The unique or enhanced properties of manufactured nanomaterials (MNs) suggest that their use in nanoenabled products will continue to increase. This will result in increased potential for human and environmental exposure to MNs during manufacturing, use, and disposal of nanoenabled products. Scientifically based risk assessment for MNs necessitates the development of reproducible, standardized hazard testing methods such as those provided by the Organisation of Economic Cooperation and Development (OECD). Currently, there is no comprehensive guidance on how best to address testing issues specific to MN particulate, fibrous, or colloidal properties. This paper summarizes the findings from an expert workshop convened to develop a guidance document that addresses the difficulties encountered when testing MNs using OECD aquatic and sediment test guidelines. Critical components were identified by workshop participants that require specific guidance for MN testing: preparation of dispersions, dose metrics, the importance and challenges associated with maintaining and monitoring exposure levels, and the need for reliable methods to quantify MNs in complex media. To facilitate a scientific advance in the consistency of nanoecotoxicology test results, we identify and discuss critical considerations where expert consensus recommendations were and were not achieved and provide specific research recommendations to resolve issues for which consensus was not reached. This process will enable the development of prescriptive testing guidance for MNs. Critically, we highlight the need to quantify and properly interpret and express exposure during the bioassays used to determine hazard values.

A magnetic cell separation technique (MACS) was developed for isolating and characterizing peanut lectin agglutinin positive (PNA(+)) cells from rainbow trout gills. Percoll density separated mitochondria-rich (MR) cells were serially labeled with PNA-FITC and an anti-FITC antibody covalently coupled to a 50-nm iron particle and then applied to a magnetic column. PNA(+) MR cells were enriched to >95% purity. Transmission electron microscopy analysis of both the PNA(+) and PNA negative (PNA(-)) fraction showed that PNA binds to MR chloride cells while the PNA(-) cell fraction is comprised of MR cells with features characteristic of pavement cells. Western blotting demonstrated that both PNA(+) and PNA(-) fractions had high levels of Na(+)-K(+)-ATPase and Sco1 expression; however, relative expression of H(+)-ATPase in PNA(+) and PNA(-) cells demonstrated that untreated fish had twofold higher H(+)-ATPase levels in PNA(-) cells relative to the PNA(+) cells. Furthermore, hypercapnic acidosis significantly increased the relative H(+)-ATPase expression on PNA(-) cells only, whereas metabolic alkalosis had no significant effect.

Fluorescently labeled peanut lectin agglutinin (PNA-FITC) was used to identify a subtype of mitochondria-rich (MR) cells in the gills of freshwater rainbow trout. In situ binding of PNA-FITC was visualized by inverted fluorescence microscopy and found to bind to cells on the trailing edge of the filament epithelium as demonstrated by differential interference contrast optics. The amount of PNA-FITC binding on the filament epithelium increased with cortisol pretreatment concomitant with an increased chloride cell fractional area as demonstrated by scanning electron microscopy. Dispersed gill cells were isolated by trypsinization and separated using a discontinuous Percoll density gradient. Cells migrating to the 1.06-1.09 g/ml interface were found to be MR as demonstrated by staining with the vital mitochondrial dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide and transmission electron microscopy (TEM). However, only approximately 40% of the MR cells were found to bind PNA-FITC. Cortisol pretreatment increased the relative numbers of MR cells isolated from the dispersed gill cell population, but the relative proportions of PNA binding cells remained unchanged. Ultrastructural analysis of isolated cells in the TEM demonstrated that the MR cell fraction was comprised of a mixed population of chloride cells and pavement cells.

Background and Purpose-Hyperosmotic mannitol therapy is widely used in the clinical setting for acute and subacute reduction in brain edema, to decrease muscle damage in compartment syndrome, and to improve renal perfusion. Though beneficial rheological effects commonly are attributed to mannitol, its direct effects on endothelial cells are poorly understood. Methods-We studied the effect of hypertonic and hypotonic stress on bovine aortic endothelial (BAE) cells, using mannitol, urea, and sodium chloride and medium dilution in vitro. Results-Exposure to incremental osmolar concentrations of 300 mOsm of each osmotic agent increased apoptosis in BAE cells (mannitolХNaClϾurea). Induced programmed cell death was detected by DAPI staining of intact cell nuclei, and by TUNEL and DNA fragmentation ladder assays. Mannitol-induced apoptosis exhibited dose dependence (42% of cells at 300 mOsm [PϽ0.0001] compared with 1.2% of control cells) and was also observed in bovine smooth muscle cells. Mannitol-induced apoptosis was attenuated Ϸ50% in the presence of cycloheximide or actinomycin D. Hypertonic mannitol and NaCl, but not urea, increased tyrosine phosphorylation of the focal adhesion contact-associated proteins paxillin and FAK. Hypotonic medium, which did not lead to apoptosis, increased protein tyrosine phosphorylation of FAK but not of paxillin. Addition of mannitol or NaCl also produced sustained increases in c-Jun NH2-terminal kinase (JNK) activity. In addition, hypertonic mannitol increased intracellular free [Ca 2ϩ ] in a dose-dependent manner. Chelation of intracellular Ca 2ϩ with quin2-AM (10 mol/L) inhibited mannitol-induced apoptosis Ϸ50%, as to a lesser extent did inhibition of tyrosine kinase activity with herbimycin (1 mol/L). Conclusions-We have shown that hypertonic mannitol exposure induces endothelial cell apoptosis, accompanied by activation of tyrosine and stress kinases, phosphorylation of FAK and paxillin, and elevation of intracellular free [Ca 2ϩ ]. The apoptosis is attenuated by inhibition of transcription or translation, by inhibition of tyrosine kinases, or by intracellular Ca 2ϩ buffering. These data suggest that clinical use of the osmotic diuretic mannitol may exert direct deleterious effects on vascular endothelium. (Stroke. 1998;29:2631-2640.)

Acid-sensing ion channels (ASICs) are epithelial Na + channels gated by external H + . Recently, it has been demonstrated that ASICs play a role in Na + uptake in freshwater rainbow trout. Here, we investigate the potential involvement of ASICs in Na + transport in another freshwater fish species, the zebrafish (Danio rerio). Using molecular and histological techniques we found that asic genes and the ASIC4.2 protein are expressed in the gill of adult zebrafish. Immunohistochemistry revealed that mitochondrion-rich cells positive for ASIC4.2 do not co-localize with Na + /K + -ATPase-rich cells, but co-localize with cells expressing vacuolar-type H + -ATPase. Furthermore, pharmacological inhibitors of ASIC and Na + /H + -exchanger significantly reduced uptake of Na + in adult zebrafish exposed to low-Na + media, but did not cause the same response in individuals exposed to ultra-low-Na + water. Our results suggest that in adult zebrafish ASICs play a role in branchial Na + uptake in media with low Na + concentrations and that mechanisms used for Na + uptake by zebrafish may depend on the Na + concentration in the acclimation medium.

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Rainbow trout (Salmo gairdneri) fitted with dorsal aortic cannulae were exposed in a flow-through soft water system to three acidities (pH 5.2, 4.8, or 4.4) and two concentrations of Ca (45 or 410 μequiv.∙L−1), in the presence (105 μg∙L−1) or absence of Al. Blood was sampled for respiratory gases, ions, metabolites, and hematology before and at 4, 18, 28, 42, and 66 h exposure. Two toxic mechanisms of Al and acidity were seen: (i) ionoregulatory toxicity, which was caused by Al at pH 5.2 and 4.8 and by acidity at pH 4.4, and (ii) respiratory toxicity, which was caused solely by Al, and was greatest at higher pH. Ionoregulatory toxicity involved decreases in plasma Na+ and Cl−, red cell swelling, and hemoconcentration. Respiratory toxicity involved reduced blood oxygen tension, elevated blood carbon dioxide tension, and increases in blood lactate. Blood acidosis was a combination of respiratory acidosis (due to CO2 accumulation in the blood; higher pH exposures) and metabolic acidosis (probably due to differential Na+ and Cl− loss into the external, acidic environment; lower pH exposures). Higher water Ca reduced ionoregulatory disturbances due to acidity alone but not those due to Al at higher pH. Higher water Ca also reduced respiratory disturbances at lower pH but not at higher pH. The results are discussed with reference to the chemistry of Al and changes in the gill epithelium associated with acid and Al exposure.

Thirteen states in the United States allow the spreading of O&G wastewaters on roads for deicing or dust suppression. In this study, the potential environmental and human health impacts of this practice are evaluated. Analyses of O&G wastewaters spread on roads in the northeastern, U.S. show that these wastewaters have salt, radioactivity, and organic contaminant concentrations often many times above drinking water standards. Bioassays also indicated that these wastewaters contain organic micropollutants that affected signaling pathways consistent with xenobiotic metabolism and caused toxicity to aquatic organisms like Daphnia magna. The potential toxicity of these wastewaters is a concern as lab experiments demonstrated that nearly all of the metals from these wastewaters leach from roads after rain events, likely reaching ground and surface water. Release of a known carcinogen (e.g., radium) from roads treated with O&G wastewaters has been largely ignored. In Pennsylvania from 2008 to 2014, spreading O&G wastewater on roads released over 4 times more radium to the environment (320 millicuries) than O&G wastewater treatment facilities and 200 times more radium than spill events. Currently, state-by-state regulations do not require radium analyses prior to treating roads with O&G wastewaters. Methods for reducing the potential impacts of spreading O&G wastewaters on roads are discussed.