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Shoot apices of healthy non-flowering plants of Myriophyllum spicatum (in house breeding at B.T.) are potted in standardised, arificial sediment, supplemented with additional nutrients and manteined in Smart and Barko medium.
After roots formation, plants are exposed to a series of test concentrations added to the water column. Alternatively exposure via sediment may be simulated by spiking the artificial sediment with the test substance and transplanting plants into the treated sediment. The test is carried out under static conditions. Total shoot length, main shoot length, fresh and dry weight are measured.
A toxic reference substance is tested twice a year to check the sensitivity of the organisms.
Range finding test includes 5 concentrations of the test substance and 1 untreated control (2 replicates/concentration and 3 replicates for the control) with 3 shoots per pot and 1 pot/replicate.
Limit test is carried out with 1 concentration (100 mg/L or at solubility limit) of the test substance and 1 untreated control (6 replicates/concentration or control) with 3 shoots per pot and 1 pot/replicate.
Dose-response test includes at least 5 concentrations of the test substance and
1 untreated control (4 replicates/concentration and 6 replicates for the control) with 3 shoots per pot and 1 pot/replicate.
Analytical checks, to verify the exposure concentrations in the medium, are performed for static test at time 0 and at 14 days on control and on each test substance sample. Where relevant or required, samples to be analyzed include: water, pore water and sediment.
The analysis are performed using a method validated according to SANCO/825/00 rev. 8.1 and SANCO/3029/99 rev. 4 guidance documents.
Specific growth rate and yield for each measured variable: EC50 at 14 days as the main endpoint and as other endpoints: EC10, EC20, LOEC/NOEC.
Study includes GLP management and reporting.
REFERENCES AND GUIDELINES
OECD Guideline fo Testing of Chemicals, No. 239 (26th September 2014) - Water-sediment Myriophyllum spicatum 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.