2.2.2. Daphnia magna Acute Toxicity Test

Acute immobilization test with Daphnia magna (freshwater fleas) was carried out according to the Organization for Economic Cooperation and Development (OECD) guidelines for the testing of chemicals, Tests No. 202 [20], which forms the basis of the kits manufacturer’s protocol, with slight modification. The basic principle of this test is based on whether the test solutions are toxic to the daphnids; signified by motility and mortality rates.

Freshwater was simulated using standard concentrated salt solutions (batch number ISOD070319). Sodium hydrogen carbonate (NaHCO₃), calcium chloride dihydrate (CaCl₂.2H₂O), magnesium sulfate heptahydrate (MgSO₄.7H₂O), and potassium chloride (KCl) concentrated solutions were used to simulate the freshwater medium for the daphnids’ hatching and exposure study. Ephippia were hatched within 72 h period at 28 °C under constant illumination with the aid of cool white light. After the 72-h hatching period, the daphnids were fed with dry algae (spirulina) for a period of 2 h (this was to annul external effects, such as starvation, prior to exposure to lamivudine), after which they were harvested into a 30-well plate containing the test solutions. Test solutions of lamivudine (10 and 100 µg/L) were prepared in freshwater using serial dilutions from a 1000 µg/mL stock solution of lamivudine. Freshwater (containing the same amount of methanol used in the preparation of the test solutions) was used as the control. Freshwater used for both control and exposure studies was aerated with the aid of an aquarium air pump before exposure. Four replicate analyses were conducted for each test solution where five active daphnids were placed in each well (making a total of 20 daphnids) and exposed over a period of 24 and 48 h, with survival rates from which the lethal dose (LD50) was obtained.