Butyl 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:

From 2018-02-06 to 2019-02-18

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20141015
- Expiration date of the lot/batch: 2019-01-31
- Purity test date: 2017-01-31
- Purity: 99.35%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. Tightly closed container in a dry and well-ventilated place
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data

Analytical monitoring:

yes

Details on sampling:

- Sampling method:
For the determination of the actual test item concentrations, duplicate samples were taken from each test concentration and the control at the start and at the end of the two 24-hour test medium renewal periods. For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling. Aged samples from the two highest test concentrations of JEFFSOL® AG-1700 (dilution 1:3.2 and undiluted filtrate), were taken only from the first renewal period as all daphnids were immobile (i.e. dead) at Day 1. Nevertheless the fresh samples from these two highest test concentrations were also taken at the start of the second test interval for confirmation of the dosage.
- Sample storage conditions before analysis: All samples were stored frozen (at -20 ± 5 °C) immediately after sampling. Based on analytical pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
* Pre-experiment
To determine the solubility of the poorly soluble test item (water solubility < 100 mg/L) and the optimum time period to reach equilibration between dissolved and undissolved test item a stirring pre-experiment was performed:
Individual suspensions of the test item with a loading rate of 100 mg/L were mixed into ISO-Medium using intensive stirring for 3, 24, 48, 72 and 96 hours. After stirring, the suspensions were filtered through a 0.45 µm membrane filter. The filter was saturated by 200 mL test medium and the negative pressure of the filtration unit was reduced to a minimum to avoid losses of the test item.
Intensive stirring for 3 hours showed no significant increase of the concentration of dissolved test item. Additionally, an aliquot of the filtered test medium sampled after 24 hours stirring period (45 mg/L test item) was incubated for 48 hours at room temperature in an open and in a closed vessel. The test item concentrations in these aged test medium were 23 and 43 mg/L, corresponding to 52 and 96 % of the initially measured values, respectively. Thus the test item was demonstrated to be volatile. These results suggested to perform the range finding test and the main test in a closed system.
Additionally, the filtered test medium sampled after 24 hours stirring period (45 mg/L test item) was filtered a second time to check adsorption onto the filter. The test item concentration in this double filtered test medium was 44 mg/L, corresponding to 97 % of the initially measured value. Thus there were no losses due to adsorption onto the filter material.

* Range-Finding Test: The highest test concentration (undiluted filtrate) was prepared by applying 99 µL of the test item to 1000 mL of test water using intense stirring for 3 hours at room temperature in the dark in a closed vessel. After stirring, the suspension was filtered through a 0.45 µm membrane filter (Whatman, NC45). The filter was saturated by 200 mL test medium and the negative pressure of the filtration unit was reduced to a minimum to avoid losses of the test item. The undiluted filtrate was used as highest test concentration. It was further used in a serie of dilutions with test water to prepare the test media with the lower test concentrations.

* Definitive Test: Due to the low water solubility of the test item, a suspension with the loading rate of 100 mg/L was prepared at the start of the test and at the start of the renewal period after 24 hours (on Day 0 and Day 1, respectively). On both preparation dates, 99 µL of the test item was applied to 1000 mL of test water. This volume is equivalent to a loading rate of 100 mg/L, considering the relative density of the test item of 1.01. The resulting test medium was intensively stirred for 3 hours at room temperature in the dark in a closed vessel. The stirring time was based on a stirring pre-experiment which showed, that the maximum amount of dissolved test item was reached after this stirring time. After stirring, the suspension was filtered through a 0.45 µm membrane filter (Whatman, NC45). The filter was saturated by 200 mL test medium and the negative pressure of the filtration unit was reduced to a minimum to avoid losses of the test item. No auxiliary solvent or emulsifier was used. This undiluted filtrate with a loading rate of 100 mg/L, was used as highest test concentration and was used in a serie of dilutions with test water to obtain the following dilutions: 1:3.2, 1:10, 1:32 and 1:100. Additionally, a control (test water only) was run in parallel. The test media were prepared just before the start of the test (i.e., introduction of the daphnids to the test media) and prior to the test media renewal after 24 hours.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All solutions were clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water-flea
- Strain: Straus
- Source: in-house laboratory culture with a known history
- Age at study initiation (mean and range, SD): < 24 h (from parental daphnids of more than 2 weeks old, maximum age 4 weeks)
- Method of breeding: Culture conditions ensured that reproduction was by parthenogenesis.
- Feeding during cultivation: 3 times a week with mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension, daily
- Feeding during test: no

ACCLIMATION
- Acclimation period: not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

not reported

Test temperature:

20-21°C

pH:

At t=0h: 7.8-7.9
At t=24h: 7.8-8.0
At t=48h: 7.8

Dissolved oxygen:

At t=0h: 8.4-8.6
At t=24h: 8.4-8.7
At t=48h: 8.1-8.2

Salinity:

not relevant

Nominal and measured concentrations:

Final test:
Nominal concentrations: undiluted filtrate and its dilutions: 1:3.2, 1:10, 1:32 and 1:100
Measured concentrations (mg/L) at t=0h: 1.29, 4.14, 12.8, 41.4
Measured concentrations (mg/L) at t=24h (start): 1.34, 4.11, 13.4, 43.7
Measured concentrations (mg/L) at t=24h (end): 1.09, 3.63, 12.1, 37.4
Measured concentrations (mg/L) at t=48h: 1.21, 3.76, n.d., n.d.

Mean Measured* concentrations during 48-h test period (mg/L): 1.2, 3.9,12*, 39*

n.d.: Not determined since all daphnids were immobile (i.e. dead) after 24 hours exposure time.
* Mean measured (geometric mean) calculation only based on the first renewal period since all daphnids were immobile (i.e. dead) after 24 hours exposure time.

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Type (delete if not applicable): tightly sealed with glass stoppers
- Material, size, headspace, fill volume: 60 mL, glass beaker filled with 60 mL,
- Aeration: not during the test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Test medium: adjusted ISO medium

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility was defined as those organisms which are not able to swim within 15 seconds after gentle agitation of the test container and was monitored after 24 and 48 hours of continuous treatment with the test substance.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.0, 10 and 100 mg/L (setup: combined range finder/limit test)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% C.L.: 3.2 - 5.3 mg/L

Details on results:

- Mortality of control: none
- Other adverse effects control: no
- Abnormal responses: none
- 48-hour EC0 and NOEC: 1.2 mg/L
- 48-hour EC100: 1.2 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Mortality: no
- EC50/LC50: 24-h EC50 = 6.8 mg/L.

Reported statistics and error estimates:

The 48 hour EC50 and their 95% confidence limits were calculated by Weibull Analysis using linear maximum likelihood regression. The lowest concentration tested (dilution 1:100) was not taken into account at the Weibull Analysis calculation, because it was below the determined 48 hour NOEC and, thus, not analyzed.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test substance to Daphnia magna was determined in a 48 hour static test according to OECD guideline 202. Based on geometric mean measured concentrations, the 48-h EC50 was 4.0 mg/L (95% C.L.: 3.2 - 5.3). The results of the test can be considered reliable without restriction.

Description of key information

The study of Dupont (2018), investigating the acute toxicity of butyl benzoate in Daphnia magna according to OECD guideline 202, was considered as the key study for endpoint coverage. An EC50 of 4 mg/L (based on geometric measured test concentrations) was obtained after an exposure of 48 hours in a static test in freshwater. As the test item is a volatile substance, the test was performed using glass tubes completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system). A semi-static test design with a test medium renewal after 24 hours was used. It can be concluded that the test item had acute toxic effects on Daphnia magna in a 48-hour semi-static test in a closed system.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

4 mg/L