Acute Toxicity Evaluation of Chemical Compounds on Daphnids

Daphnids were maintained in glass beakers in OECD media (final concentrations 0.29 g CaCl₂.2H₂O/L, 0.123 g MgSO₄.7H₂O/L, 0.065 g NaHCO₃/L, 0.0058 g KCl/L, 2 μg Na₂SeO₃/L, pH 7.7) at a density of 80 adults per 4 L of media and under a 16h:8h of light:dark photoperiod at 20 °C [7 (link)]. For experiments, neonates (<24 h) were collected from the third brood of their mothers and cultured until four days old, and then used for experiments. Typically, in experiments with daphnids, acute toxicity is performed with neonates (<24 h); however, in several cases this has proven to be not reproducible, mainly because of the time window of neonate selection which extends to up to 24 h, thus resulting in a less homogenous population for experiments affecting toxicity [8 (link)]. Furthermore, as acute exposures are performed in the absence of food, the animals experience an additional stress of starvation which we avoided by allowing them to grow over a period of four days prior to exposure to the chemicals. Based on the selection of 24 h exposure periods, the chemicals were only added once following the general outlined procedure of the OECD guidelines [9 ]. The chemicals used in this study were aluminium (CAS 16828-11-8), lithium (CAS 7447-41-8), acetylsalicylic acid (CAS 50-78-2), diltiazem (CAS 33286-22-5), metformin (CAS 115-70-4), propranolol (CAS 318-98-9), glyphosate (CAS 1071-83-6) and nicotine (CAS 54-11-5). All chemicals were of highest purity >99.9%.
For exposures, 15 four-day-old animals were exposed to each chemical separately in a final volume of 100 mL OECD media with four replicates per concentration tested. Toxicity curves were plotted for 24 h exposures and EC values were calculated. A mixture was constructed for all chemicals and further assessed for its toxicity (Figure 1). All plots were calculated using the Four parameter logistic (4PL) model, following the equation Span = Top − Bottom and Y = Bottom + (Top-Bottom)/(1 + 10^((LogIC50-X)*HillSlope)), using the GraphPad software. The parameters top and bottom were commonly fixed to 100 and 0, accordingly.