Tall-oil fatty acids oligomeric reaction products with triethylenetriamine and gylcidyl tolyl ether

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:

This study was conducted between 11 July 2017 and 09 August 2017

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:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: Tall-oil fatty acids oligomeric reaction products with triethylenetetramine and glycidyl tolyl ether
CAS Number: 1628940-03-3
Physical state/Appearance: dark yellow viscous liquid
Batch: Ei 4015
Purity: not applicable – complex mixture
Expiry Date: 15 November 2021
Storage Conditions: room temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on
10 July 2017 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 2.8 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

13.7 mg/L

Based on:

test mat.

Initial conc.:

10 mg/L

Based on:

IC (inorganic carbon)

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Experimental Design and Study Conduct
Preliminary Solubility Work
Information provided by the Sponsor indicated that the solubility of the test item was unknown. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.
The following preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation:
i) Ultrasonication and High Shear Mixing: A nominal amount of test item (100 mg) was dispersed in 1 liter of deionized reverse osmosis purified water with the aid of shaking by hand for approximately 1 minute prior to ultrasonication for 30 minutes. This formed a cloudy dispersion. This was then subjected to high shear mixing (approximately 7500 rpm, 30 minutes) and formed a slightly cloudy dispersion.
This work confirmed that the test item was insoluble in water. Therefore the following additional solubility work was conducted to ascertain the best method to employ in the biodegradation test.
ii) Ultrasonication: A nominal amount of test item (50 mg) was dispersed in 400 mL of mineral media with the aid of ultrasonication for 15 minutes. The volume was then adjusted to a final volume of 3 liters with mineral media. This formed a slightly cloudy brown dispersion with a few particles of test item visible on the surface. After 24, 48 and 72 hours of magnetic stirring a cloudy dispersion was formed with a few particles of test item visible on the surface.
iii) High Shear Mixing: A nominal amount of test item (50 mg) was dispersed in
400 mL of mineral media with the aid of high shear mixing (approximately
7500 rpm, 15 minutes). The volume was then adjusted to a final volume of 3 liters with mineral media. This formed a clear colorless media column with particles of test item visible on the surface. After 24, 48 and 72 hours of magnetic stirring a cloudy dispersion was formed with a few particles of test item visible on the surface.
iv) Adsorption onto an Inert Support: A nominal amount of test item (50 mg) was weighed onto 500 mg of silica gel. The silica gel was dispersed in 400 mL of mineral media with the aid of high shear mixing (approximately 7500 rpm,
15 minutes). The volume was then adjusted to a final volume of 3 liters with mineral media. This formed a cloudy dispersion with silica gel/particles of test item visible dispersed throughout and on the surface. After 72 hours of magnetic stirring the appearance of the dispersion remained unchanged.
v) Adsorption onto an Inert Support: A nominal amount of test item (50 mg) was weighed onto a filter paper. The filter paper was added to 3 liters of mineral media. This formed a clear colorless media column with the test item visible adhered to the filter paper at the bottom of the vessel.
vi) Addition onto an Inert Support: A nominal amount of test item (50 mg) was weighed onto a glass slide. The glass slide was added to 3 liters of mineral media. This formed a clear colorless media column with the test item visible adhered to the glass slide on the bottom of the vessel.
vii) Preliminary Solution in a Volatile Solvent: The addition of a test item solvent stock to glass fibre filter paper was attempted. A nominal amount of test item (1000 mg) was dissolved in acetone (10 mL) with the aid of shaking by hand for approximately 1 minute and ultrasonication for 3 minutes and formed a clear yellow solution. An aliquot (450 µL) of this solvent stock solution was dispensed to filter paper. The solvent was allowed to evaporate to dryness for approximately 15 minutes. The filter paper was then added to 400 mL of mineral medium and subjected to high shear mixing (approximately 7500 rpm, 5 minutes). The volume was then adjusted to 3 liters with mineral medium. This formed a cloudy dispersion containing many pieces of broken up filter paper visible dispersed throughout and on the surface. After 24, 48 and 72 hours of magnetic stirring this formed a very cloudy dispersion containing many pieces of broken up filter paper visible dispersed throughout and on the surface.
viii) Preliminary Solution in a Non-Volatile, Non-Degradable Solvent: A nominal amount of test item (100 mg) was dispersed in silicone oil (10 mL) with the aid of shaking by hand for approximately 1 minute followed by ultrasonication for 5 minutes. A cloudy dispersion was formed.
From the preliminary solubility work and following the recommendations of the International Standards Organisation (ISO 10634, (1995)) it was concluded that the best testable dispersion was found to be obtained when using the high shear mixing and solvent method of preparation.

Test Item Preparation
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.
A nominal amount of test item (822 mg) was dissolved in 10 mL of acetone to give a 822 mg/10 mL solvent stock solution. An aliquot (500 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 13.7 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.
A filter paper* was added to each control vessel in order to maintain consistency between the test and procedure control vessels. Acetone (500 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 to each vessel.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.
As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

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. 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 (500 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 to 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 (500 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
13.7 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 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).
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.
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 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 32.1 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 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 prior to acidification with hydrochloric acid, using a Hach HQ40d Flexi handheld meter.

IC Analysis
See "Details of Analytical Methods" (above)

Reference substance:

benzoic acid, sodium salt

Test performance:

The total CO2 evolution in the inoculum control vessels on Day 28 was 27.70 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.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0

Sampling time:

28 d

Remarks on result:

other: Not Readily Biodegradable

Details on results:

Definitive Test
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. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3. The pH values of the test preparations on
Days 0 and 28 are given in Table 4.

Validation Criteria and Biodegradation
The total CO2 evolution in the inoculum control vessels on Day 28 was 27.70 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.
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 Replicate 1 and procedure control Replicate 1.
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 37% biodegradation after 14 days and 47% 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.
Sodium benzoate attained 77% biodegradation after 14 days and 89% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

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	1.75	1.52	2.10	1.75	1.75	1.75	3.85/1.63*	1.75	3.15	1.75	1.75	2.10	1.75	1.75
2	3.59	-	8.70	-	23.20	-	18.91	-	8.35	-	5.22	-	11.37	-
6	7.50	-	11.42	-	35.87	-	27.33	-	14.30	-	6.92	-	24.45	-
8	13.87	-	13.30	-	37.61	-	27.29	-	12.61	-	13.07	-	24.65	-
10	15.16	-	15.05	-	33.63	-	37.28	-	14.02	-	14.48	-	24.62	-
14	17.00	-	17.11	-	40.35	-	40.23	-	15.87	-	16.09	-	39.44	-
21	21.86	-	20.51	-	44.73	-	47.55	-	18.93	-	17.35	-	40.45	-
28	22.74	-	22.62	-	47.60	-	45.59	-	20.27	-	19.93	-	44.13	-
29	22.15	2.78	22.82	2.78	47.54	2.78	50.66	2.78	20.71	2.78	22.49	2.78	50.29	3.13

R₁– R₂         =   Replicates 1 and 2

Abs             =  CO₂absorber vessels

*               =   Results from re-analysis of original sample

-              =      No Value determined

Table 2            Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	50	2	9
6	74	4	25
8	63	0	18
10	68	0	16
14	77	0	37
21	83	0	32
28	80	0	36
29*	89	0	47

*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		9.53^**		-0.35		0
Test Item 10 mg C/L R2		9.29^**		-0.49		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            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 ControlR1	7.6	-	7.6
Inoculum Control R2	7.5	-	7.6
Procedure Control R1	7.6	-	7.7
Procedure Control R2	7.7	7.6	7.6
Test Item R1	7.6	-	7.7
Test Item R2	7.6	-	7.7
Toxicity Control	7.7	7.6	7.8

 R₁– R₂    =      Replicates 1 and 2

-                          =     No adjustment necessary

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

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 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 temperatures of between 22 and 24 °C for 28 days.

Following the recommendations of the International Standards Organisation (ISO 10634, (1995)), the test item was dissolved in an auxiliary solvent prior to being adsorbed onto a filter paper and subsequent dispersal in test media. 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 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.

Description of key information

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. The registered substance is a UVCB/multiconstituent and therefore biodegradation studies should be treated with caution, due to the differing conversion rate of each chemical species/component into CO2.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed