hydrogenation products of (esterification products of 2-ethylhexan-1-ol with (Estolide formation products of oleic acid and Fatty acids, C8-18 and C18-unsatd. (branched or linear)).

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 29th, 2015 - February 27th, 2015

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Inoculum
Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht was used as inoculum with a concentration corresponding to 30 mg dry solids per litre. The treatment plant clarifies predominantly domestic wastewater and has a capacity of 600.000 inhabitant equivalents. Sampling date of activated sludge was on January 27th, 2015. Dry solid of the activated sludge was determined as 4.5 g/L by weight measurements after drying at 105°C (mean of triplicate measurements). The activated sludge was washed twice with tap water and one time with mineral medium by settling the sludge, decanting the supernatant and resuspending the sludge.

Duration of test (contact time):

29 d

Initial conc.:

20 mg/L

Based on:

TOC

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Method
The CO2-evolution test determines the ready biodegradability of test items according to the Modified Sturm Test. A solution or suspension of the test item in a mineral medium, corresponding to 10 - 20 mg TOC/L, is inoculated with activated sludge (30 mg d. s./L). The test vessels are aerated by the passage of carbon dioxide-free air and are incubated under aerobic conditions in diffuse light for 28 days. Degradation is followed by determining the carbon dioxide produced and absorbed to sodium hydroxide via IC-measurement (IC = inorganic carbon). The amount of carbon dioxide produced from the test item less the amount derived from the blank inoculum is expressed as a percentage of ThCO2 (theoretical amount of CO2). The pass level for ready biodegradability is 60% of ThCO2 and must be reached within a 10-d window. The 10-d window begins when the degree of biodegradation reaches 10%. The test method is suitable for water soluble and non-soluble test items which are non-volatile and not toxic under the test conditions.

Solutions
-Mineral medium
A:
---Potassium dihydrogenphosphate KH2PO4 8.50 g
---Dipotassium hydrogenphosphate K2HPO4 21.75 g
---Disodium hydrogenphosphate dihydrate Na2HPO4 * 2 H2O 33.40 g
---Ammonium chloride NH4Cl 0.50 g
---are dissolved in demineralised water and made up to 1 litre.
B:
---Calcium chloride dihydrate CaCl2 * 2H2O 36.4 g
---is dissolved in demineralised water and made up to 1 litre.
C:
---Magnesium sulfate heptahydrate MgSO4 * 7H2O 22.5 g
---is dissolved in demineralised water and made up to 1 litre.
D:
---Iron (III) chloride hexahydrate FeCl3 * 6H2O 0.25 g
---is dissolved in demineralised water, stabilised with one drop of concentrated HCl and made up to 1 litre.

For preparation of the mineral medium 10 mL of solution (A) is mixed with 900 mL demineralised water, 1 mL each of solutions (B), (C) and (D) are added and the volume is made up to 1 litre.

CO2-absorption medium
72.14 g NaOH was dissolved in 9 L deionised water in closed recipients (0.2 M NaOH). The inorganic carbon concentration of the 0.2 M NaOH was determined.

Test item
Due to the insolubility of the test item in water, the test item was added directly into the test vessels. Carrier material was inert PE film. 38.9 – 39.1 mg of the test item was weighed on PE film and added into each vessel. This corresponds to a test concentration of 20 mg/L organic carbon. PE-film was used as an inert carrier material.

Reference substance
A stock solution of 10 g/L sodium benzoate in water was prepared. 5.15 mL of this stock solution were added into the reference vessels corresponding to a concentration of 20 mg/L organic carbon.

Toxicity control
39.0 mg of the test item and 5.15 ml of the reference stock solution were added to the toxicity control vessel corresponding to a concentration of 40 mg/L organic carbon.

Experimental set up
The CO2-free air production system consists of an air compressor, three 1000 mL gas wash bottles filled with dry soda lime in series followed by one bottle filled with 0.1 M NaOH (sodium hydroxide). At the end of the system was one gas wash bottle filled with demineralised water, followed by an empty one to catch any drops of condensation water. A colour change of the soda lime from white to blue indicates that the CO2 absorption capacity is depleted. The CO2-free air is passed on to an air distributor with two input and 22 output channels and through PE-tubes.

In total three reactors containing the test item, three reactors containing only inoculum (blank), three reactors containing the reference compound and one reactor containing test item and reference compound (toxicity control) were set up.

Gas wash bottles (2000 mL volume) with lateral connecting pieces for butyl rubber septums were used as reactors. The liquid volume was fixed as 1500 mL each. Mixing was performed by a magnetic stirrer with 2 cm stir bars. 38.9 – 39.1 mg of the test item were added into the three test vessels (corresponding to a TOC concentration of 20.0 mg/L). The reference compound (5.15 mL of a 10 g/L stock solution) was added to the reference vessels. 39.0 mg of the test item and 5.15 ml of the reference stock solution were added to the toxicity control vessel.

The CO2 produced in the reactors was absorbed in two 250 mL gas wash bottles in series each filled with 200 mL 0.2 M NaOH. Sampling was performed through the lateral connecting pieces through the butyl rubber septum using 5 mL PE syringes.

Procedure
10 mL activated sludge was filled up to 1500 mL with 1490 mL mineral medium corresponding to 30 mg/L dry solids. The system was sealed and aerated with CO2-free air overnight. The reactors were kept mixed with magnetic stirrers. On the next day, after filling the absorber wash bottles with 0.2 M NaOH, the test item and reference compound were introduced and the test started. The aeration rate was kept at a rate of 30 - 100 mL / min (1.6 - 5.5 bubbles / second) and determined visually daily on working days. The determination by counting the gas bubbles over a defined period using a stop watch was made at day 11 and 28. The CO2-free air production system, the air-tightness of the whole experimental set-up, the aeration of the absorber flasks and the magnetic stirrers were controlled daily on working days.

At the beginning of the study the IC concentration of the 0.2 M NaOH used for the CO2- absorption flasks was determined as 2.9 ppm. The IC in the reactor at the beginning of the test showed insignificant amounts of IC (0.5 mg/L). On the 4th, 7th, 11th, 14th, 21st and 28th day 4 mL NaOH from the first of two CO2-absorber flasks connected in line was sampled and the IC's were determined. The vials were immediately closed with sealing film in order to avoid CO2 uptake from the air. On the 28th day 2 mL of 4M hydrochloric acid (HCl) was added into each reactor to release the CO2 dissolved in water. On day 29 the IC was determined in both CO2-absorber flasks in line.

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (CO2 evolution)

Value:

89.5

Sampling time:

28 d

Details on results:

Summary of the results
Test item:
The degradation extent of the test item at the end of the test was 89.5% (28 d after acidification, mean value of three replicates).
The mean degradation extent on day 11 was already higher than 60% (81.1%, mean value of three replicates). Therefore the criteria for ready biodegradation was met (10-d window).
The test item is ultimately biodegradable and reached the criterion for ready biodegradation.

Toxicity control:
The degradation extent in the toxicity control was 66.5% within 14 days, the test substance had no inhibitory effect on the inoculum.

Reference item:
The reference compound sodium benzoate reached the pass levels for ready biodegradability within 4 days.

Blank:
The mean CO2-evolution of the blank flasks was 33.3 mg/L on day 28 after acidification.

Before adding the test item, the IC in the reactor was determined, but only insignificant amounts of IC (0.6 mg/L) were found. The IC-concentration of the NaOH in the second CO2-absorber flasks in line, used as protective flasks, was below 10 ppm and was not considered in the data processing, because CO2 absorption from room air was its source.
The temperature was 20 – 23.°C throughout the whole study. The aeration rate was in the tolerated range of 1.6 – 5.5 bubbles/second (counted bubbles: 2.6 – 5.0 bubbles/second).
The degradation extents after acidification are lower than the one before acidification. This is caused by a higher CO2-evolution after the acidification in the blanks than in the test vessels.

Reactor	Day	0	4	7	11	14	21	28	29
7	Test flasks	0	56.6	79.6	92	94.1	104.7	102.6	99.6
8		0	51.6	72.1	76	77.2	82	80.2	78.2
9		0	37.8	63.1	75.3	79.5	91.4	92.3	90.7
4	Reference Flasks	0	72.2	81.1	84.6	88.4	93.7	93	91.9
5		0	73.4	84.8	91.5	91.2	98.6	99.1	97.3
6		0	84	95.7	99.1	99.7	106.9	104.1	103
19	Toxicity control: Test item	0	39.8	51.3	62.8	66.5	85.4	96	96.4

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The degradation extent of the test item at the end of the test was 89.5% (28 d after acidification, mean value of three replicates).
The mean degradation extent on day 11 was already higher than 60% (81.1%, mean value of three replicates). Therefore the criteria for ready biodegradation was met (10-d window).

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental Starting Date: 23 May 2012 Experimental Completion Date: 20 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained on 23 May 2012 from the final effluent stage of the Severn Trent Water PIc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Details on study design:

TEST CONDITIONS
- Composition of medium: The mineral medium was composed as follows:
Solution a was made up of the following:
KH2PO4 8.50 g/L
K2HPO4 21.75 g/L
Na2HPO4.2H20 33.40 g/L
NH4CI 0.50 g/L
pH = 7.4

Solution b CaCh 27.50 g/L
Solution c MgS04.7H2O 22.50 g/L
Solution d FeCl).6H2O 0.25 g/L

To 1 liter (final volume) of purified water* was added the following volumes of solutions a - d:
10 mL of Solution a
1 mL of Solution b
1 mL of Solution c
1 mL of Solution d

- Solubilising agent (type and concentration if used): not applicable
- Test temperature: The test was conducted in diffuse light at a temperature of 22 ± 1 ºC.
- pH: pH values for the test item were measured between 7.5 and 7.9 during the 28 day test period.
- pH adjusted: no
- Continuous darkness: The test was conducted in diffuse light.

Test item preparation:
An amount of test item (50 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh Sigma Lot No STBB8297V) prior to dispersal in 350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes). The high shear mixer head was then washed with mineral media (50 mL) and the washings and silica gel/test item/mineral medium dispersion added to the test vessel prior to the addition of the inoculum (5 mL) and adjusting to a final volume of 500 mL to give a final concentration of 100 mg/L.

TEST SYSTEM
The following test preparations were prepared and inoculated in 500 mL bottles:
a) Three replicate bottles containing inoculated mineral medium to act as the inoculum control plus 100 mg silica gel.
b) Two replicate bottles containing inoculated mineral medium plus 100 mg silica gel and the reference item, aniline, at a concentration of 100 mg/L.
c) Three replicate bottles containing inoculated mineral medium plus 100 mg silica gel and the test item at a concentration of 100 mg/L.
d) Two replicate bottles containing inoculated mineral medium plus 100 mg silica gel and the reference item, aniline, at a concentration of 100 mg/L and the test item at a concentration of 100 mg/L to act as toxicity control vessels.

All vessels were inoculated with the prepared inoculum at a rate of 1% vivo.

SAMPLING
The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.

As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask (see Figure 1). The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer's own battery backed memory was collected on the hard disk drive of a non-dedicated computer.

The remaining vessels which were not sampled were discarded and are not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes, three replicate bottles containing inoculated mineral medium to act as the inoculum control plus 100 mg silica gel.
- Toxicity control: For the purposes of the test, a toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the test.

An amount of test item (50 mg) was adsorbed onto the surface of 100 mg of granular silica gel (230-400 mesh Sigma Lot No. STBB8297V) prior to dispersal in 350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 15 minutes). The high shear mixer head was then washed with mineral media (50 mL) and the washings and test item/silica gel/mineral medium dispersion were then added to the test vessel with an aliquot (50 mL) of the 1000 mg/L aniline stock solution. The volume was adjusted to 500 mL with mineral medium to give a final concentration of 100 mg test item/L plus 100 mg aniline/L.

- Reference substance:
For the purposes of the test, a reference item, aniline (C6H5NH2), 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 (50 mL) of this stock solution was diluted with mineral medium (445 mL) and the inoculum (5 mL), to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.

STATISTICAL METHODS:
None mentioned in test report

Reference substance:

aniline

Preliminary study:

Not applicable

Test performance:

The mean BOD of the inoculated mineral medium (control) was 54.42 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The pH of the inoculated test item vessels on Day 28 ranged from 7.8 to 7.9 and hence satisfied the validation criterion given in the OECD Test Guidelines.

The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

The toxicity control (test item and aniline) should attain ≥ 25% degradation by Day 14 for the test item to be considered as non-inhibitory.

The percentage degradation of aniline calculated from oxygen consumption values must be ≥60% after 14 days. Test items giving a measured BOD value which is ≥60% of the ThOD value (within 28 days) are regarded as readily biodegradable. This level must be reached within 10 days of the degradation rate exceeding 10%.

Parameter:

% degradation (O2 consumption)

Value:

72

Sampling time:

28 d

Details on results:

The BOD values and the degree of degradation calculated from the oxygen consumption data for the inoculum control, test item, procedure control and the toxicity control are given in Table 1 (please see the additional information on results, including tables section below):

Results with reference substance:

The percentage degradation of aniline calculated from oxygen consumption values must be ≥ 60% after 14 days. The reference substance aniline was recorded to of degraded 68% after 28 days.

Table 1     BOD and Biodegradation Values:

Sample Description		ThOD (mg O2/L)	Day 7		Day 14		Day 28
			BOD (mg O2/L)	Degradation (%)	BOD (mg O2/L)	Degradation (%)	BOD (mg O2/L)	Degradation (%)	Mean Biodegradation (%)
Inoculum Control	R1	-	20.08	-	31.90	-	50.94	-	-
	R2	-	18.62	-	31.70	-	57.90	-
Procedure Control		309	91.54	23	225.36	63	264.82	68	-
Test Item	R1	298	152.64	45	231.00	67	272.52	73	72
	R2	298	105.92	29	166.10	45	263.22	70
Toxicity Control		607	126.12	18	423.24	64	462.86	67	-

R₁– R₂= Replicates 1 and 2

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable, but failing 10-day window

Conclusions:

The test item attained 72% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No. 301F the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. However, the test item has exhibited the potential for rapid degradation. In terms of the classification and labelling requirements (EU Directive for Dangerous Substances, L110A), the test item may be considered as readily biodegradable as evidence of >70% degradation has been shown over a 28-day period in a standard biodegradation study.

Executive summary:

Introduction:

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, "Ready Biodegradability; Manometric Respirometry Test" referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (q)).

Methods:

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at 22 ± 1 °C for 28 days. Following the recommendations of the International Standards Organisation (ISO 1995) and published literature (Handley et ai, 2002), the test item was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test item in the test medium and to increase the surface area of the test item exposed to the test organisms.

The degradation of the test item was assessed by the measurement of daily oxygen consumption values on Days 0 to 28. Control solutions with inoculum and the reference item, aniline, together with a toxicity control were used for validation purposes.

Results:

The test item attained 72% degradation after 28 days. Under the strict terms and conditions of OECD Guideline No. 301F the test item cannot be considered to be readily biodegradable as the test item failed to satisfy the 10-Day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation rate exceeding 10%. However, the test item has exhibited the potential for rapid degradation. In terms of the classification and labelling requirements (EU Directive for Dangerous Substances, L110A), the test item may be considered as readily biodegradable as evidence of >70% degradation has been shown over a 28-Day period in a standard biodegradation study.

Description of key information

In the two studies referenced, the test item showed high biodegradation. in the 2015 study it was shown that the test item achieved the 10 day window, thus the substance should be considered readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable