Study the Toxicity of Chitosan Nanowaste in Freshwater Fishes

The bio-degradable and low toxicity of chitosan was generally used to formulate nanoparticle for drug delivery. Reducing size of chitosan as nanomaterial (CsNM) to formulate chitosan nanoparticle (CsNP) is the general strategy to increase delivery efficiency. The CsNM is a positively charged nanomaterial and assembly with negatively charged carriers or materials to formulate nanoparticle. The unpacked CsNM is generally as the nontoxic material and released to the environment directly. Especially in nano-technological industry, whether amounts of chitosan nanowaste could be directly released to the environment? The toxicity of chitosan is well studies but the toxicity of CsNM is still unclear. In our preliminary data, we have found the highly toxicity of CsNM in zebrafish ZFL cells, larvae and adult fish but the toxicity is not observed in chitosan nanoparticle (CsNP) or undigested chitosan solution. The yolk of zebrafish larvae is broken within 30 minutes by incubating with 100 ppm of CsNM diluted with ddH2O. Before broken of yolk, the zebrafish larvae show dysphoria and seizures. Some epithelial cells are scraped from epidermis in zebrafish larvae. Amounts of sodium is efflux from the cell measuring by scanning ion-electrode technique (SIET). In adult zebrafish, incubation with 100 ppm of CsNM also cause death within 30 minutes. The adult zebrafish show hypoxia before death. Histochemistry staining analysis of tissue of death adult zebrafish show the damages in gill lamellar, epidermis, intestine and muscle. Further analyzed the toxicity of CsNM in ZFL cells by trypan blue staining and scanning electronic microscope (SEM) show the amounts of damage in cell membrane. Furthermore, toxicity of CsNM diluted by river water also show toxicity in zebrafish larvae. Collectively, we suggest that CsNM might cause death of zebrafish larvae and adult fish by damaging epidermis and gill lamellar. The nanowaste from formulation of CsNP might neutralize before emission to the river. To study the toxicity of CsNM related to the incubation concentration and time in freshwater fishes, we propose this three-year proposal to elucidate. In first and second year, we will evaluate the toxicity of CsNM in zebrafish larvae and adult fish by incubating with different dosages and incubation time in ddH2O and river water. The transgenic zebrafish which are stable expressing fluorescent proteins in epidermis, intestine, muscle and gill will be used to real-time analyze the toxicity of CsNM. In second and third year, we will use the Tilapia larvae and adult fish to study the toxicity of CsNM in ddH2O and river water. In the third year, we will study the neutralization strategies of CsNM using biocompatible materials to reduce the impacts on freshwater fishes before emission. This project will provide the evaluable and real-time monitoring platform to analyze the toxicity of nanowaste in transgenic zebrafish. We suggest that the other positively charged nanomaterial might use this platform to evaluate the toxicity before emission.