Hexanoic acid, 2-ethyl-, C12-15-alkyl esters

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Test item information:
- Identification: Hetester FAO
- Appearance: Clear colourless liquid
- Batch: P7275
- Purity/Composition: UVCB
- Test item storage: At room temperature
- Stable under storage conditions until: 31 December 2018 (expiry date)

Additional information
- Test Facility test item number: 209079/A
- Purity/Composition correction factor: No correction factor required
- Test item handling: No specific handling conditions required
- Chemical name (IUPAC), synonym or trade name: C12-15 alkyl Octanoate
- CAS number: 90411-66-8
- Volatile: Not indicated
- Specific gravfity/density: 0.860 at 25 ºC
- Solubility in water: Insoluble
- Stability in water: Stable

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Test Item – Preparation and Test Loading Rates:
The batch of Hetester FAO tested was a clear colourless liquid (UVCB). No correction was made for the purity/composition of the test item.
The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the dark brown test-bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates (see Table 1). Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
Any residual volumes were discarded.

- Reference Item – Preparation and Test Concentrations:
The batch of activated sludge was checked for sensitivity by testing the reference item 3,5- dichlorophenol.
A 1 g/L stock solution was prepared by dissolving 250.8 mg 3,5-dichlorophenol in Milli-RO water and making up to a total volume of 250 mL. The pH as used for the test was 7.9. The 3,5-dichlorophenol stock solution was stored in a freezer (≤-15℃) until use. The reference item solution was defrosted at room temperature and diluted to reach the test concentrations. Volumes of 05, 1.6, 5.0 and 16 mL from the stock solution were added to the test medium resulting in the final test concentrations of 1.0, 3.2, 10 and 32 mg/L respectively.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Micro-organisms in activated sludge.

Source
Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

Preparation of the sludge
The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.5 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (= sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Approximately 10 minutes

Hardness:

0.4 g CaCl2.2H2O

Test temperature:

20 °C

pH:

7.5 ± 0.5

Dissolved oxygen:

The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.

Salinity:

Not applicable.

Conductivity:

Not reported.

Nominal and measured concentrations:

10, 100, and 1000 mg/l (loading rates)

Details on test conditions:

- Rationale: Recognized by international guidelines as the recommended test system.
- Contact time: 3 hours, during which aeration and stirring took place.
- Vessels: All glass open bottles/vessels.
- Milli-RO water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
- Synthetic medium (=sewage feed):
• 16 g peptone
• 11 g meat extract
• 3 g urea
• 0.7 g NaCl
• 0.4 g CaCl2.2H2O
• 0.2 g MgSO4.7H2O
• 2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

- Air supply: Clean, oil-free air.
- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- Test set up: See paragraph 4.7.1.
- Performance of the test: The synthetic medium (16 mL) made up to 50 mL with Milli- RO and 200 mL test item solution were mixed (total volume 250 mL). The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in two temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
- Oxygen recording: Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

- Medium: Adjusted ISO-medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
• CaCl2.2H2O: 211.5 mg/L
• MgSO4.7H2O: 88.8 mg/L
• NaHCO3: 46.7 mg/L
• KCl: 4.2 mg/L

Reference substance (positive control):

yes

Remarks:

3,5- dichlorophenol

Key result

Duration:

190 min

Dose descriptor:

EC50

Remarks:

ELR50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Key result

Duration:

190 min

Dose descriptor:

NOEC

Remarks:

NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Details on results:

- Inhibition of the Respiration Rate:
Detailed study results are presented in Table 4 of the attachment.
The combined limit/range-finding test showed no inhibition at 10 and 100 mg/L, and an average of 2% inhibition of the respiration rate at 1000 mg/L. Therefore, the ELR50 was above the highest loading rate tested (1000 mg/L).
No statistically significant inhibition of the respiration rate was observed at 1000 mg/L (the mean value was considered). Therefore, the NOELR was considered to be the highest loading rate.
There was no oxygen uptake from abiotic processes.

- Determination of Effect Parameters:
Table 3 shows the effect parameters based on Hetester FAO loading rates.

- Experimental Conditions:
The pH values in the individual vessels are presented in Table 4 of the attachment.
The pH in all test vessels, before addition of sludge was between 7.5 and 7.6. After the 3 hour exposure period the pH was between 7.3 and 8.1.
The temperature continuously measured in the temperature control vessels ranged between 20 and 22°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

Results with reference substance (positive control):

The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity.

Table 3; Effect Parameters

Parameter	Hetester FAO Loading rate (mg/L)
NOELR	1000
ELR50	>1000

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test Hetester FAO was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR).
The ELR50 was above 1000 mg/L.

Executive summary:

The objective of the study was to evaluate Hetester FAO for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the ELR₅₀ and/or the no- observed effect loading rate (NOELR).

The influence of Hetester FAO on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based onthe OECD guideline No. 209, 2010.

The batch of Hetester FAO tested was a clear colourless liquid (UVCB).No correction was made for the purity/composition of the test item.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test-bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested.Responses were compared to the controls.

Nostatistically significant inhibition of the respiration rate of the sludge was recordedat a loading rate of 1000 mg Hetester FAO per litre.Therefore, the ELR₅₀ was above the highest concentration tested (1000 mg/L).

There was no oxygen uptake from abiotic processes.

The batch of activated sludge was tested for sensitivity with the reference item 3,5- dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this present test, Hetester FAO was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR).

The ELR₅₀ was above 1000 mg/L.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L