Stability of citrate-capped silver nanoparticles in exposure media and their effects on the development of embryonic zebrafish (Danio rerio)

Abstract

The stability of citrate-capped silver nanoparticles (AgNPs) and the embryonic developmental toxicity were evaluated in the fish test water. Serious aggregation of AgNPs was observed in undiluted fish water (DM-100) in which high concentration of ionic salts exist. However, AgNPs were found to be stable for 7 days in DM-10, prepared by diluting the original fish water (DM-100) with deionized water to 10 %. The normal physiology of zebrafish embryos were evaluated in DM-10 to see if DM-10 can be used as a control vehicle for the embryonic fish toxicity test. As results, DM-10 without AgNPs did not induce any significant adverse effects on embryonic development of zebrafish determined by mortality, hatching, malformations and heart rate. When embryonic toxicity of AgNPs was tested in both DM-10 and in DM-100, AgNPs showed higher toxicity in DM-10 than in DM-100. This means that the big-sized aggregates of AgNPs were low toxic compared to the nano-sized AgNPs. AgNPs induced delayed hatching, decreased heart rate, pericardial edema, and embryo death. Accumulation of AgNPs in the embryo bodies was also observed. Based on this study, citrate-capped AgNPs are not aggregated in DM-10 and it can be used as a control vehicle in the toxicity test of fish embryonic development.

Fig. 1. Size monitoring of AgNPs in embryo media over seven days

AgNPs suspension was prepared in cuvettes for the DLS measurement and the cuvettes were maintained at room temperature during the monitoring period. Agitation or sonication was not performed after fresh preparation. A; 10 nm sized-AgNPs, B: 100 nm sized-AgNPs

Fig. 2. Size monitoring of AgNPs in embryo culture media with vortexing or sonication

AgNPs suspension was prepared in cuvettes and the size was measured using DLS. When the cuvettes were left standing for 1 day at room temperature, sedimentation was observed in some cuvettes. The cuvettes were vortexed for 15 second and the size of AgNPs in the suspension was measured. The same AgNPs suspension was sonicated for 30 seconds and measured again (VWR Model B1500A-MTH, 50W 42 KHz). The experiments were performed and representative result was shown (n=3). A; 100 nm sized-AgNPs, B: 10 nm sized-AgNPs

Fig. 3. Mortality of embryonic zebrafish exposed to AgNPs

The proportion of embryos surviving at 120 hpf for each treatment type was shown. Data are shown as the mean ± SEM of four experimental replicates (n=4). 100 % media means undiluted original culture media and 10 % media means diluted the original culture media by one to ten.

Fig. 4. Embryonic hatching after AgNP exposures

The proportion of embryos hatched by 72 hpf for each treatment type. Data are shown as mean ± SEM of four experimental replicates (n=4).

Fig. 5. Decreased heart rate of zebrafish embryos to AgNP exposure

Graphs are the differences in mean heart rate from control embryos at 48 hpf for each treatment type. Individual means were calculated for each of four experimental replicates and the mean of the control animals were subtracted from each of the replicates and a new mean was calculated from those values. Plotted is mean ± SEM of four experimental replicates (n=4).

Fig. 6. Morphological impacts of embryonic exposure to AgNPs

Graph shows the proportion of embryos surviving at 120 hpf that also had pericardial edema (PE) for each treatment type. Plotted is mean ± SEM of four experimental replicates (n=4).

Fig. 7. Effects of 10 nm sized-AgNPs on the developmental stage of zebrafish embryos

Embryos were cultured in 10 % diluted embryo media (DM-10) and a representative photo is shown (5X). C stands for control group and T for AgNPs-treated group. AgNPs in treated group seemed to be accumulated in the core of embryos with a black color. C1 and T1 at 8 hpf, C2 and T2 at 1 dpf, C3 and T3 at 2 dpf, C4 and T4 at 3 day post fertilization (dpf).