Reaction mass of [2,2,4-trimethyl-1-(2-methylpropanoyloxy)pentan-3-yl] benzoate and [2,2,4-trimethyl-3-(2-methylpropanoyloxy)pentyl] 2-methylpropanoate and (3-benzoyloxy-2,2,4-trimethylpentyl) benzoate

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:

10.01.2013-07.02.2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes

Specific details on test material used for the study:

Description: clear colourless liquid
Batch: V046212201
Purity: not supplied
Expiry date: 30 April 2014
Storage conditions: room temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained on 10 January 2013 from the final effluent stage of the Severn Trent Water Plc seage treatment plant at Loughborough, Leciestershire, UK, which treats predominantly domestic sewage.
The sample effluent was allowed to stand and settle and the supernatent maintained on aeration in temperature controlled room at 21+-1 C prior to use.
The total viable count of the inoculum was 6.75 x 10^5 colony forming units/mL which was within the range of 10^3 to 10^6.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Pre-study solubility work
Information indicated that the water solubility of the test itme was negligeble. Therefore a pre-study solubility/dispersibility work was performed (study report appendix 3)

Experimental preparation
An amount of test item (50mg) was dispersed in 350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes). The test item/mineral medium dispersion was 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.
A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guideline.
Inoculum control vessels were prepared containing mineral medium (495 mL) and inoculum (5 mL)

Reference item
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. 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.

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 itme (50 mg) was dispersed in 350 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm, 5 minutes). The test item/mineral medium dispersion was then added to the test vessel with an aliquot (50 mL) of the 1000 mg/L aniline stock solution (see Section 3.5.3). 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.

Preparation of 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
b) Two replicate bottles containing inoculated mineral medium plus aniline at a concentration of 100 mg/L.
c) Three replicate bottles containing inoculated mineral medium and the test item at a concentration of 100 mg/L
d) Two replicate bottles containing inoculated mineral medium, 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% v/v.
On day 0 the test and reference items were added and the pH of all vessels measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.
All inoculum control, test item, procedure control and toxicity control vessels were placed in the CES Muli-Channel Aerobic Respirometer.
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 elctrolyzing 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 test was conducted in diffuse light at a temperature of 20+-1 C. On three occasions, the temperature was outside this range. However as this occured at the start of the study and the temperature settled after the initial few days of the study and all validation criteria were met, this was considered to have no adverse effect on the study.
On day 28, two inoculum control, one procedure control, two test item and one toxicity control vessels were sampled for pH.
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 witout jeopardizing the integrity of the test.

Reference substance:

aniline

Preliminary study:

Information indicated that the water solubility of the test itme was negligeble. Therefore a pre-study solubility/dispersibility work was performed (study report appendix 3)

Key result

Parameter:

% degradation (O2 consumption)

Value:

94

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 adn the toxicity control are given in Table 1 in the report. The daily BOD values and percentage degradation values are given in Appendices 4 and 5 in the report. The calculated BOD value for the procedure control is given in Appendix 6 and the Chemical Oxygen Demand for the test item is given in Appendix 7 in the report. BOD curves and biodegradation curves are given in Figures 2 and 3 in the report.
Daily temperature readings are given in Table 2 and pH values of each individual vessel on Day 0 and 28 are given in Table 3.
The mean BOD of the inoculated mineral medium (control) was 49.17 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 7.7 to 8.2 and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between extreme BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Gudielines.
The test item attained 94% degrdation after 28 days, calculated from the oxygen consupmtion values, and satisfied the 10-day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under strict terms and conditions of OECD Guideline No.301F.
The toxicity control attained 45% degradation after 14 days and 62% degradation after 28 days thereby confirming that the test itme was not toxic to the sewage treatment micro-organisms used in the test.
Aniline (procedure control) attained 60% degradation after 14 days and 61% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
The carbon content from the inoculums was 0.07 mg/L which is lower than 10% of that from the test item.
The total viable count of the inoculum was 6.75 x 10^5 colony forming units/mL which was within the range of 10^3 to 10^6.

Key result

Parameter:

ThOD

Value:

3.09 other: mg O2/mg test material

Key result

Parameter:

COD

Value:

2.66 other: mg O2/mg test material

Key result

Parameter:

BOD5

Value:

>= 290.64 - <= 300.46 other: mg O2/L

Results with reference substance:

Aniline attained 60% degradation after 14 days and 61% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 94% degradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Executive summary:

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 Commision Regulation (EC) No. 440/2008, US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 (Paragraph (q)) and ISO 9408:1999 Water quality - Evaluation of ultimate aerobic biodegradability of organic compounds in aquaeos medium by determination of oxygen demand in a closed respirometer.

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 20 +- 1 C for 28 days.

The degradation of the test item was assessed by the measurement of daily oxigen 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 itme attained 94% degradation after 28 days and satisfied the 10 -day window validation criterion, whereby 6% degradation must be attained within 10 days of the degradation rate exceeding 10%. The test itme can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Description of key information

The test item attained 94% degradation after 28 days and satisfied the 10-day window validation criterion, whereby 60% degradation must be attained within 10 days of the degradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Type of water:

freshwater