**4. Conclusions**

The results of this study provide several insights into a nanoparticle engineered to be an environmentally friendly alternative to solid silver core nanoparticles. Our data shows that the use of lignin as the nanoparticle core could be a viable alternative, as it did not pose a significant toxicological hazard to our test organism. Since our reported toxicity was similar to other findings when compared on the basis of silver content, the toxicity of silver-enabled nanoparticles may be predictable, based on the silver concentration of the particle. Ionic silver and PDAC alone were the most toxic components of the formulation, which may be attributed to their higher diffusivity and propensity to interact with cell membranes relative to silver and/or PDAC associated with the particle. The inclusion of PDAC not only adds antimicrobial activity to the particle, but also seems to delay the release of silver ions, so in situations where time release of antimicrobial agents is desired, stabilizing the particles with PDAC may be warranted. This data also encourages further development of similar nanomaterials to minimize their impact on the environment, as well as testing the current particle under environmentally relevant conditions to evaluate toxicity. One way of reducing the environmental impact of these engineered nanomaterials is to design them in a way to minimize the release of soluble components, or to replace these components with less toxic ingredients. We are presently investigating the use of an alternative nanoparticle coating which is biologically derived, that may have the potential to be less toxic in comparison to PDAC.

## **5. Associated Content**

#### *Supplemental Information Is Available for This Publication*

Representative images of zebrafish with and without significant developmental impacts; Average zeta potential and hydrodynamic diameter measurements for particle-containing formulations over a five day period; Metadata associated with zeta potential measurements; Concentration-response curves for formulation components and dialyzed materials based on zebrafish mortality at 120 hpf; Modeled concentration-response curve for the reference material silver nitrate based on zebrafish mortality at 120 hpf; Visual MINTEQ output for all silver-containing formulations.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2079-6382/7/2/40/s1, Figure S1: Representative images of zebrafish with and without significant developmental impacts, Figure S2: Average zeta potential and hydrodynamic diameter (HDD) of particle formulations over a five day period in fishwater. Figure S1a,b are average zeta potential measurements for the formulation components (a) and the dialyzed formulations (b), and Figure S1c,d are the average HDD measurements for the formulation components (c) and the dialyzed formulations (d), Figure S3: Concentration-response comparisons for formulation components (a) and dialyzed materials (b) based on zebrafish mortality at 120 hpf (a) Significant differences (*p* ≤ 0.05) existed between materials in the formulation component treatments. The lignin particle exhibited the lowest toxicity, followed by silver, and PDAC. (b) No significant differences (*p* > 0.05) existed in the dialyzed sample treatments. Comparisons included the two full formulations (NP + Ag + PDAC) and the three NP + Ag formulations, Figure S4: Concentration–response curve for silver nitrate based on zebrafish mortality at 120 hpf, Figure S5: Frequency of delayed hatching in zebrafish embryos exposed to Ag+ as silver nitrate in fishwater. Asterisk represents significant increase (*p* ≤ 0.05) relative to unexposed (control) fish embryos, Figure S6: Frequency of delayed developmental progression in zebrafish embryos exposed to lignin nanoparticles in fishwater. Asterisk represents significant increase (*p* ≤ 0.05) relative to unexposed (control), Table S1: Metadata associated with zeta potential measurements.

**Author Contributions:** Alexander P. Richter and Orlin D. Velev conceived the materials, Cathryn G. Conner synthesized and provided the materials, Bryan J. Harper and Stacey L. Harper designed the experiments and contributed to the analysis of the data and Cassandra E. Nix performed the toxicological experiments, contributed to the data analysis and wrote the paper.

**Acknowledgments:** We would like to thank Alicea Meredith, Lindsay Denluck and Teresa Peterson for their assistance on the project and the Sinnhuber Aquatic Research Laboratory (Grant #P30 ES000210) for providing the zebrafish embryos. This work was supported by the United States Department of Agriculture National Institute of Food and Agriculture (USDA-NIFA), Grant 2013-67021-21181, partially supported by an Oregon State University Agricultural Research Foundation Grant (ARF8301A), as well as a gran<sup>t</sup> from the National Institute of Environmental and Health Sciences (Grant # R01ES017552) to Stacey Harper. Alexander P. Richter thanks the Lemelson Foundation and the Lemelson-MIT program for support. We also acknowledge the support provided to Cathryn G. Conner by the Molecular Biotechnology Training Program (MBTP) sponsored by the National Institutes of Health and the Graduate School at North Carolina State University (5 T32 GM008776-15).

**Conflicts of Interest:** The authors declare no conflict of interest.
