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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Mar - 03 Mar 2000
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study.
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
according to guideline
EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: Duplicate samples were taken from the main stock solutions of control and each test level at 0 and 24 h (fresh) and pooled replicates after 24 and 48 h (expired).
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 2-Ethyl hexyl benzoate is poorly soluble in water and potentially biodegradable. The test substance was initially dispersed in dimethylformamide to give a stock solution of 100 mg/mL. A 500 µL aliquot of this solvent stock was carefully added to the surface of 5 L of autoclaved Elendt M4 medium to produce an even surface layer without forming a dispersion. This was then left to stir in darkness on a Chiltern magnetic stirrer gently so no visible vortex was seen at the water surface to produce a saturated solution. Serial dilutions of this saturated solution were prepared with autoclaved diluent medium to give additional exposure levels of 3.2, 5.6, 10, 18, 32 and 56% saturation.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Waterflea
- Strain: Straus
- Source: In-house culture, originally obtained from the Institute National de Recherche Chimique Appliqué, France.
- Age at study initiation (mean and range, SD): < 24 h
- Feeding during test: None

- Stock cultures: Daphnia magna were maintained in glass vessels containing approx. 500 – 800 mL of Elendt M4 culture medium with a 16 h light/8 h dark photoperiod at 19 °C.
- Type and amount of food: Suspension of Chlorella vulgaris
- Feeding frequency: Daily
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Test temperature:
20 - 21 °C
8.3 - 8.8
Dissolved oxygen:
6.1 - 6.9 mg/L
Nominal and measured concentrations:
Nominal: 0, 3.2, 5.6, 10, 18, 32, 56 and 100% saturation
Measured: < LOD, 0.009, 0.014, 0.023, 0.031, 0.053, 0.069 and 0.125 mg/L (geometric mean)
Details on test conditions:
- Test vessel: Jars
- Type (delete if not applicable): loosely covered
- Material, size, fill volume: Glass, 250 mL, 125 mL
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): Test medium was renewed after 24 h.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- Source/preparation of dilution water: Elendt M4 medium was used.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature, pH and dissolved oxygen were measured daily in each vessel.

- Photoperiod: 16 h light/8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Immobilisation was observed after 24 and 48 h.

- Spacing factor for test concentrations: 1.8
- Range finding study
- Test concentrations: 0, 1, 10 and 100% saturation
- Results used to determine the conditions for the definitive study: Immobilisation was 20, 40 and 13% at 1, 10 and 100% saturation, respectively.
Reference substance (positive control):
48 h
Dose descriptor:
Effect conc.:
> 0.125 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
- Mortality of control: No immobilisation was observed in the control.
- Effects observed: 5% immobilisation was observed at 3.2 and 10% nominal saturation values.
- Analytics: Analysis confirmed there was significant loss of the dissolved test compound for the test solutions over the 24 h period between renewal.
- Effect concentrations exceeding solubility of substance in test medium: Yes

Description of key information

No adverse effects up to the limit of water solubility (0.4 mg/L measured in aqua dest; 0.125 mg/L in M4 test medium) for Daphnia magna (OECD 202)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.125 mg/L

Additional information

Short-term toxicity of the substance to aquatic invertebrates was investigated in one study according to OECD guideline 202 and GLP (Barrett, 2001). The test was conducted with Daphnia magna as test organisms that were exposed under semi-static conditions. Groups of twenty daphnids, less than 24 h old, were exposed to 2-ethylhexyl benzoate in Elendt M4 medium for 48 h at nominal concentrations of 0, 3.2, 5.6, 10, 18, 32, 56 and 100 % saturation. Analytical measurements confirmed there was significant loss of the dissolved test compound for the test solutions over the 24 h period between renewal. Geometric mean concentrations of 0, 0.009, 0.014, 0.023, 0.031, 0.053, 0.069 and 0.125 mg/L were established during the test. As no significant immobilization was noted at the maximum concentration tested, which was the limit of solubility under test conditions, the EC0 (48 h) and EC50 (48 h) were assumed as 0.125 mg/L and > 0.125 mg/L (based on geometric mean measured concentrations), respectively.