*4.6. Daphnia magna Toxicity Bioassay*

In ecotoxicology, the planktonic crustacean *Daphnia magna* Staus is specified for use in the "OECD Guidelines for the Testing of Chemicals". Test No. 202: "*Daphnia* sp., Acute Immobilization Test" is an acute toxicity study, in which young daphnids, aged less than 24 h at the beginning of the test, are exposed to different concentrations of the chemical under test for 48 h and the half-maximal effective concentration (EC50) determined [95].

In this study, the young *D. magna* Straus organisms were selected according to their size from a parthenogenetic culture. All compounds were tested at six concentrations ranging from 2 to 50 μg/mL, in duplicate, with each replicate having 10 individuals. The concentration range was selected based on the compounds' solubilities and a pre-screening bioassay. *L*-Valine, compound **3**, and a 1% DMSO solution were used as controls. All determinations were carried out using the same conditions (25 ◦C, a long day photoperiod of 16 h light/8 h dark cycle) in a Sanyo MLR-351H climate test chamber (Sanyo, San Diego, CA, USA) [48,96,97]. After 24 and 48 h of exposure, the lethality was evaluated. LC50 was calculated for each compound based on interpolating on lethality curves which were obtained using the least square fit method. The 95% confidence intervals (95% CI) were calculated and the goodness of fit was also evaluated. The calculations were performed using GraphPad Prism v5.1 software (GraphPad Software, Inc., La Jolla, CA, USA). The prediction of LC50 at 48 h was made using GUSAR online application (Institute of Biomedical Chemistry, Moscow, Russia, http://www.way2drug.com/gusar/) [98].
