Development of a fluoride chronic effects benchmark for aquatic life in freshwater.

Canada has an interim water-quality guideline for fluoride for protection of freshwater aquatic life that dates from 2002, and 1 Canadian province has a different interim water-quality guideline for fluoride that dates to 1995. The United States does not have a national benchmark for fluoride in freshwater, and only 1 US state has such a benchmark. There are no other national or regional benchmarks for fluoride chronic toxicity in freshwater. In the present study, available data on the acute and chronic toxicity of fluoride to freshwater aquatic life were compiled and reviewed. Acute toxicity was reported to occur at concentrations ranging from 11.5 to >800 mg/L fluoride (F(-) ). The majority of chronic effects occur at concentrations between 1.8 mg/L and 195 mg/L. A total of 10 chronic studies representing 16 species (5 fish, 7 invertebrates, and 4 algae/aquatic plants) were used to derive a chronic effects benchmark of 1.94 mg/L F(-) , applying the species sensitivity distribution approach.

In vitro biotransformation rates in fish liver S9: effect of dosing techniques.

In vitro biotransformation assays are currently being explored to improve estimates of bioconcentration factors of potentially bioaccumulative organic chemicals in fish. The present study compares thin-film and solvent-delivery dosing techniques as well as single versus multiple chemical dosing for measuring biotransformation rates of selected polycyclic aromatic hydrocarbons in rainbow trout (Oncorhynchus mykiss) liver S9. The findings show that biotransformation rates of very hydrophobic substances can be accurately measured in thin-film sorbent-dosing assays from concentration-time profiles in the incubation medium but not from those in the sorbent phase because of low chemical film-to-incubation-medium mass-transfer rates at the incubation temperature of 13.5 °C required for trout liver assays. Biotransformation rates determined by thin-film dosing were greater than those determined by solvent-delivery dosing for chrysene (octanol-water partition coefficient [KOW ] =10(5.60) ) and benzo[a]pyrene (KOW =10(6.04) ), whereas there were no statistical differences in pyrene (KOW =10(5.18) ) biotransformation rates between the 2 methods. In sorbent delivery-based assays, simultaneous multiple-chemical dosing produced biotransformation rates that were not statistically different from those measured in single-chemical dosing experiments for pyrene and benzo[a]pyrene but not for chrysene. In solvent-delivery experiments, multiple-chemical dosing produced biotransformation rates that were much smaller than those in single-chemical dosing experiments for all test chemicals. While thin-film sorbent-phase and solvent delivery-based dosing methods are both suitable methods for measuring biotransformation rates of substances of intermediate hydrophobicity, thin-film sorbent-phase dosing may be more suitable for superhydrophobic chemicals.

Real-time quantification of microRNAs by stem-loop RT-PCR.

A novel microRNA (miRNA) quantification method has been developed using stem-loop RT followed by TaqMan PCR analysis. Stem-loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30,000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem-loop RT-PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem-loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.