Fatty acids, coco, tetraesters with bis[2,2-bis(hydroxymethyl)butyl] adipate

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:

15 May 2012 to 14 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study; well documented study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ECHA REACH Guidance Document "Guidance on information requirements and chemical safety assessment: Chapter R.7b: Endpoint specific guidance

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,6-bis({2,2-bis[(undecyloxy)methyl]butyl}) hexanedioate

EC Number:

700-772-5

Cas Number:

1190961-28-4

Molecular formula:

N/A - too complex

IUPAC Name:

1,6-bis({2,2-bis[(undecyloxy)methyl]butyl}) hexanedioate

Test material form:

other: liquid

Details on test material:

- Physical state: straw colored liquid
- 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:

Source:
A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire UK, which treats predominantly domestic sewage.

Culture medium: The culture medium used in this study was that recommended in the OECD guidelines.

Preparation:
The activated sewage sludge sample was washed two times 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 °C 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 °C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.


Concentration:
The suspended solids concentration was equal to 3.7g/l prior to use.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

10 other: mg/L carbon

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Pre-Study Solubility Work
An amount of test item (1000 mg) was dissolved in 10 mL of acetone to give a 1000 mg/10 mL solvent stock solution. An aliquot (420 uL) ofthis solvent stock solution was dispensed onto a filter pape 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 14.0 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 test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guideline.

TEST SYSTEM
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 4 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.

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 50 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 3400 mL of mineral medium and 54.1 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 WTW pH/Oxi 3401 pH and dissolved oxygen meter prior to the volume in all the vessels being adjusted to 4 liters by the addition of mineral medium which had been purged overnight with CO2 free air.

The test vessels were sealed and C02-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by magnetic stirrer.


-The C02 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.


-Following guidance given in the ECHA Reach Guidance Document "Guidance on information requirements and chemical safety assessment: Chapter R.7b: Endpoint specific guidance" the following modifications to a standard OECD 301B test were made:

Testing in larger volumes: The test volume employed was increased from 3 liters to 4liters.
Increasing the biomass: The concentration of inoculum used was increased from 30 mg suspended solids (ss)/L to 50 mg ss/L.

Reference substance

Reference substance:

benzoic acid, sodium salt

Results and discussion

% Degradation

Parameter:

% degradation (CO2 evolution)

Value:

70

Sampling time:

28 d

Details on results:

The test item attained 70% degradation after 28 days.

BOD5 / COD results

Results with reference substance:

Sodium benzoate attained 71% degradation after 14 days and 92% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Any other information on results incl. tables

Percentage Biodegradation Values

Day	%Degradation Sodium Benzoate Procedure Control	%Degradation Test Item
0	0	0
7	40	16
14	71	56
21	81	53
28	90	78
29	92	70

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test material attained 70% degradation after 28 days. The reference substance attained 92% degradation after 28 days

Executive summary:

Introduction

 

A study was performed to assess the biodegradability of the test item in an enhanced biodegradation test in an aerobic aqueous medium.  The method followed is based upon the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; C02 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (m)) however following guidance given in the ECHA Reach Guidance Document "Guidance on information requirements and chemical safety assessment: Chapter R.7b: Endpoint specific guidance" the following modifications to a standard OECD 301B test were made:

 

Testing in larger volumes: The test volume employed was increased from 3 liters to 4liters.

Increasing the biomass: The concentration of inoculum used was increased from 30 mg suspended solids (ss)/L to 50 mg ss/L.

 

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.  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 degradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate were used for validation purposes

 

 

 

Results

The test item attained 70% degradation after 28 days.