Hexyl propionate

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:

01 Jun 2018 to 21 August 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:

April 13, 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 Propionate
Appearance: Clear colourless liquid
Purity/Composition: 99.54% (assumed 100% for testing)
Test item storage: At room temperature protected from light

Analytical monitoring:

yes

Details on sampling:

Frequency: at t=0 h and t=48 h
Volume: 6.0 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.
Hexyl Propionate: Solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L.

Vehicle:

no

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:

19-20°C

pH:

8.0-8.4

Dissolved oxygen:

8.0-9.2 mg/l

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal: Solutions containing 0, 10, 18, 32, 56 and 100% of the saturated solution prepared at a loading rate of 100 mg/L.
Measured @ 0 hours: 0.012, 8.45, 15, 25, 54.3, 92.1
Measured @ 48 hours: 0.014, 2.18, 3.83, 6.76, 8.81, 24

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 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:
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/L in 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: 23 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):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

meas. (geom. mean)

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 and the two lowest test concentrations throughout the test. At the highest test concentration, all daphnids were immobile after 24 hours of exposure and onwards.

Based on these results, samples taken from solutions containing 10 and 100% of the SS prepared at a loading rate of 100 mg/L were analysed. The measured concentrations were 5.0 and 47 mg/L, respectively, at the start of the test. These concentrations decreased to 21-26% of the initial concentrations after 48 hours of exposure (see also Table 2 of the appended Analytical Report).

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


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 test concentrations were analysed. At the start of the test, the measured concentrations were 8.5, 15, 25, 54 and 92 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS, respectively. After 48 hours of exposure, 16-29% of the initial concentrations were measured.

It should be noted that a concentration of 0.050 mg/L was originally measured in the sample of the 56 mg/L test group at the end of the test, which was significantly below of what could be expected based on the concentration profiles of the other test solutions. No reason could be identified for this and it was decided to analyse the corresponding reserve sample to confirm the result. The concentration detected in the reserve sample was 8.8 mg/L and thus at the level of what was expected. It was subsequently concluded that the original result was erroneous and the result of the reserve sample was used for the further interpreptation of the test results.
Based on these results, the average exposure concentrations were calculated and used to express effect parameters (see Table 2).

Immobility:
Table 3 shows the responses recorded during the final test.
No immobility was observed in the control and the lowest test concentration throughout the exposure period. A dose-related increase of immobilization was observed at the four highest test concentrations, resulting in 100% immobility at the highest test concentration after 24 hours and onwards.
The responses recorded in this test allowed for reliable determination of an EC50 and were in agreement with what was expected based on the results of the combined limit/range-finding test.

Determination of Effect Concentrations:
Table 4 shows the effect parameters based on average exposure concentrations, see also Appendix 1.

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 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18 22°C, constant within ±1°C).

Reported statistics and error estimates:

See table 4

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

 

Time (h)	Replicate	Hexyl Propionate, % 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	5
	B	0	0	0	5 (2)^#
	C	0			5
	D	0			5 (1)
	Total immobilised	0	0	0	20
	Effect %	0	0	0	100
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 daphnids 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.

Table 2          
Average Exposure Concentration vs.Percentage of the Saturated Solution

 

Hexyl Propionate %SS prep. at 100 mg/L	Measured concentrations (mg/L)		Average exposure conc. (mg/L)
	t=0h	t=48h
10	8.45	2.18	4.3
18	15	3.83	7.6
32	25	6.76	13
56	54.3	8.81	22
100	92.1	24	47

Table 3          
Number of Introduced Daphnids and Incidence of Immobility in the Final Test

 

Time (h)	Replicate	Hexyl Propionate,Average exposure conc.
		Control	4.3	7.6	13	22	47
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	1	0	2	5 (2)^1
	B	0	0	1	0	0 (1)	5
	C	0	0	1	2	4 (2)	5
	D	0	0	0	3^#	2	5 (1)
	Total immobilised	0	0	3	5	8	20
	Effect %	0	0	15	25	40	100
48	A	0	0	1	3	3	5
	B	0	0	2	2	2	5
	C	0	0	2	4	4	5
	D	0	0	0	3	2	5
	Total immobilised	0	0	5	12	11	20
	Effect %	0	0	25	60	55	100

( ) 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.

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

¹Oxygen was measured to be 8.6 mg/L.

Table 4          
Effect Parameters

Parameter	Hexyl Propionate Average exposure conc. (mg/L)	95%-confidence interval (mg/L)
24h-EC50	19	12 - 32
48h-EC50	14	8.0 - 24

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

Hexyl Propionate Average conc. (mg/L)	Start (t=0 h)		End (t=48 h)
	O2	pH	O2	pH
Control	8.6	8.1	9.2	8.2
4.3	8.7	8.0	8.7	8.4
7.6	8.7	8.0	8.7	8.3
13	8.9	8.0	8.6	8.3
22	9.2	8.0	8.5	8.3
47	9.2	8.0	8.0	8.3

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to Hexyl Propionate was 14 mg/L based on average exposure concentrations (95% confidence interval between 8.0 and 24 mg/L).

Executive summary:

The objective of the study was to evaluate Hexyl Propionate 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 Propionate tested was a clear colourless liquid with a purity of 99.54% 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 containing10, 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.

Samples taken from all test concentrations were analysed.At the start of the test, the measured concentrations were 8.5, 15, 25, 54 and 92 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS, respectively. After 48 hours of exposure, 16-29% of the initial concentrations were measured.

No immobility was observed in the control and the lowest test concentration throughout the exposure period. A dose-related increase of immobilization was observed at the four highest test concentrations, resulting in 100% immobility at the highest test concentration after 24 hours and onwards. Based on these results, the average exposure concentrations were calculated and used to express 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 Propionate was 14 mg/L based on average exposure concentrations (95% confidence interval between 8.0 and 24 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:

14 mg/L

Additional information

The substance is not classified for CLP as the effect levels are above the limit of water solubility (0.0631 mg/l), the registered substance is readily biodegradable, and the Log Pow is < 4 (3.9).