UC Berkeley

Chemical contaminants are being detected with increasing frequency in the environment, both as a result of better analytical monitoring techniques and from increasing contaminant concentrations. It is important to understand if un-monitored "emerging contaminant" chemicals can affect or cause toxicity in organisms of ecological importance or affect ecological health. Acute toxicity testing is a usual first step to determine contaminant effects on model organisms. In this work, the nominal acute LC₅₀ (concentration that kills fifty percent of animals tested) was established for chemicals from two classes of emerging contaminants, silver nanowires and chemical flame-retardants, on freshwater crustacean, Daphnia magna. Gene expression studies were then done with microarray technology at sub-acute 1/10 nominal LC₅₀ concentrations to determine genes differentially expressed in exposed animals as compared to control. Results were computationally analyzed with KEGG gene ontology pathway analysis to determine biological pathways affected, and with HOPACH clustering to determine similarities between different gene expression profiles. In each class of contaminant, each different exposure elicited a largely unique gene expression profile.

Further studies on characterization of the nanowires in Daphnia growth media and on nanowire uptake were conducted, and high-throughput, short-read sequencing was done on Daphnia magna RNA samples. A de novo transcriptome was assembled and used for differential gene expression analysis of sequenced RNA from control and silver nanowire-exposed daphnids. The silver nanowires investigated in this study were long and short polyvinylpyrrolidone-coated silver nanowires (PVP AgNW) and long and short silica-coated silver nanowires (SiO₂ AgNW). Ionic silver (Ag⁺) was also tested.

Silver nanowires were not as toxic as ionic silver, and toxicity varied as a function of size and of coating and in different Daphnia growth media. Toxicity could not be attributed to the concentration of dissolved silver in media, but might be caused by internal silver dosing and by a nanowire-specific effect, perhaps related to nanowire shape. The short SiO₂ coated AgNWs caused the most similar response to Ag⁺, and were also the most toxic of the nanowires. The long PVP AgNWs were the least toxic material under most conditions, and caused the most unique gene expression profile. This is the first study to show what appears to be uptake and in vivo modification of nanowires into an animal and the first to show toxic effects of a silver nanomaterial both caused by and independent of silver.

De novo assembly of short-read sequencing data resulted in a robust Daphnia magna transcriptome. Over 101,000 unique transcripts were identified, which illustrated the power of new sequencing technologies and computation algorithms to identify alternate splicing of RNA transcripts. The generated transcriptome was used as a scaffold on which short-read sequencing data were aligned to analyze differential gene expression in control versus AgNW-exposed animals. Preliminary results showed that gene expression analysis with sequencing data resulted in different numbers of differentially expressed genes than were determined with microarray techniques. A combination of four align-and-count methods were subsequently used, which all resulted in different sets of differentially expressed genes. Further work on confirming which method is best for this dataset will need to be done before the sequencing results can be further compared to microarray results. To date, it is unclear whether gene expression studies using sequencing data are more robust or informative than studies with microarray data. However, the assembled transcriptome represents a significant Daphnia magna genomic contribution.

The chemical flame-retardant nominal LC₅₀ values ranged from 58 μg/L (pentaBDE) to 3.96 mg/L (octaBDE). These chemicals are not very soluble in water, so the LC₅₀ values only represent the amount of chemical added to the exposure system, not the amount of chemical dissolved in water or the amount of chemical available to the animals. These nominal LC₅₀ values likely underestimate toxicity of FRs to Daphnia magna. Chemical flame-retardants tested were Firemaster® 550 (FM550), Firemaster® BZ54 (BZ54), pentabrominated diphenyl ether (pentaBDE), octabrominated diphenyl ether (octaBDE), triphenyl phosphate (TPP), tetrabromobenzoate (TBPH). The un-brominated analog to TBPH, phthalate di(2-ethylhexyl) phthalate (DEHP), was also tested. Gene expression profiles had little similarity between chemicals, indicating potentially unique biological effects instead of general toxicity, such as by narcosis. Additional lipidomic and metabolomic studies with FM550 and pentaBDE at 1/10 LC₅₀ showed distinct differences between effects of the two compounds. Genomic results indicate that FM550 exposure may affect transcription and translation of mRNA in Daphnia magna, but further studies are warranted to determine exact mode of toxicity.

Both chemical flame-retardants and silver nanowires are highly toxic to Daphnia magna and cause unique molecular responses. However, exposure concentrations used in this study are typically higher than concentrations detected in environmental samples. Further studies are needed at lower concentrations to confirm the lower limits of effect, and to further investigate specific modes of toxicity. Distinct biological processes were affected by exposure to each chemical and by exposure to different concentrations of one chemical, FM550. This work was limited by lack of annotation of the Daphnia genome and by limited characterization of the Daphnia proteome. Molecular studies in Daphnia were limited, as Daphnia cell lines do not exist. This work contributes, however, to basic knowledge about acute toxicity and molecular effects of environmental contaminants.