Mortality rate was already 50% in E0 and increased over subsequent generations, reaching 100% in three groups of the F3 generation groups, whilst remaining 20% in the control groups (Table 1). In the perinatal exposed group of in vitro cultured D. magna embryos, mortality was 31.4% with no mortality observed during the subsequent reproduction test in pure ADaM (Table 2).
Reproduction inhibition in tested groups remained above 50%. F1 and F2, groups returned to pure medium showed a greater inhibition than the re-exposed ones (Table 1 and Fig. 2). In adult daphnids coming from the perinatal exposed group of in vitro cultured D. magna embryos, no significant reproduction inhibition was observed ( Table 2).
Fig. 2.uIncreasing mortality (grey bars, raw percentage) and reproduction inhibition (black bars, percentage of control mean, with SD) across four generations of D. magna exposed, continuously or alternately, to 2umguL1 EFX. ** p<0.01;*** p<0.001; NP Not Performed.Figure optionsDownload full-size imageDownload as PowerPoint slide
No significant growth inhibition was observed in F0, F1 and F2 generations. In F3, due to the high mortality rate, the end point was evaluated only for the EENN3 group, which showed significant inhibition of growth (51%). In the perinatal exposed group of in vitro cultured D. magna embryos, no significant growth inhibition was observed ( Table 2).
The current study indicates that Spinorphin prolonged exposure (continuous or alternating) to 2umguL1 of EFX may have a serious impact on D. magna population, while the classic (single generation) reproduction test had previously indicated a NOEC of 5umguL1 EFX for reproduction inhibition and adult survival of D. magna ( Park and Choi, 2008). This underlines the importance of testing xenobiotic toxicity on multiple generations of daphnids. The increasing toxicity of EFX over four generations of D. magna was demonstrated not only by the progressive impairment of reproduction performances but also by the mortality rate that reached 100% in three out of four tested groups in the fourth generation. Reproduction performances of daphnids born to EFX-exposed mothers were invariably compromised, regardless of their subsequent incubation either in EFX-containing or pure medium. Apparently, the effect was even worse in the non-re-exposed groups (see Table 1). In particular, the high reproduction inhibition of the group EN1 made it impossible to continue the test along that specific arm of the experimental flow chart (Fig. 2) as the number of neonates harvested from the third brood was insufficient for this purpose.
As already indicated, in multigenerational tests a perinatal exposure of daphnids born to exposed mothers is unavoidable. Therefore, it is important to clarify whether the toxic effects observed in non-exposed; groups are simply the consequence of perinatal exposure. On this regard, it is worth noting that strong inhibitory effects on reproduction were observed also in group EENN3 which was generated by non-exposed; mothers (group EEN2). Hence, the effects were not due to a perinatal exposure of the daphnids; a fact that was confirmed by the results of the targeted test on D. magna embryos where daphnids exposed to 2umguL1 EFX only during embryo development showed a reproduction performance equal to that of the control group. More specifically, perinatal exposure to EFX seemed to act as an all-or-nothing; toxicity effect as 31.4% of embryos died, but the surviving daphnids did not show any inhibition of reproduction activity. On the other hand, embryo mortality may at least partially justify the reproduction inhibition observed in exposed groups in the multigenerational test.
Altogether, there must be damage to adult daphnids exposed to 2umguL1 EFX that is transmitted to subsequent generations. Considering that EFX may exert genotoxic (Thomé et al., 2012) or epigenetic (Csoka and Szyf, 2009) effects in eukaryotes, transmission of genetic alterations or regulations to the Daphnia offspring can be hypothesized. In the real context, even if the pharmaceutical entity disappears from the aquatic environment, due to dilution and/or degradation, the D. magna population may pay the consequences of its historical presence, hence what
Reproduction inhibition in tested groups remained above 50%. F1 and F2, groups returned to pure medium showed a greater inhibition than the re-exposed ones (Table 1 and Fig. 2). In adult daphnids coming from the perinatal exposed group of in vitro cultured D. magna embryos, no significant reproduction inhibition was observed ( Table 2).
Fig. 2.uIncreasing mortality (grey bars, raw percentage) and reproduction inhibition (black bars, percentage of control mean, with SD) across four generations of D. magna exposed, continuously or alternately, to 2umguL1 EFX. ** p<0.01;*** p<0.001; NP Not Performed.Figure optionsDownload full-size imageDownload as PowerPoint slide
No significant growth inhibition was observed in F0, F1 and F2 generations. In F3, due to the high mortality rate, the end point was evaluated only for the EENN3 group, which showed significant inhibition of growth (51%). In the perinatal exposed group of in vitro cultured D. magna embryos, no significant growth inhibition was observed ( Table 2).
The current study indicates that Spinorphin prolonged exposure (continuous or alternating) to 2umguL1 of EFX may have a serious impact on D. magna population, while the classic (single generation) reproduction test had previously indicated a NOEC of 5umguL1 EFX for reproduction inhibition and adult survival of D. magna ( Park and Choi, 2008). This underlines the importance of testing xenobiotic toxicity on multiple generations of daphnids. The increasing toxicity of EFX over four generations of D. magna was demonstrated not only by the progressive impairment of reproduction performances but also by the mortality rate that reached 100% in three out of four tested groups in the fourth generation. Reproduction performances of daphnids born to EFX-exposed mothers were invariably compromised, regardless of their subsequent incubation either in EFX-containing or pure medium. Apparently, the effect was even worse in the non-re-exposed groups (see Table 1). In particular, the high reproduction inhibition of the group EN1 made it impossible to continue the test along that specific arm of the experimental flow chart (Fig. 2) as the number of neonates harvested from the third brood was insufficient for this purpose.
As already indicated, in multigenerational tests a perinatal exposure of daphnids born to exposed mothers is unavoidable. Therefore, it is important to clarify whether the toxic effects observed in non-exposed; groups are simply the consequence of perinatal exposure. On this regard, it is worth noting that strong inhibitory effects on reproduction were observed also in group EENN3 which was generated by non-exposed; mothers (group EEN2). Hence, the effects were not due to a perinatal exposure of the daphnids; a fact that was confirmed by the results of the targeted test on D. magna embryos where daphnids exposed to 2umguL1 EFX only during embryo development showed a reproduction performance equal to that of the control group. More specifically, perinatal exposure to EFX seemed to act as an all-or-nothing; toxicity effect as 31.4% of embryos died, but the surviving daphnids did not show any inhibition of reproduction activity. On the other hand, embryo mortality may at least partially justify the reproduction inhibition observed in exposed groups in the multigenerational test.
Altogether, there must be damage to adult daphnids exposed to 2umguL1 EFX that is transmitted to subsequent generations. Considering that EFX may exert genotoxic (Thomé et al., 2012) or epigenetic (Csoka and Szyf, 2009) effects in eukaryotes, transmission of genetic alterations or regulations to the Daphnia offspring can be hypothesized. In the real context, even if the pharmaceutical entity disappears from the aquatic environment, due to dilution and/or degradation, the D. magna population may pay the consequences of its historical presence, hence what