Ethyl pyruvate

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 29 August 2017 and 26 September 2017.

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 (incl. QA statement)

Specific details on test material used for the study:

Identification: EC 210-511-2
Physical state/Appearance: very pale yellow liquid
Batch: 61112
Purity: 98.54%
Expiry Date: 16 May 2018
Storage Conditions: room temperature in the dark

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 August 2017 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

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

Details on study design:

Test Item Preparation
The test item was dissolved directly in mineral medium.
A nominal amount of test item (500 mg) was dissolved in mineral medium and the volume adjusted to 500 mL to give a 1000 mg/L stock solution. An aliquot (50 mL) of this stock solution was diluted with mineral medium (445 mL) and inoculum (5 mL) to give the final test concentration of 100 mg/L. The volumetric flasks containing the stock solution and the test concentration were inverted several times to ensure homogeneity.
A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guidelines.
Inoculum control vessels were prepared containing mineral medium (495 mL) and inoculum (5 mL).

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 10 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 aliquot (50 mL) of the 1000 mg/L test item stock solution (see Section 3.5.1) and an aliquot (50 mL) of the 1000 mg/L aniline stock solution (see Section 3.5.2) was diluted with mineral medium (395 mL) and inoculum (5 mL) to 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 and test item stock solutions were prepared correctly, a diluted, 100 mg/L stock solution (in reverse osmosis water) was also sampled for Dissolved Organic Carbon (DOC) analysis.
All remaining 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 temperatures of between 22 and 24 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.

Reference substance:

aniline

Remarks:

Information as provided by the Supplier (Acros Organics). Physical state/Appearance: pale orange/brown liquid Batch: A0358342 Purity: 99.9% Expiry Date: 01 March 2018 Storage Conditions: room temperature, over silica gel

Key result

Parameter:

% degradation (O2 consumption)

Value:

97

Sampling time:

28 d

Details on results:

The mean BOD of the inoculated mineral medium (control) was 26.24 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 7.5 to 7.6 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 10-Day window was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The test item attained 97% biodegradation after 28 days, calculated from the oxygen consumption values, and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained with 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
The toxicity control attained 69% biodegradation after 14 days and 75% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro organisms used in the test.

Results with reference substance:

Aniline (procedure control) attained 72% biodegradation after 14 days and 77% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item			Toxicity Control
		R1	R2	Mean
0	0	0	0	0	0
1	1	1	0	1	0
2	2	45	44	45	14
3	3	63	64	64	20
4	23	73	74	74	25
5	52	77	78	78	42
6	59	79	81	80	58
7	63	81	82	82	63
8	65	82	84	83	66
9	66	82	84	83	67
10	68	83	85	84	68
11	69	84	86	85	68
12	71	85	87	86	68
13	71	86	87	87	69
14	72	87	89	88	69
15	73	89	90	90	70
16	74	91	91	91	71
17	74	93	92	93	71
18	75	94	93	94	72
19	75	94	94	94	73
20	76	95	95	95	73
21	76	95	95	95	74
22	76	95	95	95	74
23	76	96	96	96	75
24	76	96	96	96	75
25	77	96	96	96	75
26	77	96	96	96	75
27	77	97	97	97	75
28	77	96	97	97	75

 

R₁– R₂    =      Replicates 1 and 2

pH Values of the Test Preparations on Days 0 and 28

Test Vessel	pH
	Day 0	Day 28
Inoculum Control R1	7.4	7.5
Inoculum Control R2	7.4	7.5
Procedure Control	7.4	7.8
Test Item R1	7.4	7.6
Test Item R2	7.4	7.5
Toxicity Control	7.4	7.1

R₁– R₂    =      Replicates 1 and 2

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test item attained 97% biodegradation after 28 days, calculated from the oxygen consumption values, and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained with 10 days of the biodegradation 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:

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 temperatures of between 22 and 24 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 97% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Description of key information

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 temperatures of between 22 and 24 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 97% biodegradation after 28 days and satisfied the 10-Day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation 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