Evaluation of tissue and cellular biomarkers to assess 2,4,6-trinitrotoluene (TNT) exposure in earthworms: effects-based assessment in laboratory studies using Eisenia andrei

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at ≥1.8 µg TNT cm^-2, whereas sublethal (weight loss) and lethal effects to earthworms were detected at ≥3.5 and 7.1 µg TNT cm^-2, respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations ( ≥55 mg TNT kg^-1 soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO₄, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.     

Storage of Wheat FoliagePrior to Enzyme-Linked Immunosorbent Assay for Detection of Wheat Soil-Borne Mosaic Virus

Plant samples, collected at various times during a growing season, are frequently stored prior to evaluating resistance to wheat soil-borne mosaic virus (WSBMV) by enzyme-linked immunosorbent assay (ELISA). Leaves of winter wheat cvs Sage and Vona, showing symptoms of WSBMV infection, were cut in half along the midrib. Each half was either: 1) refrigerated at 4 °C, 2) frozen at ?20 °C, 3) frozen at –70 °C, or 4) desiccated with CaCl₂. Relative virus antigen titres were evaluated for individual leaf halves by ELISA. ELISA absorbance means from desiccated leaf halves were consistently higher than absorbance means from corresponding leaf halves that had been frozen. This distinction suggests that virus antigen decreases during freezing but is retained during chemical desiccation. All 4 methods of storage were found to be suitable for short-term storage prior to qualitative evaluations by ELISA, but chemical desiccation was the superior method for long-term storage and for storage of foliar samples prior to quantitative evaluations by ELISA.