Hexyl benzoate

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:

16 September 2017 - 30 November 2017

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 items and mixtures, OECD series on testing and assessment number 23

Version / remarks:

14 December 2000

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Solubility in water: 15.1 mg/L at 24°C

Analytical monitoring:

yes

Details on sampling:

Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency: at t=0 h and t=24 h from the freshly prepared solutions; at t=24 h and t=48 h from the 24-hour old solutions
Volume: 2.0 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.

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

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Correction for purity: no correction for purity was made.
- Method: Direct application to the test medium.
The substance was a colourless liquid and not completely soluble in test medium at the loading rate initially prepared. 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 medium. The obtained mixture was allowed to settle for a period of 198-206 minutes (combined limit/range-finding test) or 176-210 minutes (final test). Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning (in the combined limit/range-finding study through glass wool; in the final test without glass wool) 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, except for the highest test concentration where droplets of undissolved test item were observed at the surface of the test solutions.
- Controls: test medium without test item or other additives.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, 1820
- Source: in-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock: 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.
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding of test organisms during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7) in an all-glass culture vessel at 18-22 °C. Cultures were max. 4 weeks old. After 7 days of cultivation half of the medium was renewed twice a week.
- Feeding: daily, a suspension of fresh water algae.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg CaCO3/L

Test temperature:

19-20°C

pH:

7.7-8.2 throughout the entire study, measured in fresh and old solutions

Dissolved oxygen:

8.6-9.6 mg O2/L throughout the entire study, measured in fresh and old solutions

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56 and 100% of a saturated solution prepared at a nominal loading rate of 100 mg/L
Average exposure concentrations: 0.40, 0.81, 1.2, 2.1 and 3.6 mg/L. For measured concentrations per sampling point see table 1.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Fill volume: 50 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, adjusted ISO medium
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Culture medium different from test medium: yes, M7 was used as culture medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours daily

EFFECT PARAMETERS MEASURED:
- Immobility (including mortality) at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning, after 24 hours and at the end of the test, in all concentrations and the control. Temperature of the medium: continuously in a temperature control vessel.

COMBINED LIMIT/RANGE-FINDING STUDY:
- Test concentrations: 1.0, 10 and 100% of SS prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: yes, the results of a combined limit/range-finding test showed 100% immobility at the highest concentration after 24 hours of exposure and 10% immobility at the lowest test concentration after 48 hours of exposure. No immobility was observed at a test concentration of 10% of SS prepared at 100 mg/L. Even though 15% of the daphnids became immobile in the control group, the results were still considered useful and used to determined the test concentrations in the final test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (September 2017)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95%-CI: 1.3-1.8 mg/L

Details on results:

- For details on incidence of immobility see table 2 in 'Any other information on results incl. tables'.
- No immobility was observed in the control or the two lowest test concentrations throughout the test duration. Immobility observed at the three highest test concentrations increased with increasing concentration and duration.
- Small droplets of undissolved test item were observed after 24 hours in all test concentrations, and in the highest test concentration from the start of the test. Considering the low level of daphnids trapped at the surface, it was assumed that the signs of oversaturation were not responsible for the observed immobility but that the dissolved concentrations caused the observed effects.
- No other abnormalities were observed in the control group or any of the test concentrations.

- Analyses of the samples taken from the old solutions at t=24 h and t=48 h showed that the test substance concentration decreased to 58-68% of initially measured concentrations. Therefore, the average exposure concentrations were calculated and used to determine the effect parameters.

- Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 48h-EC50: 0.41 mg/L, 95%-confidence interval: 0.34-0.46 mg/L
- Other: results fell within the historical range.

Reported statistics and error estimates:

The 24h- and 48h-EC50-values were calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (average) using the maximum likelihood estimation method. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Table 1 Average exposure concentrations versus nominal concentrations

Substance % SS prep. at 100 mg/L	Measured concentration (mg/L)				Average exposure concentration (mg/L)
	t=0h (fresh)	t=24h (old)	t=24h (fresh)	t=48h (old)
10	0.587	0.386	0.425	0.251	0.40
18	1.33	0.786	0.747	0.489	0.81
32	1.94	1.13	1.24	0.815	1.2
56	3.09	1.99	2.27	1.45	2.1
100	5.27	3.12	3.83	2.61	3.6

Table 2 Incidence of immobility in the final test

Time (h)	Replicate	HEXYL BENZOATE; Average conc. (mg/L)
		Control	0.40	0.81	1.2	2.1	3.6
0	A	5	5	5	6	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	6	5	5
	Total introduced	20	20	20	22	20	20
24	A	0	0	0 (1)	1	5^#	5
	B	0	0	0 (1)	1 (1)	4	5
	C	0	0	0	0 (1)	5	5 (2)*
	D	0	0	0 (1)	1	5	5
	Total immobilised	0	0	0	3	19	20
	Effect %	0	0	0	14	95	100
48	A	0	0	0	1	5	5
	B	0	0	0	1	5	5
	C	0	0	0	1	5	5
	D	0	0	0	1	5	5
	Total immobilised	0	0	0	4	20	20
	Effect %	0	0	0	18	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.

* Three daphnids observed to be stuck to each other, trying unsuccessfully to move away from each other.

Validity criteria fulfilled:

yes

Remarks:

1). In the control, no daphnids became immobilised or showed other signs of disease or stress. 2). The oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels (i.e. >= 8.6 mg/L).

Conclusions:

The 48h-EC50 of the substance towards Daphnia magna was 1.5 mg/L based on average exposure concentrations (95% confidence interval between 1.3 and 1.8 mg/L).

Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline No. 202 and GLP under semi-static conditions.

Based on the results of a combined limit/range-finding test, the following nominal concentrations were tested in the definitive test: 10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 mg/L.

For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed to the substance for 48 hours.The incidence of immobility was recorded for each test and control group at 24 hours and at 48 hours. Samples were taken from all concentrations at the start and the end of both 24-hour renewal periods and analysed with a validated UPLC-UV method.

Measured concentrations ranged from 58% to 68% in the old solutions. Based on these results, the average exposure concentrations (geometric means) were calculated to be 0.40, 0.81, 1.2, 2.1 and 3.6 mg/L.

All acceptability criteria were met and the study was considered valid.

The 48h-EC50 value to Daphnia magna was 1.5 mg/L based on average exposure concentrations.

Description of key information

The 48h-EC50 of the substance towards Daphnia magna was 1.5 mg/L based on average exposure concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.5 mg/L

Additional information

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline No. 202 and GLP under semi-static conditions.

Based on the results of a combined limit/range-finding test, the following nominal concentrations were tested in the definitive test: 10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 mg/L.

For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed to the substance for 48 hours. The incidence of immobility was recorded for each test and control group at 24 hours and at 48 hours.Samples were taken from all concentrations atthe start and the end of both 24-hour renewal periods andanalysed with a validated UPLC-UV method.

Measured concentrations ranged from 58% to 68% in the old solutions. Based on these results, the average exposure concentrations were calculated to be 0.40, 0.81, 1.2, 2.1 and 3.6 mg/L.

All acceptability criteria were met and the study was considered reliable.

The 48h-EC50 value to Daphnia magna was 1.5 mg/L based on average exposure concentrations.