The fishes are exposed to the test substance for 96 hours under static or semi-static conditions. Temperature is controlled and from 12 to 16 hours photoperiod applied. Mortalities are recorded at 24, 48, 72 and 96 hours and the concentration which kill 50% of the fishes determined.

TEST SYSTEM
Oncorhynchus mykiss
A toxic reference substance is tested twice a year to check the sensitivity of the organisms.

STUDY DESIGN
Range finding test includes at least 5 concentrations of the test substance and 1 untreated control (7 replicates/concentration or control) with 1 fish/replicate.
Limit test is carried out with 1 concentration (100 mg/L or at solubility limit) of the test substance and 1 untreated control (7 replicates/concentration or control) with 1 fish/replicate.
Dose-response test includes at least 5 concentrations of the test substance and 1 untreated control (7 replicates/concentration or control) with 1 fish/replicate.

Analytical checks, to verify the exposure concentrations in the medium, are performed  for static test at time 0 and at time 96 hours on control and on each test substance sample.
For semi-static test the analysis are carried out at time 0, 24 hours, 48 hours, 72 hours (in spent and fresh solutions) and at 96 hours on control and on each test substance sample.

The analysis are performed using a method validated according to SANCO/825/00 rev. 8.1 and SANCO/3029/99 rev. 4 guidance documents.

ENDPOINTS
Mortality: LC₅₀ at 96 hours as the main endpoint and as other endpoints: LC₁₀, LC₂₀, LOEC/NOEC.
Study includes GLP management and reporting.

REFERENCES AND GUIDELINES
OECD Guideline fo Testing of Chemicals, No. 203 (17^th July 1992) – Fish, acute toxicity test.
SANCO/825/00 rev. 8.1 (16/11/2010) – Guidance document on pesticide residue analytical methods.
SANCO/3029/99 rev. 4 (11/07/2000) – Residues: guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (Part A, section 4) and Annex III (Part A, section 5) of Directive 91/414.