There is a pressing need to develop rapid whole animal-based testing assays to assess the potential toxicity of engineered nanomaterials. To meet this challenge, the embryonic zebrafish model was employed to determine the toxicity of fullerenes. Embryonic zebrafish were exposed to graded concentrations of fullerenes [C 60 , C 70 , and C 60 (OH) 24 ] during early embryogenesis and the resulting morphological and cellular responses were defined. Exposure to 200 μg/L C 60 and C 70 induced a significant increased in malformations, pericardial edema, and mortality; while the response to C 60 (OH) 24 exposure was less pronounced at concentrations an order of magnitude higher. Exposure to C 60 induced both necrotic and apoptotic cellular death throughout the embryo. While C 60 (OH) 24 induced an increase in embryonic cellular death, it did not induce apoptosis. Our findings concur with results obtained in other models indicating that C 60 (OH) 24 is significantly less toxic than C 60 . These studies also suggest that that the embryonic zebrafish model is well-suited for the rapid assessment of nanomaterial toxicity.

Due to its unique physicochemical and optical properties, C 60 has raised interest in commercialization for a variety of products. While several reports have determined this nanomaterial to act as a powerful antioxidant, many other studies have demonstrated a strong oxidative potential through photoactivation. To directly address the oxidative potential of C 60 , the effects of light and chemical supplementation and depletion of glutathione (GSH) on C 60 -induced toxicity were evaluated. Embryonic zebrafish were used as a model organism to examine the potential of C 60 to elicit oxidative stress responses. Reduced light during C 60 exposure significantly decreased mortality and the incidence of fin malformations and pericardial edema at 200 and 300 ppb C 60 . Embryos co-exposed to the glutathione precursor, N-acetylcysteine (NAC), also showed reduced mortality and pericardial edema; however, fin malformations were not reduced. Conversely, co-exposure to the GSH synthesis inhibitors, butathionine sulfoximine (BSO) and diethyl maleate (DEM), increased the sensitivity of zebrafish to C 60 exposure. Co-exposure of C 60 or its hydroxylated derivative, C 60 (OH) 24 , with H 2 O 2 resulted in increased mortality along the concentration gradient of H 2 O 2 for both materials. Microarrays were used to examine the effects of C 60 on the global gene expression at two time points, 36 and 48 hours post fertilization (hpf). At both life stages there were alterations in the expression of several key stress response genes including glutathione-S-transferase, glutamate cysteine ligase, ferritin, α-tocopherol transport protein and heat shock protein 70. These results support the hypothesis that C 60 induces oxidative stress in this model system.

With production and use of carbon nanoparticles increasing, it is imperative that the toxicity of these materials be determined; yet such testing requires specific and selective analytical methodologies that do not yet exist. Quantitative liquid-liquid extraction was coupled with liquid chromatography/electrospray ionization mass spectrometry for the quantitative determination of fullerenes from C60 to C98. Isotopically enriched, 13C60, was used as an internal standard. The method was applied to determine the loss of C60 from exposure water solution and uptake of C60 by embryonic zebrafish. The average recovery of C60 from zebrafish embryo extracts and 1% DMSO in aqueous-exposure solutions was 90 and 93%, respectively, and precision, as indicated by the relative standard deviation, was 2 and 7%, respectively. The method quantification limit was 0.40 microg/L and the detection limit was 0.02 microg/L. During the toxicological assay, loss of C60 due to sorption to test vials resulted in the reduction of exposure-solution concentrations over 6 h to less than 50% of the initial concentration. Time-course experiments indicated embryo uptake increased over course of the 12-h exposure. A lethal concentration that caused 50% mortality was determined to be 130 microg/L and was associated with a zebrafish embryo concentration, LD50, of 0.079 microg/g of embryo.

Polybrominated diphenyl ethers (PBDEs) have become ubiquitous environmental contaminants with potential for bioaccumulation and maternal-fetal transfer that has led to regulatory bans and/or phasing out of several technical mixtures of PBDEs. In the present study, six PBDE congeners (BDE 28, BDE 47, BDE 99, BDE 100, BDE 153, BDE 183) were evaluated for developmental effects on embryonic zebrafish. These congeners were chosen because they are environmentally relevant and cover a wide range of physical-chemical properties. Alterations in behavior, physical malformations, and mortality were scored daily until 168 h postfertilization (hpf). A concentration-dependent increase in spontaneous movement indicated an early onset of behavioral responses to PBDE exposures. Spontaneous movement was affected the most by BDE 47 and BDE 28, whereas BDE 183 did not alter behavior at any concentration tested. Swimming rates were significantly increased by BDE 28 at 96 and 120 hpf, but decreased swimming activity at 168 hpf. Additionally, BDE 47 significantly decreased the swimming rate at 168 hpf. Other endpoints included malformations and mortality. Congeners with fewer bromines (BDE 28, 47, 99, and 100) also induced a curved body axis starting around 120 hpf, which was followed by mortality. BDEs 153 and 183, however, did not elicit these adverse effects. A relationship was found between log K(OW) and median lethal concentration (LC50) and median effective concentration (EC50). Structure-activity relationships in this study suggest that PBDE acute toxicity results from a receptor-mediated effect and further studies are necessary to determine these pathways.

The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories.