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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 May - 27 May 2000
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
adopted in 1992
according to guideline
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
adopted in 1992
according to guideline
EPA OPP 72-1 (Fish Acute Toxicity Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: Duplicate medium samples (100 mL) were taken for analysis from each replicate vessel of the solvent control and the test concentration at 0, 24, 72 and 96 h.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was dissolved in tetrahydrofuran (THF) to give a stock solution of 5.0 mg/L. This stock solution was dispensed automatically by the dosing apparatus into the flowing test water to give the desired test concentrations. The solvent stock solution was prepared twice during the exposure period and stored at approx. 4 °C until required.
- Controls: Diluent water with 100 µL THF/L
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tetrahydrofuran
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At 72 and 96 hours a layer of undissolved test compound was noted on the water surface in all the replicate exposure vessels.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
- Common name: Rainbow trout
- Source: Fish were obtained from Donnington Fish Farm, Stow-on-the-Wold, UK on 9 May 2000.
- Length at study initiation: 4.4 cm (SD = 0.1 cm)
- Weight at study initiation: 1.26 g (SD = 0.16 g)
- Feeding during test: None

- Acclimation period: 15 - 23 May 2000
- Acclimation conditions (same as test or not): 14 - 17 °C, 8.1 - 9.2 mg O2/L
- Type and amount of food: Fry pellets
- Feeding frequency: Daily until 48 h before test start
- Health during acclimation (any mortality observed): No mortality occurred in the 7 days before test start.
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
157 - 187 mg/L CaCO3
Test temperature:
15 °C constant
7.5 - 7.9
Dissolved oxygen:
8.6 - 9.7 mg/L
Nominal and measured concentrations:
Nominal: 0 and 0.5 mg/L
Measured: < LOD, 0.66 mg/L
Details on test conditions:
- Test vessel: Aquarium
- Material, size, fill volume: Glass, 25 x 46 x 25 cm, 20 L
- Aeration: No aeration of the aquaria, but aeration of the dilution water before delivery to the test system
- Renewal rate of test solution (frequency/flow rate): 260 - 300 mL/min
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3
- Biomass loading rate: 0.63 g/L (static volume) and 0.034 g/L of test solution passing through the chamber in 24 h

- Source/preparation of dilution water: Tap water, filtered, dechlorinated, softened, reverse osmosis treated
- Pesticides: All measured pesticides were < 0.02 µg/L.
- Chlorine: 0.01 - 0.08 mg/L
- Alkalinity: 110 - 116 mg/L CaCO3
- Conductivity: 220 - 223 µS
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature, pH and oxygen were recorded daily for each vessel. Alkalinity and conductivity were measured at test start and test end in all vessels.

- Photoperiod: 16 h light/8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Mortality was observed at 15 min, 3, 6, 24, 48, 72 and 96 h.
- Sublethal effects were also recorded.

Reference substance (positive control):
96 h
Dose descriptor:
Effect conc.:
> 0.66 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Mortality of control: One fish escaped from one of the solvent control tanks.
- Mortality or adverse effects in the test solution: None observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At 72 and 96 hours a layer of undissolved test compound was noted on the water surface in all the replicate exposure vessels. In replicate 3 it was believed the layer was disturbed during sampling and dispersed into the water column resulting in a non-homogenous sample containing increased test material. The layer of undissolved test material is considered to have formed as a result of the continuous addition of the test substance to already saturated test medium.
- Effect concentrations exceeding solubility of substance in test medium: Yes
- Analytics: Measured concentrations ranged from 69 - 85% of nominal at 0 h, 75 - 93% of nominal at 24 h, 98 - 356% of nominal at 72 h and 92 - 325% of nominal at 96 h.

Description of key information

No adverse effects up to the limit of water solubility (0.4 mg/L measured in aqua dest; 0.66 mg/L in fish test medium) for Oncorhynchus mykiss (OECD 203)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.66 mg/L

Additional information

Short-term toxicity of 2-ethylhexyl benzoate to fish was investigated in one study with rainbow trout (Oncorhynchus mykiss) according to OECD guideline 203 and GLP (Barrett, 2001). Three replicates of ten juvenile fish were exposed to a single concentration of the test substance of nominally 0.5 mg/L dispersed in water. This concentration was determined to be the limit of solubility for the test substance under the conditions of this study. Three replicate control and solvent control vessels were also prepared. The measured concentrations ranged from 69% - 85% at test start to 92% - 325% at test end. The latter value is assumed to be the result of the continuous addition of the test substance to an already saturated test medium (a layer of undissolved test material was noted from 72 hours on the water surface). The mean measured concentration was thus calculated as 0.66 mg/L. As no mortality was observed in these saturated solutions throughout the study, the LC0 (96 h) and LC50 (96 h) were assumed as 0.66 mg/L and > 0.66 mg/L (based on mean measured concentrations), respectively.