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We investigated the ability of chitosan/double-stranded RNA polyplex nanoparticles to silence genes in Caenorhabditis elegans in different environmentally analogous media. Using fluorescence microscopy, we were able to rapidly assess gene knockdown and dsRNA uptake under numerous conditions. Scanning transmission electron micrographs of polyplexes confirms heterogeneous distribution of chitosan and RNA in single particles and a wide range of particle morphologies. High pH and the presence of natural organic matter inhibited the ability of polyplex nanoparticles to silence genes, but were unaffected by the presence of inorganic nitrate and phosphate. Environmental media did not affect particle size in any specific pattern, as determined by dynamic light scattering and fluorescence correlation spectroscopy. The efficacy of polyplexes seems to be closely tied to zeta potential, as all treatments that resulted in a net negative zeta potential (high pH and high natural organic matter) failed to achieve gene knockdown. These results support earlier work that emphasized the importance of charge in gene carriers and will aid in the development of effective gene silencing biological control agents. 

After release into the aquatic environment, engineered nanomaterials (ENMs) undergo complex chemical and physical transformations that alter their environmental fate and toxicity to aquatic organisms. Hyalella azteca are sediment-dwelling amphipods predicted to have a high exposure level to ENMs and have previously shown to be highly sensitive to ZnO nanoparticles (NPs). To investigate the impacts of environmentally transformed ZnO NPs and determine the route of uptake for these particles, we exposed H. azteca to ZnSO 4 , ZnO NPs, and environmental aged ZnO NPs which resulted in three types of particles: 30 nm ZnO–Zn 3 (PO 4 ) 2 core–shell structures (p8-ZnO NPs), micron scale hopeite-like phase Zn 3 (PO 4 ) 2 ·4H 2 O (p6-ZnO NPs), and ZnS nano-clusters (s-ZnO NPs). Treatments included freshwater, saltwater (3 ppt), and the presence of sediment, with a final treatment where animals were contained within mesh baskets to prevent burrowing in the sediment. Dissolution was close to 100% for the pristine ZnO NPs and phosphate transformed NPs, while s-ZnO NPs resulted in only 20% dissolution in the water only exposures. In the freshwater exposure, the pristine and phosphate transformed ZnO NPs were more toxic (LC 50 values 0.11–0.18 mg L −1 ) than ZnSO 4 (LC 50 = 0.26 mg L −1 ) and the s-ZnO NPs (LC 50 = 0.29 mg L −1 ). Saltwater treatments reduced the toxicity of ZnSO 4 and all the ZnO NPs. In the presence of sediment, water column concentrations of Zn were reduced to 10% nominal concentrations and toxicity in the sediment with basket treatment was similarly reduced by a factor of 10. Toxicity was further reduced in the sediment only treatments where the sediments appeared to provide a refuge for H. azteca . In addition, particle specific differences in toxicity were less apparent in the presence of sediment. Bioaccumulation was similar across the different Zn exposures, but decreased with reduced toxicity in the saltwater and sediment treatments. Overall, the results suggest that H. azteca is exposed to ZnO NPs through the water column and NP transformations in the presence of phosphate do not reduce their toxicity. Sulfidized ZnO NPs have reduced toxicity, but their similar level of bioaccumulation in H. azteca suggests that trophic transfer of these particles will occur.