Hexyl butyrate

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:

19 May 2018 to 25 June 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 Butyrate
Appearance: Clear colourless liquid
Purity/Composition: 99.26%
Test item storage: At room temperature protected from light
Test Facility test item number: 209412/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC, synonym or trade name): Hexyl butanoate
CAS number: 2639-63-6
EC number: 220-136-6
Volatile: Not indicated
Solubility in water: 20.3 mg/L at 20°C
Stability in water: Stable

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report (Appendix 3).

Frequency: 1. At the start of the test and after 24 hours from the freshly prepared solutions.
2. At the first renewal (t=24h) and the end of the test from the 24-hour old solutions.

Volume: 12 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.

At the end of each refreshment period, the replicates were pooled at each concentration before sampling.

Additionally, reserve samples of 12 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Measurements and Recordings:

Immobility (including mortality): At 24 hours and at 48 hours.

pH and dissolved oxygen: At the beginning, after 24 hours of exposure 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.

Vehicle:

no

Details on test solutions:

The batch of Hexyl Butyrate tested was a clear colourless liquid with a purity of 99.26% 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.

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 three hours. 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.

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: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.

Breeding:
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.

Maximum age of the cultures: 4 weeks

Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.

Temperature of medium: 18-22°C

Feeding: Daily, a suspension of fresh water algae.

Medium: M7, as prescribed by Dr. Elendt-Schneider
(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
The following salts and vitamins were added to freshly prepared test medium to reach the following concentrations:

Salts: H3BO3 0.73 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.092 mg/L
LiCl 0.078 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.039 mg/L
Na2MoO4.2H2O 0.016 mg/L
NaBr 0.0041 mg/L
CuCl2.2H2O 0.0043 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0033 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00059 mg/L
Na2EDTA.2H2O 0.64 mg/L
Na2SiO3.5H2O 7.6 mg/L
NaNO3 0.28 mg/L
KH2PO4 0.15 mg/L
K2HPO4 0.19 mg/L

Vitamins: Thiamine hydrochloride 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L



[copy and paste test organism section of report]

Things to include:
- Strain/clone:
- Type of breeding method: [parthenogenesis]
- Source:
- Age of parental stock (mean and range, SD):
- Feeding during test:
- Food type:
- Amount:
- Frequency:

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test temperature:

18-22 °C

pH:

6-9

Dissolved oxygen:

≥3 mg/L at the end of the test

Nominal and measured concentrations:

Nominal: 4.6, 10, 22, 46, 100% of Saturated Solution prep. at 100 mg/L
Average exposure conc.: 0.40, 0.68, 1.4, 2.6, 6.6 mg/l

Details on test conditions:

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 saturated solution prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• 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/L in the combined range-finding test.
• Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.
• The test solutions were not renewed after 24 hours.

Test Procedure and Conditions:
Test duration: 48 hours
Test type: Semi-static with renewal of test solutions after 24 hours.
Test vessels: 100 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.
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 60 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 30 minutes after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate (see Appendix 2)

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

See "Any other information on results incl. tables".

Results with reference substance (positive control):

See appendix 2

Reported statistics and error estimates:

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

Combined Limit/Range-Finding Test

Table1 shows the responses recorded during the combined limit/range-finding test.

No immobility was observed in the control or the two lowest test concentrations during the test period. At the highest test concentration, immobility increased to 45 and 100% after 24 and 48 hours of exposure, respectively.

Based on these results, samples taken from solutions containing 10 and 100% of the SS were analysed. The measured concentrations were 2.9 and 32 mg/L, respectively, at the start of the test. These concentrations decreased to 0.99-2.0% of the initial concentrations at the end of the test (see also Table 2 of the appended Analytical Report).

Due to the strong decrease of the measured concentrations during the test period, it was decided to perform a final test with renewal of the test solutions after 24 hours of exposure.

All test conditions were maintained within the limits prescribed by the study plan.

 

Table 1
Incidence of Immobility in the Combined Limit/Range-Finding Test

Time (h)		Replicate		Hexyl Butyrate;%SS prep. at 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		1
		B		0		0		0		3 (4)^#
		C		0						2 (4)
		D		0						3 (3)
		Total immobilised		0		0		0		9
		Effect %		0		0		0		45
48		A		0		0		0		5
		B		0		0		0		5
		C		0						5
		D		0						5
		Total immobilised		0		0		0		20
		Effect %		0		0		0		100

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

^#Microscopic observation revealed no test item was attached to the daphnids.

 

Final Test

Measured Test Item Concentrations       

The results of analysis of the samples taken during the final test are described in Table 3 of the appended Analytical Report.

Samples taken from all concentrations were analysed. Analysis of the samples taken at the start of the final test showed measured concentrations between 0.91 and 8.4 mg/L in solutions containing 4.6 to 100% of the SS prepared at a loading rate of 100 mg/L. During the 24-hour period until renewal, the measured test item concentrations were at 16-111% relative to the initial concentrations. At the start of the second renewal period, the measured concentrations were between 0.92 and 5.6 mg/L in solutions containing 4.6 to 100% of the SS prepared at a loading rate of 100 mg/. These concentrations decreased to 23-63% of the initial concentrations at the end of the test.

Based on these results, the average exposure concentrations were calculated (seeTable2) and used to determine the effect parameters.

It should be noted that the concentrations measured at the start of each renewal phase were generally lower than the concentration measured at the start of the combined limit range-finding test in the undiluted SS. The water solubility of the test item was determined to be 20 mg/L. The concentration at the start of the combined limit range-finding test was above this value and it is likely that the solution was oversaturated, which would also explain the strong decrease of the concentrations during the test period. The concentrations measured at the starts of the renewal periods in the final test is considered to be more representative of the test item solubility in test medium.

Table2
Average Exposure Concentration Versus Nominal Concentration

Hexyl Butyrate %SS prep. at 100 mg/L	Measured concentrations (mg/L)				Average exposure conc. (mg/L)
	t=0h (fresh)	t=24h (old)	t=24h (fresh)	t=48h (old)
4.6	0.91	0.15	0.92	0.21	0.40
10	1.2	0.51	0.84	0.42	0.68
22	2.2	1.2	1.9	0.69	1.4
46	3.7	2.9	3.0	1.4	2.6
100	8.4	9.3	5.6	3.5	6.6

Immobility

Table 3 shows the responses recorded during the final test.

No immobility was observed in the control or the three lowest test concentrations during the test period, except for a single daphnid after 24 hours of exposure to 0.40 mg/L and a single daphnid after 48 hours of exposure to 1.4 mg/L. At the two highest test concentrations, a dose and time related increase of immobility was observed, resulting in 100% immobility at both concentration levels at the end of the test.

It should be noted that immobility and/or entrapment of up to 10% is allowed in the control, and therefore effects <10% were considered biologically not relevant. The responses recorded in this test allowed for reliable determination of an EC₅₀.

 

 

Table3
Incidence of Immobilityin the Final Test

Time (h)		Replicate		Hexyl Butyrate; Average conc. (mg/L)
			Control		0.40		0.68		1.4		2.6		6.6
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		4^1		5
		Total introduced		20		20		20		20		19		20
24		A		0		0		0		0		3 (2)		4
		B		0		1		0		0		3 (5)^#		5
		C		0		0		0		0		2 (2)		2
		D		0		0		0		0		2 (3)		4
		Total immobilised		0		1		0		0		10		15
		Effect %		0		5		0		0		53		75
48		A		0		0		0		1		5		5
		B		0		0		0		0		5		5
		C		0		0		0		0		5		5
		D		0		0		0		0		4		5
		Total immobilised		0		0		0		1		19		20
		Effect %		0		0		0		5		100		100

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

^#Microscopic observation revealed no test item attached to the daphnids.;¹See deviations.

Determination of Effect Concentrations          

Table 4 shows the effect parameters based on average concentrations, see also Appendix 1.

Table 4
Effect Parameters

Parameter	Hexyl Butyrate Average conc. (mg/L)	95%-confidence interval (mg/L)
24h-EC50	2.5	1.4-6.6
48h-EC50	1.8	1.6-2.0

Experimental Conditions

The results of measurement of pH, temperature (°C) and oxygen concentrations (mg/L) are presented inTable5. These test conditions remained within the limits prescribed by the study plan (pH: 6.0-8.5, not varying by more than 1.5 unit; oxygen: ³3 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 2°C).

Table 5
Oxygen Concentrations (mg/L), pH and Temperature (°C) During the Final Test

Hexyl Butyrate Average conc. (mg/L)	Start (t=0 h)		t=24h old			t=24h fresh			End (t=48 h)
	pH	O2	pH	T	O2	pH	T	O2	pH	O2
Control	8.2	9.8	8.2	20	8.8	8.0	21	8.9	8.2	8.8
0.40	8.2	9.7	8.1	20	8.6	8.0	21	8.9	8.2	8.8
0.68	8.2	9.8	8.1	20	8.6	7.9	21	8.8	8.1	8.8
1.4	8.2	9.6	8.0	20	8.6	7.8	21	8.8	8.1	8.7
2.6	8.2	9.6	7.9	20	8.7	7.7	21	8.8	8.0	8.7
6.6	8.2	9.2	7.8	20	8.4	7.5	21	8.8	7.9	8.8

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to Hexyl Butyrate was 1.8 mg/L based on average exposure concentrations (95% confidence interval between 1.6 and 2.0 mg/L).

Executive summary:

The objective of the study was to evaluate Hexyl Butyrate for its ability to generate acute toxic effects on the mobility of Daphnia magna during 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 Butyrate tested was a clear colourless liquid with a purity of 99.26% 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 preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to solutions containing 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L. The total exposure period was 48 hours, test solutions were renewed after 24 hours of exposure and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of both renewal periods from the fresh and 24-hour old solutions.

Samples taken from all concentrations were analysed. Analysis of the samples taken at the start of the final test showed measured concentrations between 0.91 and 8.4 mg/L in solutions containing 4.6 to 100% of the SS prepared at a loading rate of 100 mg/L. After the 24-hour period until renewal, the measured test item concentrations were at 16-111% relative to the initial concentrations. At the start of the second renewal period, the measured concentrations were between 0.92 and 5.6 mg/L in solutions containing 4.6 to 100% of the SS prepared at a loading rate of 100 mg/L. These concentrations decreased to 23-63% of the initial concentrations at the end of the test.Based on these results, the average exposure concentrations were calculated to be 0.40, 0.68, 1.4, 2.6 and 6.6 mg/Lin solutions containing 4.6 to 100% of the SS prepared at a loading rate of 100 mg/L, respectively,and were used to determine the effect parameters.

No immobility was observed in the control or the three lowest test concentrations during the test period, while at the two highest test concentrations, a dose and time related increase of immobility was observed, resulting in 100% immobility at both concentration levels at the end of the test.

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 Butyrate was 1.8 mg/L based on average exposure concentrations (95% confidence interval between 1.6 and 2.0 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:

1.8 mg/L