Delayed toxicity of three fluoroquinolones and their mixtures after neonatal or embryonic exposure, in Daphnia magna

Fluoroquinolones (FQs) are antibacterial drugs, used both in human and veterinary medicine, that are currently considered as emerging micropollutants. This study investigated the delayed toxic effects of enrofloxacin (ENR), flumequine (FLU), levofloxacin (LEV) and their binary mixtures in D. magna. For this purpose, a 10-day follow-up in pure medium was added to the standard D. magna immobilization test. During this follow-up, phenotypic alterations were evidenced, which were related to scarce or zeroed egg production and early mortality. Consequently, the EC50 (s) recalculated at the end of the follow-up were always remarkably lower than those obtained after the 48 h immobilization test: ENR 3.13 vs. 16.72 mg L-1; FLU 7.18 vs. 25.35 mg L-1; LEV 15.11 vs. > 40 mg L-1. To analyse the possible interactions within the binary mixtures, the method of nonlinear additive isoboles was applied. The three compounds showed invariably to follow the principle of concentration addition. Furthermore, as previous experiments showed toxicity of FLU and ENR after embryonic exposure of D. magna at a concentration of 2 mg L-1, an additional two embryonic tests were conducted with identical design: one with 2 mg L-1 LEV and the other with a ternary mixture containing 0.66 mg L-1 of each of the three FQs. The embryos were exposed for three days in vitro to the drug solutions and were then reconducted to pure medium for 21 days observation. Both the tests ended-up with only non-significant effects on growth and reproduction, confirming the lower toxicity of LEV, when compared to ENR and FLU, and the absence of any evident synergistic interaction among the three FQs. Overall, these studies have shown two relevant features related to the toxicity of the three FQs: (1) they give rise to delayed toxic effects in D. magna that are undetectable by the standard immobilization test; (2) their interaction in mixtures follow the principle of Concentration Addition. Both these indications concern the Environmental Risk Assessment of FQs and may be of interest to regulatory authorities.