Anal. Chem. 1996, 68, 162-169
Quantitative Enzyme-Linked Immunosorbent Assay for Determination of Polychlorinated Biphenyls in Environmental Soil and Sediment Samples Jeffre C. Johnson* and Jeanette M. Van Emon
United States Environmental Protection Agency, National Exposure Research Laboratory, Characterization Research DivisionsLas Vegas, Las Vegas, Nevada 89119
An enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of Aroclors 1242, 1248, 1254, and 1260 in soil and sediments was developed and its performance compared with that of gas chromatography (GC). The detection limits for Aroclors 1242 and 1248 in soil are 10.5 and 9 ng/g, respectively. The assay linear dynamic range is 50-1333 ng/g. Cross-reactivity of the assay with 37 structurally related potential cocontaminants in environmental soil samples was examined; none of the chlorinated anisoles, benzenes, or phenols exhibited >3% cross-reactivity, with 500
mental occurrence. The results of cross-reactivity determinations are presented in Table 2, expressed as I50 values. The data show that none of the potential cross-reactants exhibits >3% cross-reactivity relative to Aroclor 1248, based on the ratio of the I50 value for cross-reactants relative to the I50 value for Aroclor 1248. These data show that the PCB ELISA can be used with environmental samples which may contain significant levels (e.g., gmg/kg) of these compounds. Assay Detection Limit. The assay detection limit is defined as three standard deviations above the zero standard. For Aroclor 1248, the detection limit is 1.34 ng/mL ( 0.87 (n ) 12). For Aroclor 1242, the detection limit is 1.57 ng/mL ( 0.62 (n ) 7). This corresponds to 8.95 ng/g (ppb) and 10.5 ng/g for Aroclors 1248 and 1242 in soil, respectively, based on analysis of 5 g soil samples. Assay Working Range. The ELISA standard curve was linear from 3 to 200 ng/mL when optical density is plotted against the log of concentration. This corresponds to a range of 20-1333 ng/g in soil. The calibration curve was defined by analysis of 10 standards over the assay working range. This range represents the maximum working range; slight nonlinearity was occasionally observed for the boundary concentrations as a result of interassay variation. When variation was too great, the boundary points were omitted such that the points which were retained gave correlation coefficients of 0.99 or greater, resulting in a slight narrowing of the working range. Extraction Efficiency. The methanol shake procedure was initially chosen on the basis of several criteria. In addition to high extraction efficiency, it was desired that the procedure remove the PCBs using a solvent compatible with the ELISA, thus avoiding the need for a solvent exchange step. Finally, it was hoped that the extraction procedure would complement the ELISA procedure in terms of labor and time required for its performance. Previous work by Spittler16 indicates that a water/methanol/ hexane mixture gave 80%-105% recovery of Aroclor 1242 from a real (nonspiked) dry sediment using a shake procedure. The water apparently served to wet the samples, allowing extraction by the methanol. Following extraction by the methanol, the PCBs were partitioned into the hexane layer. In the present study, the samples were extracted wet, essentially eliminating the need to add water. In addition, the PCB ELISA tolerated up to 20% methanol with no performance degradation, whereas hexane resulted in assay degradation after addition of only a few volume percent. Since the hexane in the previous work served only to partition the PCBs into a GC-friendly solvent, it was decided that methanol alone would be a good solvent system. Extraction efficiency of the methanol shake method was evaluated apart from the ELISA measurement step by spiking (16) Spittler, T. M. In Environmental Sampling for Hazardous Wastes; Schweitzer, G. E., Santolucito, J. A., Eds.; ACS Symposium Series 267; American Chemical Society: Washington, D.C., 1984; pp 37-42.
Table 2. Cross-Reactivity of Related Compounds compound
I50, ng/mL
compound
I50, ng/mL
anisole, 2-chloroanisole, 4-chloroanisole, 2,6-dichloroanisole, 3,5-dichloroanisole, 2,3,4trichloroanisole, 2,4,6-trichlorobenzene, chlorobenzene, 1,2dichlorobenzene, 1,3-dichlorobenzene, 1,4-dichlorobenzene, 1,2,3-trichlorobenzene, 1,2,4-trichlorobenzene, 1,3,5-trichlorobenzene, 1,2,3,4-tetrachlorobenzene, 1,2,4,5-tetrachlorobiphenyl butyric acid, 4-(2,4,5-trichlorophenoxy)-
>20 000 >20 000 >20 000 >20 000 >20 000 >20 000 >20 000 >20 000 >20 000 >20 000 >20 000 >20 000 >20 000 >20 000 8 920 >20 000 5 300
DDE DDT phenol, 2-chlorophenol, 4-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 2,6-dichlorophenol, 3,4-dichlorophenol, 3,5-dichlorophenol, 2,3,4-trichlorophenol, 2,3,5-trichlorophenol, 2,3,6-trichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, pentachloro-
>1 400 000 >205 500 >20 000 >20 000 >20 000 7 990 8 740 >20 000 98 000 1 290 3 940 000 >20 000 >20 000 914 >20 000 1 280 >20 000
carbon-14 ring-labeled 2,2′,5,5′-tetrachlorobiphenyl into several different sets of soil samples. Although this PCB congener does not exactly correspond to Aroclor mixtures themselves, it is a major congener of Aroclors 1242 and 124817,18 and was the most appropriate of the commercially available labeled compounds. These spiked soils were then extracted and the extracts counted using liquid scintillation. Thirty 5 g clean soil samples obtained from the U.S. Army Toxic and Hazardous Environmental Material Agency were spiked with the radiolabeled tetrachlorobiphenyl using spiking procedure A. Ten of these samples were spiked to 0.1 mg/kg, and 20 samples were spiked to 0.21 mg/kg with respect to the radiolabeled tetrachlorobiphenyl. Half of this second group of 20 samples was spiked with additional Aroclor 1248, using spiking procedure A, to give a total PCB concentration of 1.0 mg/kg. The second half of this group of twenty samples was spiked with additional Aroclor 1248, again with spiking procedure A, to give a total PCB concentration of 10 mg/kg. The samples, along with three blanks, were extracted using the methanol shake procedure, and the extracts were counted by liquid scintillation. Recoveries of 85%, 89%, and 90% were obtained for the 0.1, 1, and 10 mg/kg spiked samples, respectively, with relative standard deviations (RSDs) of 0.2%, 1.5%, and 0.6%, respectively (n ) 10 for all three sets). A second set of samples consisting of Aroclor 1242 soil standard reference materials (SRMs) at 0.5, 1.5, 8, 25, and 45 mg/ kg and Aroclor 1248 soil SRMs at 33.9 and 282 mg/kg, all in duplicate, were spiked to 0.21 mg/kg with the radiolabeled tetrachlorobiphenyl using spiking procedure A. The samples were dried, extracted using the methanol shake procedure, and counted. A mean recovery of 89% was obtained, with a RSD of 3.7% (n ) 14). Finally, 18 environmental samples obtained from the Abandoned Indian Creek Outfall Superfund Site (to be described below) and contaminated with Aroclor 1248 were dried, crushed, and spiked to 0.21 mg/kg with the radiolabeled tetrachlorobiphenyl, again using spiking procedure A. The mean extraction efficiency for these samples was found to be 92%, with a RSD of 4%. Aroclor 1248 concentration in this latter group of samples ranged from
