1,2,3,4-Butanetetracarboxylic acid, tetramethyl ester, reaction products with 1,2,2,6,6-pentamethyl-4-piperidinol and β,β,β',β'-tetramethyl-2,4,8,10-tetraoxaspiro[5.5]undecane-3,9-diethanol

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:

The study was conducted between 22 November 2016 and 20 December 2016

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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:

Identification: LA-63P
Physical state/Appearance: extremely pale yellow powder
Batch: 112Z5
Purity: 100%
Expiry Date: 30 June 2017
Storage Conditions: room temperature in the dark

Reference Item
Information as provided by the Supplier (Fisher Scientific).
Identification: Aniline
Physical state/Appearance: yellow/orange liquid
Batch: 1479758
Purity: 99.99%
Expiry Date: 29 January 2017
Storage Conditions: room temperature

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic (adaptation not specified)

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained on 16 November 2016 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at temperatures of between 20 and 21 ºC prior to use.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Biological Oxygen Demand (BOD)

Details on study design:

Mineral Medium
The mineral medium used in this study was that recommended in the OECD Guidelines.

Experimental Design and Study Conduct
Preliminary Solubility Work
Information provided by the Sponsor indicated that the water solubility of the test item was less than 0.1 g/L at 25 °C. Therefore preliminary solubility/dispersibility work was performed in order to determine the most suitable method of preparation.

Test Item Preparation
The test item was dispersed directly in mineral medium.
An amount of test item (50 mg) was dispersed in mineral medium (350 mL) and subjected to high shear mixing (approximately 7500 rpm, 15 minutes) prior to the addition of inoculum (5 mL) and adjusting to a final volume of 500 mL with mineral media to give the test 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 Guidelines.

Reference Item Preparation
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.

Toxicity Control
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 dispersed in mineral medium (350 mL) and subjected to high shear mixing (approximately 7500 rpm, 15 minutes) prior to the addition of an aliquot (50 mL) of the 1000 mg/L aniline stock solution and inoculum (5 mL) to a final volume of 500 mL with mineral media give the test concentration of 100 mg test item/L and 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 and the reference item, 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.

Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.
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 Hach HQ40d Flexi handheld meter prior to the addition of the inoculum and the volume in all the vessels being adjusted to 500 mL by the addition of mineral medium.
In order to confirm that the aniline stock solution was prepared correctly, a diluted, 100 mg/L stock solution (in reverse osmosis water) was also sampled for Dissolved Organic Carbon (DOC) analysis.
All inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-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. 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 test was conducted in diffuse light at a temperature of 22 ± 1 ºC.
On Day 28, two inoculum control, one procedure control, two test item and one toxicity control vessel were sampled for pH analysis.
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.

Assessments
Oxygen Consumption Measurements
The daily Biological Oxygen Demand (BOD) values for the inoculum control, test item, procedure control and the toxicity control were recorded..

Physico-chemical Measurements
The temperature of the water bath was recorded daily.

pH Measurements
In order to confirm whether the pH of the test preparations changed, the pH was measured using a Hach HQ40d Flexi handheld meter on Days 0 and 28.

Reference substance:

aniline

Test performance:

The mean BOD of the inoculated mineral medium (control) was 46.56 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 7.6 to 7.7 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.

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Details on results:

The test item attained 0% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

There were statistically significant difference between the control and test item Day 28 BOD values. Care should therefore be taken in the interpretation of these results as the test item was considered to have exhibited an inhibitory effect at the test concentration employed in the study.

The toxicity control attained 27% biodegradation after 14 days and 26% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test. The slight decrease in biodegradation between Days 14 and 28 was considered to be due to biological variation.

Results with reference substance:

Aniline (procedure control) attained 75% biodegradation after 14 days and 78% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

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 as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Although the toxicity control attained 27% biodegradation by Day 14 of the test, care should be taken in the interpretation of these results as the test item was considered to have exhibited an inhibitory effect at the test concentration employed in the study as significant differences were shown between the inoculum control and test item biological oxygen demand values.

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 OCSPP 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 temperature 22 ± 1 ºC for 28 days.

The biodegradation 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 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Although the toxicity control attained 27% biodegradation by Day 14 of the test, care should be taken in the interpretation of these results as the test item was considered to have exhibited an inhibitory effect at the test concentration employed in the study as significant differences were shown between the Day 28 inoculum control and test item biological oxygen demand values.

Description of key information

The test item attained 0% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Although the toxicity control attained 27% biodegradation by Day 14 of the test, care should be taken in the interpretation of these results as the test item was considered to have exhibited an inhibitory effect at the test concentration employed in the study as significant differences were shown between the inoculum control and test item biological oxygen demand values.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed