of the test preparations was recorded on Days 0, 5, 12, 19 and 26

Calculation of Carbon Content:
The test item contains 60.4% carbon (data supplied by the Sponsor) and so for a concentration of 10 mg C/L the total organic carbon present was 30 mg C.

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 atoms / 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 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,
The values of Replicates R1 and R2 are meaned for the control, test and standard materials before substitution in the equation.
%ThCo2(=%degradation)= [(mg IC in test flask - mg IC in control) / mg TOC as test material] x 100.
The percentage degradation from the results of the DOC analysis, 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:
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 control x 100/%C of Co2 x 1/test volume.

= mg IC in control x 100/27.29 x 1/3

STATISTICAL METHODS:
Statistical analysis of the Day 29 IC values for the control and test material vessels was carried out using a Student’s t-test to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
Sodium benzoate

Results and discussion

Test performance:
Definitive test:
An initial test was carried out, however by Day 6 the agreement between replicate vessels for the inoculum control, procedure control and test item was not acceptable and the test was halted. The test was subsequently repeated.

Following the recommendations of the International Standards Organisation (ISO 1995) the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper. High shear mixing was also applied to break up the filter paper containing the test item. This method was used in order to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to test organisms.

Validation Criteria
The total CO2 evolution in the inoculum control vessels on Day 28 was 28.33 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.

*Please note all tables can be found in the any other information on results section which can be found below.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
14
Sampling time:
28 d
Details on results:
Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1*. Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2* and the biodegradation curves are presented in Figure 1**. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3*. Observations made on the contents of the test vessels are given in Table 4*.

*Please note all tables can be found in the any other information on results section which can be found below.

** Please note figure 1 can be found in the illustration section below.

Biodegradation:
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 14% biodegradation 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 49% biodegradation after 14 days and 52% biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 81% biodegradation after 14 days and 85% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Table 1     Inorganic Carbon Values on Each Analysis Occasion

Day

Inorganic Carbon (mg IC)

Inoculum Control

Procedure Control

Test Item

Toxicity Control

R1

R2

R1

R2

R1

R2

R1

Abs1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

Abs 1

Abs 2

0

0.35

0.47

0.47

0.58

0.58

0.70

0.35

0.47

0.35

0.58

0.58

0.47

0.35

0.58

2

6.61

-

9.74

-

23.43

-

23.66

-

7.66

-

7.66

-

28.30

-

6

8.88

-

13.61

-

33.56

-

32.87

-

12.11

-

11.65

-

38.64

-

8

13.07

-

16.17

-

39.33

-

39.56

-

16.05

-

15.71

-

45.52

-

10

13.91

-

14.13

-

38.65

-

37.85

-

14.36

-

14.82

-

36.14

-

14

14.05

-

18.47

-

40.35

-

40.80

-

18.02

-

19.61

-

45.79

-

21

20.05

-

21.86

-

44.39

-

43.49

-

23.89

-

21.63

-

51.49

-

28

22.07

-

24.30

-

47.04

-

45.70

-

28.33

-

26.32

-

55.67

-

29

23.16

2.78

25.49

2.67

49.32

2.90

49.77

2.90

29.95

2.78

26.72

2.90

55.78

2.44

 


R1– R2          =   Replicates 1 and 2

Abs              =  CO2absorber vessels

Table2     Percentage Biodegradation Values

Day

% Biodegradation

Procedure Control

Test Item

Toxicity Control

0

0

0

0

2

51

0

34

6

73

2

46

8

83

4

52

10

81

2

37

14

81

9

49

21

77

6

51

28

77

14

54

29*

85

14

52


*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)

Test Item

10 mg C/L R1

9.50**

-0.18

0

Test Item

10 mg C/L R2

9.39**

-0.36

0


R1– R2     =  Replicates 1 and 2

*       Corrected for control values. Negative values are due to measured 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

Table 4     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

Inoculum Control

R1

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout

 

R2

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout

Procedure Control

R1

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. No undissolved reference item visible

 

R2

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. No undissolved reference item visible

Test Item

R1

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface

 

R2

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface

Toxicity Control

 

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface. No undissolved reference item visible

Light brown cloudy dispersion with small broken up pieces of filter paper dispersed throughout. Few fine particles of test item on surface. No undissolved reference item visible

 


R1– R2= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: The test substance was not readily biodegradable
Conclusions:
The test item attained 14% biodegradation 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; CO2Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 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 mineral medium in sealed culture vessels in the dark at approximately 21 °C for 28 days.

 

Following the recommendations of the International Standards Organisation (ISO 1995), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media by the use of high shear mixing. Using this method the test item is evenly distributed throughout the test medium and the surface area of test item exposed to the test organisms is increased thereby increasing the potential for biodegradation.

 

The biodegradation 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 14% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.