In current nanotoxicology research, there is a lack of standardization in reporting of zebrafish exposure media and sonication practices. This lack of standardization in use and reporting methods has made reproducing nanoparticle (NP) toxicity results between labs a common issue. It is important to have a standardized way of reporting of exposure media recipe used as media can impact the agglomeration behavior of NPs which can directly impact toxicity results. The objective of this study was to characterize and implement a fish water media recipe to replace the fish water currently used in our laboratory. We conducted a literature review to see how and if exposure media was being reported and determined that a majority of labs did not report media used and in ones that did only a few had enough detail to replicate the recipe. We chose 5 media recipes to analyze along with ultrapure water, and my lab’s current exposure media (Instant Ocean’s simulated fresh water) in order to determine the stability of the particles and media’s impact on agglomeration size after sonication. When the 7 medias containing TiO2 NPs were characterized and measured using dynamic light scattering (DLS), a difference in agglomeration behavior was observed between each media. A difference was also seen in HDD between placing the samples directly into the DLS after sonication, time 0, and allowing the sample to rest for 48 hours before measurement.
It is also important to have a standardized way of reporting sonication methods in nanotoxicology as the range of energies found in papers spans from 450J to 1728000J which directly impacts the agglomerates size and resulting toxicity. We hypothesized that when we were able to calculate and hold energy constant during sonication that we would see a decrease in the HDD of CeO2 and TiO2 NPs. We chose to investigate three commonly used ultrasonication systems (cup horn, probe and bath). We calibrated the three systems using calorimetry calibration to hold each treatment to an equivalent energy of 8400J. The bath sonicator has a single programmed amplitude and the cup horn and probe sonicators have programmed amplitudes of 20%, 30% and 40%. This was to see if when energy was held constant, whether the agglomerates size would be similar no matter the ultrasonication system used. We saw that when energy was held constant between the three systems there was no statistical difference between the different sonicators. We also saw a statistically significant difference between NPs that were sonicated vs unsonicated in the 3 medias with both NPs. We chose to use NPs CeO2 and TiO2 in our lab’s simulated fresh water, ultrapure water and recipe 10 medium chosen from the Fish Exposure Media study. We also wanted to observe the effects of energy input and its impact on HDD. To do this we varied the energy using ultrapure water to determine whether an increase in energy would decrease the NP agglomerate size. The HDD of CeO2 decreased as sonication energy was increased while TiO2 did not exhibit this same energy dependent agglomeration.