A microscaled mercury saturation assay for metallothionein in fish.

A mercury (Hg) saturation assay for measuring metallothionein (MT) in fish liver was modified by optimizing binding conditions to minimize the mercury and tissue consumed. The revised method uses stable Hg at low concentrations instead of 203Hg. At the reduced Hg concentrations used, MT concentrations in livers homogenized in saline appeared to increase systematically with dilution in both bluegill sunfish (Lepomis macrochirus) and largemouth bass (Micropterus salmoides). This error suggested a binding limitation due to sulfhydryl oxidation or competition for and removal of mercury by non-MT proteins. Homogenizing tissues in trichloroacetic acid (TCA) eliminated the interference. To further evaluate the method, the protocol was tested in the laboratory and field. Metallothionein in bluegill injected with 0.6 mg/kg zinc chloride increased at a rate of 0.03 nmole MT/g liver/h (r2 = 0.53, p = 0.001). Linearity improved when data were corrected for protein content (r2 = 0.74, p < 0.0001). Metallothionein levels in bluegill from a coal ash-contaminated environment were significantly increased over that of hatchery-reared sunfish (F = 20.17, p = 0.0003). The microscaled procedure minimizes concerns related to radioisotope use and waste generation while retaining the high sensitivity of the 203Hg assay.

Using field data and weight of evidence to develop water quality criteria.

In the United States, ambient aquatic life water quality criteria are derived using guidelines developed in 1985 that include a clear and consistent methodology using data from standard toxicity tests. The methodology from these guidelines has been successful, but a broader methodology is needed because some effects of pollutants do not lend themselves to conventional toxicity testing. Criterion assessment is proposed as that methodology. In criterion assessment, a specific environmental goal is translated into a measurable benchmark of effect that is used together with a modeled exposure-response relationship to estimate a range of exposures that will achieve the specific goal. The model of the exposure-response relationships and the benchmark effect are developed from field data and laboratory data using multiple analytical methods. Then the model is solved for the effect, thereby estimating the criterion, an upper threshold for acceptable exposures. The resulting candidate criteria are synthesized to select criteria and other benchmark values, such as remedial goals. The criterion assessment process is illustrated using the US Environmental Protection Agency Framework for Developing for Suspended and Bedded Sediments Water Quality Criteria, which recommends developing alternative candidate criterion values and then evaluating them to select a final criterion. Candidate criteria may be derived from models of field observations, field manipulations, laboratory tests, or empirical and theoretical models. Final selection of a criterion uses a weight-of-evidence comparison that engenders confidence because causal associations are confirmed on the basis of different assumptions, independent data sets, and varied statistical methods, thereby compensating for the concerns raised by individual studies and methods. Thus, it becomes possible to specify criteria for agents with biological or physical modes of action, as well as those with chemical modes of action, to best achieve environmental goals.

Shifts in relative tissue delta15N values in snowy egret nestlings with dietary mercury exposure: a marker for increased protein degradation.

Shifts in tissue nitrogen isotope composition may be a more sensitive general indicator of stress than measurement of high-turnover defensive biomolecules such as metallothionein and glutathione. As a physical resource transmitted along the trophic web, perturbations in protein nitrogen metabolism may also help resolve issues concerning the effects of contaminants on organisms and their consequential hierarchical linkages in ecotoxicology. Snowy egret nestlings (Egretta thula) fed mercury-contaminated diets of constant nitrogen isotope composition exhibited increased relative delta15N values in whole liver (p = 0.0011) and the acid-soluble fraction (ASF) of the liver (p = 0.0005) when compared to nestlings fed a reference diet. When nitrogen isotope data were adjusted for the source term of the diet, liver mercury concentrations corresponded with both whole liver relative 15N enrichment (r2 = 0.79, slope 0.009, p < 0.0001) and relative 15N enrichment in the acid-soluble fraction of the liver (r2 = 0.85, slope 0.026, p < 0.0001). Meanwhile, significant differences were not observed in hepatic levels of the metal-binding peptides metallothionein and glutathione despite a nearly 3-fold difference in liver mercury content. Because increases in tissue delta15N values result from increased rates of protein breakdown relative to synthesis, we propose that the increased relative liver delta15N values reflect a shift in protein metabolism. The relationship between ASF and mercury was significantly stronger (p < 0.0001) than that for whole liver, suggesting that the relationship is driven by an increase in bodily derived amino acids in the acid-soluble, free amino acid pool.

Trace element speciation in largemouth bass ( Micropterus salmoides) from a fly ash settling basin by liquid chromatography-ICP-MS.

Analytical techniques used to examine the chemical speciation of multiple trace elements are important for the investigation of biological systems. Size exclusion chromatography (SEC) coupled to ICP-MS was used to investigate the speciation of Se, As, Cu, Cd and Zn in tissue extracts from a largemouth bass (Micropterus salmoides) collected from a coal fly ash basin and results were compared to a largemouth bass collected at a reference site. Using a Biosil SEC column, with an effective separation range of 100-7 KDa, Cu, Zn and Cd were shown to be bound to metallothionein (MT) in the liver, gill and, to a lesser extent, gonad tissue extract. In liver, muscle and gill of the ash basin bass, Se was predominantly present as low molecular weight species. Only in the gonad extract was the major fraction of Se associated with high molecular weight species. For the liver and gill extracts, further SEC-ICP-MS on a column with an effective separation range of 7000-500 Da was performed, but Se species still eluted near the total volume of the column suggesting a low molecular weight organic or inorganic species. Ion chromatography (IC)-ICP-MS using an AS7 column and HNO(3) gradient elution indicated that the Se and As species in the liver and gill extracts had similar retention times but these retention times did not correspond to retention times for As(III), As(V), dimethylarsenate, arsenobetaine, Se(IV), Se(VI), seleno-methionine, or seleno-cystine.