Soybean oil, maleated, ester with triethanolamine

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 20 September 2016 Experimental completion date 18 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Version / remarks:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

Deviations:

no

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Version / remarks:

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical state/Appearance: clear amber viscous liquid

Purity: 100% product
Expiry Date: 24 June 2018
Storage Conditions: room temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, adapted

Details on inoculum:

The activated sewage sludge sample was washed twice by settlement and re-suspension 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 3.0 g/L prior to use.
* Rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven

Duration of test (contact time):

28 d

Initial conc.:

10 other: mg carbon/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

Determination of carbon dioxide produced

Details on study design:

Reference Item

Information as provided by the Supplier (Sigma-Aldrich).

Identification: Sodium benzoate
Physical state/Appearance: white granular solid
Batch: SLBM8408V
Purity: >99.5%
Expiry Date: 11 May 2017
Storage Conditions: room temperature over silica gel

Test System and Supporting Information

Inoculum

A mixed population of activated sewage sludge micro-organisms was obtained on
19 September 2016 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 re-suspension 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 3.0 g/L prior to use.

Medium

The mineral medium used in this study (see Annex 2) was that recommended in the OECD Guidelines.

Experimental Design and Study Conduct

Preliminary Solubility Work

Information provided by the Sponsor indicated that the test item was dispersibile in water. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation (see Annex 3).

Test Item Preparation

The test item was dispersed directly in mineral medium.

An amount of test item (44.1 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium.
The volume was adjusted to 3 litres to give a final concentration of 14.7 mg/L, equivalent to 10 mg carbon/L.

A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Reference Item Preparation

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 10 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 litres 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.





* Rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven


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 amount of test item (44.1 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (15 minutes) prior to dispersal in inoculated mineral medium. An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3 litres to give a final concentration of 14.7 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Preparation of Test System

The following test preparations were prepared and inoculated in 5 litre test culture vessels each containing 3 litres 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 10 mg carbon/L.

d) The test item 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).

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.

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 temperatures of between 22 and 24 oC, 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 Hach HQ40d Flexi handheld 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 litres 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 water.
Assessments

Observations

The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.

pH Measurements

The pH of the test preparations was determined on Day 0 and on Day 28 using a Hach HQ40d Flexi handheld meter.

Data Evaluation

Calculation of Carbon Content

The test item contains 68.15% 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 Divided by mol wt of sodium benzoate x 100
7 x 12.011 Divided by 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 Biodegradation

The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and reference items before substitution into the following equation:
% ThCO 2 (= % biodegradation *) = mg IC in test flask – mg IC in control flask divided by mg TOC added as test chemical x 100
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 divided by %C of CO2 x 1 divided by test volume
= mg IC in control x 100 divided by 27.29 x 1 divided by 3

* The conversion factor for carbon to carbon dioxide is 3.67

Validation Criteria

The results of the degradation test are considered valid if in the same test the reference item yields ≥ 60% degradation (in a 10-Day window) 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 litres, 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.

Major Computerized Systems
Shimadzu TOC TOC measurement Delta control system Building management

Reference substance:

other: Sodium benzoate (C6H5COONa)

Key result

Parameter:

% degradation (CO2 evolution)

Value:

19

Sampling time:

28 d

Remarks on result:

other: The test item attained 19% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Details on results:

Data Evaluation

Calculation of Carbon Content

The test item contains 68.15% 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 Divided by mol wt of sodium benzoate x 100

7 x 12.011 Divided by 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 Biodegradation

The percentage biodegradation or percentage of Theoretical Amount of Carbon Dioxide (ThCO2) produced is calculated by substituting the inorganic carbon values, given in Table 1, into the following equation. The values of Replicates R1 and R2 are meaned for the inoculum control, test and reference items before substitution into the following equation:

% ThCO 2 (= % biodegradation *) = mg IC in test flask – mg IC in control flask divided by mg TOC added as test chemical x 100

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 divided by %C of CO2 x 1 divided by test volume
= mg IC in control x 100 divided by 27.29 x 1 divided by 3

* The conversion factor for carbon to carbon dioxide is 3.67
Validation Criteria

The results of the degradation test are considered valid if in the same test the reference item yields ≥ 60% degradation (in a 10-Day window) 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 litres, 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.

Major Computerized Systems
Shimadzu TOC TOC measurement Delta control system Building management

Table1            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	2.10	2.10	2.10	2.10	2.10	2.10	2.10	2.10	2.10	2.22	2.10	2.10	2.10	2.22
2	7.42	-	6.96	-	22.16	-	25.29	-	11.72	-	9.86	-	27.38	-
6	14.65	-	14.65	-	32.76	-	32.29	-	24.68	-	18.11	-	47.06	-
8	19.03	-	17.54	-	39.10	-	44.38	-	23.97	-	24.19	-	60.77	-
10	19.61	-	18.24	-	43.66	-	41.15	-	25.42	-	27.59	-	55.63	-
14	24.82	-	21.31	-	43.41	-	44.88	-	29.24	-	28.79	-	59.73	-
21	30.76	-	27.27	-	50.93	-	56.90	-	34.25*	-	34.59*	-	69.74	-
28	34.83	-	30.80	-	52.64	-	60.14	-	39.65	-	37.41	-	64.18	-

R1– R2    =  Replicates 1 and 2 Abs=  CO2 absorber vessels

*        = Results from analysis of frozen sample

Table 2          Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	55	12	34
6	60	23	54
8	78	19	71
10	78	25	61
14	70	20	61
21	83	18	68
28	79	19	52

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	10.08**	0.19	2
Test Item 10 mg C/L R2	10.10**	0.14	1

R1– R2    =  Replicates 1 and 2

*           Corrected for 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
	Day 0 Pre-Adjustment	Day 0 Post-Adjustment	Day 28
Inoculum Control R1	7.7	7.6	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	7.7	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	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
Procedure Control	R1	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
	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
Test Item	R1	Light brown dispersionwith few particles of test item visible dispersed throughout	Light brown dispersionwith few particles of test item visible dispersed throughout	Light brown dispersionwith few particles of test item visible dispersed throughout	Light brown dispersionwith few particles of test item visible dispersed throughout	Light brown dispersion with few particlesof test item visible dispersed throughout
	R2	Light brown dispersionwith few particles of test item visible dispersed throughout	Light brown dispersionwith few particles of test item visible dispersed throughout	Light brown dispersionwith few particles of test item visible dispersed throughout	Light brown dispersionwith few particles of test item visible dispersed throughout	Light brown dispersion with few particlesof test item visible dispersed throughout
Toxicity Control		Light brown dispersion with few particles of test item visible dispersed throughout. No undissloved reference item visible	Light brown dispersion with few particles of test item visible dispersed throughout. No undissloved reference item visible	Light brown dispersion with few particles of test item visible dispersed throughout. No undissloved reference item visible	Light brown dispersionwith few particles of test item visible dispersed throughout. No undissloved reference item visible	Light brown dispersion with few particles of test item visible dispersed throughout. No undissloved reference item visible

R1– R2= Replicates 1 and 2

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Remarks:

The test item attained 19% biodegradation after 28 days

Conclusions:

The test item attained 19% 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; 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)).

 

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 temperatures of between 22 and 24^oC for 28 days.

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 19% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

The test item attained 19% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines 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 temperatures of between 22 and 24^oC for 28 days.

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 19% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.