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:
2015-12-08 - 2016-04-13 (Experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals (1992) No. 301B "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)
Version / remarks:
Method C.4-C of Commission Regulation (EC) No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: pellets
Details on test material:
- Storage condition of test material: Room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): A mixed population of activated sewage sludge micro-organisms was obtained on 14 March 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- Laboratory culture: 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 (Rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven) 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.
- Concentration of sludge: The suspended solids concentration was equal to 3.6 g/L prior to use.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
5 other: mg C/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: The mineral medium used in this study was that recommended in the OECD Guidelines.
- Additional substrate:
- Solubilising agent (type and concentration if used): Following preliminary solubility work and 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.
- Test temperature: 22-24°C
- pH: 7.4 ± 0.2
- pH adjusted: yes
- Aeration of dilution water: yes, CO2-free air
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 5 liter test culture vessels each containing 3 liters of solution
- Number of culture flasks/concentration: duplicates
- Method used to create aerobic conditions: 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.
- Measuring equipment: The samples were analyzed for IC using a Shimadzu TOC-VCSH or TOC-LCSH TOC analyzer. Samples (50 or 135 μL) were injected into the IC channel of the TOC analyzer. IC analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid or 2M HCl using zero grade air as the carrier gas. Calibration was by reference solutions of sodium carbonate (Na2CO3). Each analysis was carried out in triplicate.
- Details of trap for CO2 and volatile organics if used: 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 water.

SAMPLING
- Sampling frequency: Samples (2 mL) were taken from the first CO2 absorber vessels on Days 0, 2, 6, 8, 10, 14, 21, 28 and 29. The second absorber vessels were sampled on Days 0 and 29.
- Sample storage before analysis: All samples were analyzed for IC immediately.

CONTROL AND BLANK SYSTEM
- Inoculum blank: An inoculated control, in duplicate, consisting of inoculated mineral medium plus a filter paper.
- 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 (455 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 6.8 mg test item/L plus 17.1 mg sodium benzoate/L equivalent to a total of 15 mg carbon/L.

STATISTICAL METHODS:
Statistical analysis of the Day 29 IC values for the inoculum control and test item 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

Results and discussion

Preliminary study:
Preliminary Solubility Work
From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO, 1995) it was concluded that the best testable dispersion was found to be obtained when the test item was dissolved in chloroform and an aliquot added to a GF/A filter paper. The paper dried and dispersed in approximately 400 mL of mineral medium using high shear mixing (approximately 7500 rpm for 5 minutes) and then made to 3 liters with mineral medium.
Test performance:
Sodium benzoate attained 71% biodegradation after 14 days and 75% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
% Degradation
Parameter:
% degradation (CO2 evolution)
Value:
0
Sampling time:
28 d
Details on results:
RESULTS

Definitive Test
An initial study was conducted at a concentration of 10 mg C/L. The toxicity control (containing both the test item and sodium benzoate) attained greater than 25 % degradation after 14 days, thereby satisfying the validation criteria given in the OECD Guidelines, indicating the test item to be none inhibitory to the activated sewage sludge micro-organisms. However, examination of the CO2 evolution values showed the values obtained for the replicate test item vessels to be significantly lower than the values obtained for the replicate control vessels. This showed that the test item may have caused an adverse effect to the activated sewage sludge micro-organisms.
Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test item, at a reduced concentration of 5 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at temperatures of between 22 and 24 °C, in darkness.

Validation Criteria
The total CO2 evolution in the inoculum control vessels on Day 28 was 29.14 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 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.

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 a decrease in all replicate vessels with the exception of inoculum control R1. 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 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
Statistical analysis of the Day 29 IC values for the control and test item vessels showed there were no statistically significant differences (P≥ 0.05) between the control and the test item. The test item was therefore considered not to have a toxic effect on the sewage sludge micro-organisms used in the study and this was confirmed by the toxicity control results.
The toxicity control attained 42% biodegradation after 14 days and 35% 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. The slight decrease in biodegradation between days 14 and 28 was considered to be due to sampling/analytical variation.
Sodium benzoate attained 71% biodegradation after 14 days and 75% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

BOD5 / COD results

Results with reference substance:
Sodium benzoate attained 71% biodegradation after 14 days and 75% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Table1 Inorganic Carbon Values on Each Analysis Occasion

Day

Inorganic Carbon(mgIC)

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

2.80

2.45

2.80

2.45

2.80

2.33

2.80

2.45

2.45

2.10

2.80

2.10

2.45

2.10

2

8.81

-

9.05

-

22.51

-

23.55

-

7.31

-

7.89

-

21.58

-

6

16.95

-

15.80

-

35.75

-

35.41

-

13.61

-

11.19

-

33.91

-

8

16.51

-

15.82

-

35.20

-

40.82

-

13.65

-

11.93

-

32.11

-

10

17.78

-

17.55

-

37.62

-

42.07

-

16.30

-

16.76

-

37.96

-

14

21.08

-

23.46

-

42.16

-

44.88

-

16.89

-

15.98

-

41.14

-

21

23.66

-

27.27

-

51.38

-

53.63

-

21.97

-

21.18

-

47.55

-

28

20.72

-

26.99

-

49.62

-

50.63

-

23.18

-

21.73

-

44.69

-

29

20.71

2.78

25.61

2.78

43.98

2.78

47.54

2.78

19.37

2.78

21.04

2.78

38.53

3.13

R1- R2  = Replicates1 and 2

Abs = CO2 absorber vessels

 

 

Table 2 Percentage Biodegradation Values

Day

% Biodegradation

Procedure Control

Test Item

Toxicity Control

0

0

0

0

2

47

0

28

6

64

0

39

8

73

0

35

10

74

0

45

14

71

0

42

21

90

0

49

28

88

0

46

29*

75

0

35

*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

5.02**

-0.17

0

 

5 mgC/L R1

 

 

 

 

Test Item5mg C/L R2

5.11**

0.03

1

 

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 pH Values of the Test Preparations on Days 0 and 28

Test Vessel

pH

Day0Pre-Adjustment

Day 0 Post-Adjustment

Day28

Inoculum Control R1

7.7

7.5

7.6

Inoculum Control R2

7.7

7.5

7.5

Procedure Control R1

7.7

7.5

7.6

Procedure Control R2

7.7

7.5

7.6

Test Item R1

7.8

7.5

7.5

Test Item R2

7.7

7.5

7.5

Toxicity Control

7.7

7.5

7.6

R1- R2  = Replicates 1 and 2

 

 

Table 5 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

Inoculum Control

R1

R2

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

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

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

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

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

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

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

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

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

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

Procedure Control

R1

R2

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

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

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

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

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

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

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

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

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

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

Test Item

R1

R2

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily

film of test item and foam visible on surface

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible on surface

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible on surface

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible on surface

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible on surface

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily

film of test item and foam visible on surface

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible on surface

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible on surface

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible on surface

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible on surface

Toxicity Control

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item and foam visible on surface, no undissolved reference item visible

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible

on surface, no undissolved reference item visible

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible

on surface, no undissolved reference item visible

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible

on surface, no undissolved reference item visible

Light brown cloudy dispersion with broken up pieces of filter paper

visible dispersed throughout. Slight oily film of test item visible

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:
under test conditions no biodegradation observed
Conclusions:
The study was conducted under GLP according to OECD guideline 301B on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the ready biodegradability of Octadecanoic acid, reaction products with tetraethylenepentamine.
The test item attained 0% 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:

A study was performed to assess the ready biodegradability of Octadecanoic acid, reaction products with tetraethylenepentamine 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 OCSPP 835.3110 (Paragraph (m)).

 

An initial study was conducted at a concentration of 10 mg C/L. The toxicity control (containing both the test item and sodium benzoate) attained greater than 25 % degradation after 14 days, thereby satisfying the validation criteria given in the OECD Guidelines, indicating the test item to be none inhibitory to the activated sewage sludge micro-organisms. However, examination of the CO2 evolution values showed the values obtained for the replicate test item vessels to be significantly lower than the values obtained for the replicate control vessels. This showed that the test item may have caused an adverse effect to the activated sewage sludge micro-organisms.

Therefore, following the recommendations of the Test Guidelines, in the definitive test, the test item, at a reduced concentration of 5 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at temperatures of between 22 to 24 °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. 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.

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.

 

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.