Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May 2013 - 28 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Amounts of test item (5, 50 and 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates). The pH of the test item dispersions was measured after stirring using a WTW pH/Oxi 3401 pH and dissolved oxygen meter and adjusted to between pH 7.0 and 8.0 if necessary.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): oily film visible on surface

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: A mixed population of activated sewage sludge micro-organisms was obtained on 30 May 2013 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 deg C overnight prior to use in the test. On the day of collection the activated sewage sludge (10 liters) was allowed to settle and 2 liters of the supernatant was removed to increase the suspended solids concentration. The remaining 8 liters was fed synthetic sewage sludge (400 mL). The pH of the sample on the day of the test was 7.2 measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.
The initial suspended solids concentration before removal of the supernatent was equal to 2.5 g/L. After removal of the supernatant and aeration overnight, the suspended solids concentration was repeated and was equal to 4.2 g/L. The activated sewage sludge sample was diluted with dechlorinated tap water (3 liters) and the suspended solids concentration was repeated and was equal to 3.0 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 +/- 2 deg C

pH:

7.0-7.3

Dissolved oxygen:

2.8-5.8 mg O2/L

Nominal and measured concentrations:

Nominal: 10, 100, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Material, size, headspace, fill volume: glass, 500 mL
- Aeration: yes, 0.5-1.0 L/min
- No. of vessels per concentration (replicates): 1 for 10 & 100 mg/L and 3 for 1000 mg/L
- No. of vessels per control (replicates):
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water

OTHER TEST CONDITIONS
- Light intensity: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration rate

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10x
- Range finding study
- Test concentrations: 0, 10, 100 or 1000 mg/L
- Results used to determine the conditions for the definitive study: Definitve study was not conducted based on the results of the range-finding study

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: oily film seen on surface during test
- Effect concentrations exceeding solubility of substance in test medium: yes

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 3-h EC50 = 6.0 mg/L

The dissolved oxygen concentrations after 30 minutes contact time in several of the vessels were below 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. This deviation to the study plan was considered to have had no adverse effect on the study given that all oxygen consumption values were measured/calculated over the linear portion of the traces.

No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of 20 ± 2 deg. C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference item gave a 3-Hour EC50 value of 6.0 mg/L, 95% confidence limits 4.6-7.9 mg/L.

Description of key information

One reliable study is available. In this study activated sewage sludge was exposed to aqueous dispersions of the substance at up to 1000 mg/L with the addition of synthetic sewage as a respiratory substrate for 3 hours. Based on the rate of respiration, the EC50 (3 h) was > 1000 mg/L and the NOEC (3 h) was 1000 mg/L. The substance is not harmful to microorganisms.

Additional information

One reliable study is available. In this study (Roulstone, 2013) performed under GLP according to OECD TG 209, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of 20 ± 2 deg. C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol. The 3-Hour EC50 value was > 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

The results of this study are supported by data available for the read across substances fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and their analogues is applicable based on the similarity in structure and physico-chemical properties. The substances with the CAS No. 131459-39-7 and EC 270 -290 -3 are structural analogues of one of the read-across substances (CAS No. 68424-30-6) and can therefore can also be used for read-across to EC 613 -848 -7.

The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

One study, investigating the toxicity to microorganisms, was available for Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234 -392 -1, CAS-No. 11138-60-6). This respiration inhibition test (Kuttler, 1998) with activated sludge (domestic) according to OECD 209 showed an increase about 43% of respiration rate at the nominal tested concentration of 10000 mg/L in comparison to the control. No inhibition of respiration rate was observed. Therefore the EC50 and EC10 (3h) can be stated as > 10000 mg/L (nominal).

In a static GLP-study conducted to OECD 209 (Mead, 1999) 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS-No. 131459-39-7), was mixed with activated sludge of predominantly domestic sewage. No effect on respiration rate was observed at the tested concentration (EC50 (3h) > 1000 mg/L, NOEC (3h) ≥ 1000 mg/L). Due to the low water solubility of the test substance, no toxicity was observed within the water solubility under the conditions tested.

In addition, in a third study (Comber and Coleman), a cell multiplication inhibition test with Pseudomonas putida according to Bringman and Kuehn (1980) for Fatty acids, C5-10, esters with pentaerythritol (EC 270 -290 -3, CAS-No. 68424-31-7), no inhibition on the growth was observed at the tested concentration (EC10 and EC50 (6h) > 10 mg/L (nominal)). As the test material in this study not completely dissolved in the test system, the tested concentration demonstrated the worst case.