SCREENING FOR POLYCHLORINATED DIBENZODIO

2.1 Sample preparation and analysis procedures are described in the manufacturer’s literature. In addition, tests kits are commercially available for this method and the kit instructions used to develop this method can be downloaded from the following Web address: www.capetech. com . Using the test kit from which this method was developed, the following steps are followed. 2.2 Sodium sulfate is added to a soil sample and mixed. Dimethylformamide (DMF) is added to the soil sample and the soil is extracted by shaking for two hours. The supernatant DMF extract is removed. DMF extracts are stable for weeks to months at room temperature. 2.3 Interferences are removed by chemical oxidation. Hexane is added to an aliquot of the DMF extract, then treated with 15% SO3 in concentrated H2SO4 (fuming sulfuric acid). The supernatant hexane is removed and exchanged to a water-miscible organic solvent solution. This hexane-based fuming sulfuric acid cleanup is sufficient for most samples, but in certain circumstances an additional cleanup step may be required. This is the case for samples that contain large amounts of non-volatile aliphatic oils. When the DMF extracts of such soils arecleaned using fuming sulfuric acid, the oil is not oxidized, and it remains after evaporation of the hexane, causing a biphasic system when introduced to the EIA first incubation. Such EIA samples appear opalescent or milky and their results will be invalid because the biphasic system prevents capture of analyte by the antibody. For these samples, a new aliquot of DMF extract is cleaned by carbon adsorption. In this case, the final solvent in the cleanup procedure is toluene rather than hexane. 2.4 The cleaned sample in hexane or toluene is exchanged to a water-miscible organic solvent solution for EIA analysis. PCDD/Fs have very low volatility and are retained during this solvent exchange in a small volume of keeper (Triton X-100 detergent in tetraethylene glycol [TEG]) after evaporation of the original solvent. Methanol is added to dilute this solution and the methanol-TEG-Triton mixture added directly to the EIA tubes. It should be noted that the literature value for solubility of 2,3,7,8-TCDD in methanol is 10 ppm, which is 5000 times higher than the concentration of the highest standard recommended for the kit used to develop this method. Additionally, the solubility of PCDD/Fs in methanol is augmented significantly by the addition of TEG and Triton X-100. 2.5 An accurately measured volume of negative control, standard or prepared sample is mixed with an aqueous sample diluent in test tubes with anti-dioxin antibody immobilized on the surface. The mixture is incubated at the temperature, and for the time, described in the manufacturer’s instructions. 2.6 After incubation, antibody tubes are washed and 0.5 mL of Horseradish Peroxidase (HRP) Competitor Conjugate is added to each tube using a repeater pipettor. Bound PCDD/Fs occupy the dioxin binding sites of the antibodies in proportion to the PCDD/F content of the sample and prevent binding of the competitor-HRP conjugate. After a short incubation, unbound conjugate is removed and the test tubes are washed thoroughly. 2.7 A solution of chromogenic HRP substrate and hydrogen peroxide is added to the test tubes. Color development is directly proportional to enzyme concentration and inversely related to the PCDD/F concentration in the original sample. Stop solution is added to each tube using a repeater pipettor in order to fix the amount of color development. 2.8 The test tubes are analyzed using a tube reader or spectrophotometer to measure the optical density (OD) at 450 nm. The test is interpreted by measuring the signal produced by a sample and determining the concentration from a dose-response curve constructed from standards tested at the same time.