Hexyl hexanoate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jun 2018 to 14 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures

Version / remarks:

OECD series on testing and assessment number 23, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Hexyl Caproate
Appearance: Clear colourless liquid
Purity/Composition: 99.29%
Test item storage: At room temperature

Analytical monitoring:

yes

Details on sampling:

Frequency at t=0 h and t=48 h
Volume 6 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Vehicle:

no

Details on test solutions:

The batch of Hexyl Caproate tested was a clear colourless liquid with a purity of 99.29% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.

Combined limit/range-finding test & Final test
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. The obtained mixture was allowed to settle for a period of 78-91 minutes. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Hydrolysis test
Saturated solutions were prepared in fivefold. The preparation started with individual loading rates of 100 mg/L. Subsequently, one replicate each was magnetically stirred for 1, 3, 6, 24 and 48 hours, respectively. The obtained mixtures were allowed to settle for a period of 17 minutes. Thereafter, the aqueous saturated solutions were collected by means of siphoning and analysed. All solutions were clear and colorless at the end of the preparation procedure.
Any residual volumes were discarded.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.

Source: In-house laboratory culture with a known history.

Reason for selection: This system has been selected as an internationally accepted invertebrate species.

Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20% , presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

Characteristics: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

CaCO3: 180 mg/L

Test temperature:

18-21°C, constant within ±1°C

pH:

7.1-8.1

Dissolved oxygen:

8.2-8.7 mg/l

Salinity:

Not applicable

Nominal and measured concentrations:

Solutions containing 10, 18, 32, 56 and 100% of the SS, prepared at a loading rate of 100 mg/L.

The results of analysis of the samples taken during the final test are described in Table 4 of the appended Analytical Report. Samples taken from the control and the highest test concentration were analysed. The measured concentration in the undiluted SS was 2.3 mg/L at the start of the test. This concentration decreased below the limit of detection at the end of the test. Based on these results, the average exposure concentration was calculated to be 0.14 mg/L in the undiluted SS and used to express the effect parameters.

Details on test conditions:

Test type Static
Test vessels 60 mL, all-glass.
Test medium The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
The hardness of test medium expressed as CaCO3: 180 mg/L with a pH between 6 and 9.

Combined Limit/Range-Finding Test:
The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and a SS prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the limit test with the following exceptions:
1.) Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to solutions containing 1.0 and 10% of the SS prepared at a loading rate of 100 mg/Lin the combined range-finding test.
2.) Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.

Final Test:
Number of daphnids: 20 per concentration.
Loading: 5 per vessel containing 50 mL of test solution.
Light: A daily photoperiod of 16 hours.
Feeding: No feeding.
Aeration: No aeration of the test solutions was applied.
Introduction of daphnids: 137 minutes after preparation of the test solutions.

Measurements and Recordings:
Immobility (including mortality) At 24 hours and at 48 hours.
pH and dissolved oxygen At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium Continuously in a temperature control vessel, beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate (See Appendix 1)

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Combined Limit/Range-Finding Test:
Table 1 shows the responses recorded during the combined limit/range-finding test.

No immobility was observed in the control or any of the concentrations tested throughout the test, except for one daphnid that was observed to be immobile at 24 and 48 hours of exposure in the 1.0% SS test group. Since up to 10% immobility is accepted in the control, effects below 10% were considered biologically not relevant.
All test conditions were maintained within the limits prescribed by the study plan.

Based on these results, samples taken from the control and the highest test concentration were analysed. The measured concentration in the undiluted SS was 1.2 mg/L at the start of the test. This concentration decreased to the level of what was observed in the blank QC sample (see also Table 2 of the appended Analytical Report).

Additionally, problems were encountered with the preparation of the Quality Control (QC) samples. These did not yield adequate recovery levels despite repeated preparation of the according standards. It was hypothesized that the test item may not be sufficiently stable in test medium and thus affected the recoveries during the analysis. It was subsequently decided in consultation with the sponsor to investigate the hydrolytic stability of the test item. To this end, saturated solutions were prepared and stirred for different periods of time to investigate whether a time-dependent concentration profile would be obtained this way.

Hydrolysis Test:
The analytical results of the samples taken during the hydrolysis test are described in Table 3 of the appended Analytical Report. The concentrations measured in the samples prepared with the test medium used in the present study increased from 1.1 mg/L after one hour of stirring to 2.2-2.7 mg/L after three to 48 hours of stirring. The prepared QC samples showed a sufficient recovery level.

It was thus concluded that the test item was hydrolytically relatively stable. The cause for the problems encountered with the QC samples in the preceding combined limit/range-finding test could not be determined subsequently. It was consequently decided to perform a full test with an unchanged protocol for preparing the test solutions.

Final Test:
Measured Test Item Concentrations:
The results of analysis of the samples taken during the final test are described in Table 4 of the appended Analytical Report. Samples taken from the control and the highest test concentration were analysed. The measured concentration in the undiluted SS was 2.3 mg/L at the start of the test. This concentration decreased below the limit of detection at the end of the test.

Based on these results, the average exposure concentration was calculated to be 0.14 mg/L in the undiluted SS and used to express the effect parameters.

Immobility:
Table 2 shows the responses recorded during the final test. No immobility was observed in the control and at any of the test concentrations throughout the exposure period. The responses recorded in this test allowed for reliable determination of an EC50 and were in agreement with the results of the combined limit/range-finding test.

Determination of Effect Concentrations:
Table 3 shows the effect parameters based on average exposure concentrations. Table 4 shows the effect parameters based on loading rates.

Experimental Conditions:
The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 5. These test conditions remained within the limits prescribed by the study plan (pH: 6 9, not varying by more than 1.5 units; oxygen: ≥3 mg/L at the end of the test).

The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test, and complied with the requirements as laid down in the study plan (18 22°C, constant within ±1°C).

Results with reference substance (positive control):

See Appendix 1

Reported statistics and error estimates:

The measured concentration in the undiluted SS was 2.3 mg/L at the start of the test. This concentration decreased below the limit of detection at the end of the test.

Table 1
Number of Introduced Daphnids and Incidence of Immobility in the
Combined Limit/Range-Finding Test

 

Time (h)	Replicate	Hexyl Caproate;%SS prep. at a loading rate of 100 mg/L
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	0	0
	B	0	1	0	0
	C	0			0
	D	0			0
	Total immobilised	0	1	0	0
	Effect %	0	10	0	0
48	A	0	0	0	0
	B	0	1	0	0
	C	0			0
	D	0			0
	Total immobilised	0	1	0	0
	Effect %	0	10	0	0

Table 2
Number of Introduced Daphnids and Incidence of Immobility in the Final Test

 

Time (h)	Replicate	Hexyl Caproate; %SS prep. at a loading rate of 100 mg/L
		Control	10	18	32	56	100
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0 (2)	0 (2)	0	0	0
	B	0	0	0	0	0	0
	C	0	0 (2)	0	0	0	0
	D	0	0	0	0 (1)	0	0
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0
48	A	0	0 (1)	0	0	0	0
	B	0	0	0	0	0	0
	C	0	0	0	0	0	0
	D	0	0	0	0	0	0
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Table 3
Effect Parameters Based on Average Concentrations

Parameter	Hexyl Caproate Average conc. (mg/L)
24h- & 48h-EC50	>0.14

Table 4
Effect Parameters Based on Loading Rates

Parameter	Hexyl Caproate Loading rate (mg/L)
24h- & 48h-EL50	>100

Table 5
pH and Oxygen Concentrations (mg/L) During the Final Test

Hexyl Caproate %SS prep. at a loading rate of 100 mg/L	Start (t=0 h)		End (t=48 h)
	O2	pH	O2	pH
Control	8.5	7.9	8.6	8.1
10	8.6	7.9	8.6	8.1
18	8.7	7.9	8.6	8.1
32	8.7	7.9	8.5	8.1
56	8.6	7.9	8.4	8.0
100	8.5	7.8	8.2	8.0

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to Hexyl Caproate was beyond the range of concentrations tested, i.e. exceeded an average exposure concentration of 0.14 mg/L, being considered to represent the maximum solubility of the test item in test medium at a loading rate of 100 mg/L.

Executive summary:

The objective of the study was to evaluate Hexyl Caproate for its ability to generate acute toxic effects on the mobility ofDaphnia magnaduring an exposure period of 48 hours and, if possible, to determine the EC₅₀ at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004.In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Hexyl Caproate tested was a clear colourless liquid with a purity of 99.29% and not completely soluble in test medium at the loading rate initially prepared.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

A final test was performed based on the results of a combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

No immobility was observed in the control and at any of the test concentrations throughout the exposure period.

Samples taken from the control and the highest test concentration were analysed. The measured concentration in the undiluted SS was 2.3 mg/L at the start of the test. This concentration decreased below the limit of detection at the end of the test. Based on these results, the average exposure concentration was calculated to be 0.14 mg/L in the undiluted SS and used to express the effect parameters.

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

In conclusion, the 48h-EC₅₀ for Daphnia magna exposed to Hexyl Caproate was beyond the range of concentrations tested, i.e. exceeded an average exposure concentration of 0.14 mg/L, being considered to represent the maximum solubility of the test item in test medium at a loading rate of 100 mg/L.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

In this guideline (OECD 202) study, the 48 hour EC50 of the test material (EC 228-952-4) to Daphnia magna was determined to be beyond the range of concentrations tested, i.e. exceeded an average exposure concentration of 0.14 mg/L, being considered to represent the maximum solubility of the test item in test medium at a loading rate of 100 mg/L.The test was conducted under static conditions. The result of this test is not sufficient to trigger classification and labelling under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).