Lemna sp. aquatic plants (in house breeding at B.T.) are exposed to a range of concentrations of the test substance in test medium, for 7 days in a static or semi-static system to evaluate the growth rate inhibtion or yield inhibition.

TEST SYSTEM
Lemna minor (SIS medium), Lemna gibba (AAP 20X) medium.
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 (3 replicates/concentration or control) with plants with a  total of 9 to 12 fronds/replicate
Limit test is carried out with 1 concentration (100 mg/L or at solubility limit) of the test substance and 1 untreated control (at least 6 replicates/concentration or control) with plants with a  total of 9 to 12 fronds/replicate
Dose-response test includes at least 5 concentrations of the test substance and
1 untreated control (3 replicates/concentration or control) with plants with a  total of 9 to 12 fronds/replicate.

Analytical checks, to verify the exposure concentrations in the medium, are performed for static test at time 0 and at time 7 days on control and on each test substance sample and for semi-static test at time 0, at 2 days, at 5 days and at 7 days 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
Inhibition of the growth rate and of the yield: EC₁₀, EC₂₀, EC₅₀, NOEC/LOEC after 7 days calculated by the fronds number. Dry or fresh weight.
Study includes GLP management and reporting.

REFERENCES AND GUIDELINES
OECD Guideline fo Testing of Chemicals, No. 221 (23^rd March 2006) – Lemna sp. growth inhibition test.
OECD Series on Pesticides, Number 67: “OECD guidance to the environmental safety evaluation of microbial biocontrol agents” (17^th February 2012).
Environmental Canada, EPS 1/RM/44: “Guidance Document for Testing the Pathogenicity and Toxicity of New Microbial Substances to Aquatic and Terrestrial Organisms” (March 2004).
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.