Environ. Sci. Technol. 1996, 30, 695-700
Quantitative Determination of PCBs in Soil and Water by a Magnetic Particle-Based Immunoassay T I M O T H Y S . L A W R U K , * ,† CHARLES E. LACHMAN,† SCOTT W. JOURDAN,† JAMES R. FLEEKER,‡ MARY C. HAYES,† DAVID P. HERZOG,† AND FERNANDO M. RUBIO† Ohmicron Environmental Diagnostics, 375 Pheasant Run, Newtown, Pennsylvania 18940, and Biochemistry Department, North Dakota State University, P.O. Box 5516, Fargo, North Dakota 58105
A competitive enzyme immunoassay for the quantification of polychlorinated biphenyls (PCBs) in soil and water was developed utilizing amine-terminated superparamagnetic particles as the solid phase. Aroclor 1254 was covalently attached to a bovine serum albumin carrier, and the resulting PCB-protein conjugate was used to immunize rabbits and to produce polyclonal antibodies with reactivity to a broad range of PCBs. Specificity studies indicate that the polyclonal antibody can detect Aroclors 1016, 1232, 1242, 1248, 1254, 1260, 1262, and 1268. The immunoassay has a estimated detection limit of 0.2 ppb (ng/ mL) in water and 0.5 ppm (mg/kg) in soil based on Aroclor 1254. The assay compares favorably to GC method 8080 results when soil (r ) 0.960) or water samples are evaluated (r ) 0.909). The typical within assay % CV is less than 9% in water samples, and recovery studies averaged 99% in water and 85% in soil using a 1-min extraction. This immunochemical method provides quantitative field or laboratory screening results for characterizing and delineating PCBcontaminated sites.
The principles of enzyme-linked immunosorbent assays (ELISA) have been described (9). Magnetic particle-based ELISAs have been applied to the detection of pesticide and toxic organic residues in water (10, 11), soil (12-14), fruit (15), wine (16), meat (17), and produce (18). Magnetic particle-based immunoassay has also been combined with other innovative technologies such as supercritical fluid extraction (SFE) for the analysis of PCBs in soil (6). The quantitative PCB magnetic-based immunoassay described combines an antibody specific for PCBs with an enzymelabeled PCB analog. The assay takes less than 1 h to perform, and simple sample preparation procedures are described for the analysis of water and soil samples. Previous PCB evaluations have described qualitative immunoassay systems that utilized polystyrene tubes on which the antibody is passively adsorbed (19, 20). The desorption of antibodies that are passively adsorbed to polystyrene surfaces is a major factor adversely affecting assay precision and sensitivity (21). This magnetic particlebased immunoassay eliminates the imprecision problems that may be associated with antibody-coated tubes and plates through the covalent coupling of antibody to the magnetic particle solid phase. Magnetic particle-based immunoassays for the herbicides atrazine and alachlor have been shown to be more precise than microtiter plate systems (11). The uniform dispersion of particles throughout the reaction mixture allows for nondiffusion-limited reaction kinetics and the precise addition of antibody.
Materials and Methods
Introduction Polychlorinated biphenyls (PCBs) are a class of 209 discrete congeners in which 1-10 chlorine atoms are attached to a biphenyl. PCBs were marketed under the tradename Aroclor from 1930 to 1977 for use in transformers, capacitors, printing inks, and other applications (1). PCBs do not readily degrade after disposal or dissemination in the environment and have been shown to be ubiquitous environmental contaminants, occurring in human and * Corresponding author telephone: 215-860-5115; fax: 215-8607156; e-mail address:
[email protected]. † Ohmicron Environmental Diagnostics. ‡ North Dakota State University.
0013-936X/96/0930-0695$12.00/0
animal adipose tissue and in milk, sediment, and soil (26). PCBs are classified as a probable human carcinogen, Group B2, by the EPA. This category is for chemicals for which there is sufficient evidence of carcinogenicity in animals and inadequate data in humans. The EPA has established a maximum contaminant level goal (MCLG) of zero and a maximum contaminant level (MCL) and practical quantitation limit of 0.5 ppb in drinking water (7). Regulatory limits for soil remediation vary according to state and site, but in general are 5 or 10 ppm for industrial restricted access areas and 1 or 2 ppm for residential access areas. The EPA has recently encouraged the use of innovative field testing technologies such as immunoassay to expedite and reduce the cost of site characterization, assessment, and cleanup. Immunological methods provide the environmental chemist with a cost-effective, sensitive, rapid, and reliable method suitable for both laboratory and field analysis (8).
1996 American Chemical Society
Immunochemicals. To prepare the PCB immunogen, 8 mmol of 2-mercaptoacetic acid is reacted in 50 mL of methanol with 16 mmol of sodium metal. The solution was evaporated to dryness on a rotary evaporator with a water bath at 45-50 °C. To the residue, 15 mmol of Aroclor 1254 and 100 mL of hexamethylphosphoramide (health hazard) were added and stirred in an oil bath at 80-100 °C for 18-24 h. The mixture was cooled to 60 °C and added to a stirred mixture of 800 mL of deionized water, 250 mL of methylene chloride, and 5 mL of concentrated HCl. The aqueous phase was discarded, and the organic phase was washed with 500-mL portions of water. The organic phase was dried over sodium sulfate, and the PCB hapten was
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purified by preparative silica gel thin-layer chromatography (500-1000-µm plates) using ethyl acetate as the mobile phase and acetone to elute the silica gel. The PCB hapten (0.6 mmol) was dissolved in 8 mL of anhydrous DMF and cooled in an ice bath. Triethylamine (85 µL) and isobutylchloroformate (78 µL) were added and allowed to react for 30 min. This solution was added dropwise to a cooled solution of bovine serum albumin (300 mg) in 0.2 M sodium borate, pH 8.7, and 25 mL of N,N-dimethylformamide (DMF). The immunogen mixture was stirred for 1 h in an ice bath and then for 18-24 h at room temperature and finally dialyzed against 0.2 M sodium borate, pH 8.7 (1×), and deionized water (2×). The PCBBSA immunogen was lyophilized and dissolved in sterile saline solution to a concentration of 4 mg/mL. This solution was emulsified with an equal volume of Freund’s complete adjuvant, and a total of 0.5 mL of the emulsion was injected in the hip muscle of three rabbits. After 20 and 45 days and at 30-day intervals thereafter, the rabbits were boosted with 0.5 mL of the emulsion using Freund’s incomplete adjuvant. Whole blood (30-50 mL) was obtained 10 days after each boost, allowed to coagulate, and centrifuged to obtain the antiserum, which was stored at -70 °C. The PCB antiserum was covalently attached to amineterminated superparamagnetic particles of approximately 1 µm diameter (Perseptive Diagnostics Inc., Cambridge, MA) by glutaraldehyde (Sigma Chemical Co., St. Louis, MO) activation of the solid phase as previously described (10). Superparamagnetic particles of this size separate quickly in magnetic fields but have no magnetic memory, which allows for repeated magnetic separations and resuspension of the particles. The small particle size permits the particles to stay suspended in solution for over 1 h. The particle stocks were diluted 1:1000 in Tris-buffered saline (pH 7.4) containing stabilizers and preservatives for use in the immunoassay. Chemicals and Reagents. The PCB antibody-coupled magnetic particles are commercially available in the PCB RaPID Assay kit (Ohmicron Environmental Diagnostics, Newtown, PA). This RaPID Assay kit also includes PCBhorseradish peroxidase (HRP)-labeled enzyme conjugate; calibrators containing Aroclor 1254, deionized water (wash solution), peroxide, and TMB solutions; and sulfuric acid stopping solution. Aroclors 1016, 1221, 1232, 1242, 1248, 1254, 1260, 1262, and 1268 and related congeners were purchased from ChemService Inc. (West Chester, PA). All other chemicals were reagent grade or of suitable chemical purity. Apparatus. The spectrophotometric results were determined using the RPA-I Analyzer (Ohmicron), the detailed functions of which have been previously described (10). A two-piece magnetic separation rack consisting of a test tube holder, which fits over a magnetic base containing permanently positioned rare earth magnets, is required. This two-piece design allows for a 60-tube immunoassay batch to be setup, incubated, and magnetically separated without removing the tubes from the holders (22). Gilson P-200 (Rainin, Woburn, MA) and Eppendorf repeating pipets (Eppendorf, Hamburg, GER) were used to dispense liquids. Water Sample Preparation. Water samples were collected in glass vessels with Teflon caps. Immediately upon collection, samples were diluted with an equal volume of methanol to prevent adsorptive losses to the glass containers or polystyrene tubes (3). Therefore, the water samples analyzed contained 50% methanol. The final assay results
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were multipied by the appropriate dilution factor to determine the PCB water concentration (i.e., multiply by 2 for the dilution described above). The dilution scheme described provides a detection range of 0.20-10 ppb of PCB as Aroclor 1254 in water. Samples containing greater than 10 ppb of PCB required further dilution. Soil Sample Preparation. Air-dried soils (50 g) were mixed with Aroclor 1254-fortified solutions prepared in methanol to yield soil concentrations from 0.5 to 1000 ppm. Soils were air-dried for 3 days and ground with a mortar and pestle. When analyzing soil samples, a simple extraction was performed prior to analysis. Ten grams (10 g) of soil and 20 mL of 100% methanol were added to a soil collection device (12) and capped. The collection device with the soil/methanol mixture was shaken vigorously for 1 min and allowed to settle for at least 5 min. The cap of the soil collector was then replaced with a glass fiber filter cap, and the extract was collected in a small glass vial. The filtered extract was then diluted 1:1000 (25 µL in 25 mL) in PCB zero standard and assayed as described below. Final assay results were multiplied by the appropriate dilution factor to determine the PCB soil concentration (i.e., multiply by 2000 for the dilution described above). Diluting the soil extract in the zero standard eliminates the need for solvent evaporation or exchange and reduces any possible matrix or solvent interferences in the assayed sample. The dilution scheme described provides a detection range of 500 ppb to 10 ppm of PCB as Aroclor 1254 in soil. Samples containing greater than 10 ppm of PCB required further dilution. Immunoassay Procedure. All diluted water and soil samples were assayed by adding 200 µL of sample, 250 µL of PCB-HRP conjugate, and 500 µL of anti-PCB coupled magnetic particles to a disposable polystyrene test tube in the magnetic rack tube holder and incubating for 15 min at room temperature. The magnetic rack was used to magnetically separate the reaction mixture. After separation, the magnetic particles were washed twice with 1.0 mL of deionized water to remove unbound conjugate and to eliminate any potential interfering substances. The colored product was developed for 20 min at room temperature by the addition of 500 µL of a 1:1 mixture of peroxide/TMB solution. The colored product was stopped and stabilized by the addition of 500 µL of 2 M sulfuric acid. The final concentrations of PCB for each sample were determined by measuring the absorbance at 450 nm using the RPA-I Analyzer. The RPA-I Analyzer was preprogrammed to compare the observed sample absorbances to a linear regression line using a logarithm of the concentration versus logit B/B0 standard curve (where B/B0 is the absorbance at 450 nm observed for a sample or standard divided by the absorbance at the zero standard). The calibrators were prepared in the zero standard (acetate-buffered saline preserved solution, pH 5.0, containing 50% methanol to prevent adsorptive losses) and contained Aroclor 1254 at 0, 0.25, 1.0, and 5.0 ppb. Sample concentrations were calculated by multiplying results by the appropriate dilution factor. GC Analysis for Method Comparison. Both water and soil samples were evaluated with traditional analytical methods. Municipal water, surface water, and groundwater from Pennsylvania, New Jersey, and Delaware were analytically fortified with Aroclor 1254 from 1.0 to 25.0 ppb. Samples (1 L) were extracted with hexane in a separatory funnel and analyzed by EPA SW-846 Method 8080 utilizing
TABLE 1
Precision of PCB Measurement in Water by Immunoassaya sample
replicates days n mean (ppb) % CV (within assay) % CV (between assay) % CV (total assay)
1
2
3
4
5 5 25 0.86 8.7 15.7 16.6
5 5 25 3.10 6.2 6.1 8.3
5 5 25 4.36 4.6 0.6 4.6
5 5 25 8.04 4.9 2.5 5.4
a Sample 1, surface water fortified with 1.0 ppb of Aroclor 1254; sample 2, municipal water fortified with 3.0 ppb of Aroclor 1254; sample 3, groundwater fortified with 4.0 ppb of Aroclor 1254; sample 4, runoff fortified with 8.0 ppb of Aroclor 1254. Samples were assayed in five singlicates each over 5 days. Final immunoassay results were multiplied by 2 to correct for the 1:1 dilution with methanol.
TABLE 2
Accuracy of PCB Immunoassay in Watera FIGURE 1. PCB dose-response curve. Each point represents the mean of 31 determinations. Vertical bars indicate (2 SD about the mean.
a gas chromatograph (GC) with a electron capture detection (23). The GC results were not corrected for procedural recoveries. Fifty-nine (59) soils of varying soil types were fortified with Aroclor 1254 from 0.5 to 1000 ppm as described above. In addition, 15 field-contaminated soil samples were extracted, diluted, and assayed as received. Soil samples (30 g) were extracted by sonication in hexane and analyzed by EPA SW-846 Method 8080 utilizing a gas chromatograph (GC) with a electron capture detection (23). The GC results were not corrected for procedural recoveries or soil moisture content.
Results and Discussion Dose-Response Curve and Sensitivity. Figure 1 illustrates the mean standard curve for the PCB calibrators, linearly transformed using a log/logit curve fit, collected over 31 assays with error bars representing 2 standard deviations. The error bar at each calibrator point represents the dayto-day variability from small differences in timing, temperature, or reagent age observed over the 31 assays. A calibration curve was included with every assay to correct for any day-to-day variability. The estimated assay detection limit defined as the lowest concentration that can be distinguished from zero and calculated from 90% B/B0 is 200 ppt of PCB (Aroclor 1254) in water (24). This sensitivity approaches the estimated detection limit reported for U.S. EPA Method 8080 of 65 ppt of PCB (as Aroclor 1242) using gas chromatography and an electron capture detector, which requires extensive sample preparation (23). The assay detection limit in soil was 500 ppb, which is the lowest standard concentration corrected for the dilution from extraction and extract dilution (2000-fold dilution). Quantitation with the immunoassay should be limited to within the range of the standard curve, from 0.5 to 10.0 ppb in water and from 500 ppb to 10 ppm in soil unless further sample dilution is performed. Precision. A precision study in which two surface waters, a groundwater, and a municipal water sample were fortified with Aroclor 1254 at 1.0, 3.0, 4.0, and 8.0 ppb, diluted 1:1
PCB added (ppb)
mean (ppb)
n
SD (ppb)
% recovery
+1.00 +3.00 +4.00 +8.00
0.86 3.18 4.32 7.68
12 12 12 12
0.06 0.28 0.36 0.66
86 106 108 96
average
99
a
Surface water, runoff, a municipal drinking water, and a groundwater each fortified with Aroclor 1254 at the described concentrations and assayed in duplicate in three separate immunoassays. Final immunoassay results were multiplied by 2 to correct for the 1:1 dilution with methanol.
with methanol, and assayed five times in singlicate on five different days is shown in Table 1. The within and between day variation was determined by analysis of variance (ANOVA) (25). Coefficients of variation (% CV) within and between day were less than 9% and 16%, respectively. The total % CV (n ) 25) was less than 17% at all concentrations tested. The overall variability in the soil extraction and sample dilution was also evaluated. A homogeneous soil sample fortified with 4.0 ppm of Aroclor 1254 underwent 10 separate samplings, extractions, and dilutions generating 10 immunoassay results from a single assay. The mean soil concentration determined was 3.20 ppm of PCB with a % CV of 15.2%. Accuracy in Water. The accuracy of the immunoassay was assessed by evaluating four environmental water samples each fortified with Aroclor 1254 at 1.0, 3.0, 4.0, and 8.0 ppb and analyzed in the assay. Table 2 summarizes the accuracy of the PCB immunoassay with environmental water samples. The water samples included runoff, a small stream, groundwater, and a municipal drinking water. Each sample was evaluated three times in duplicate to verify reproducibility. Added amounts of PCB were recovered quantitatively in all cases with an average assay recovery of 99%. All unfortified water samples assayed as less than the detection limit of 0.2 ppb. The accurate recovery of the spiked water samples suggests that no sample matrix problems or interferences were present in the samples tested, and the immunoassay is accurate across the range of the method. Accuracy in Soil. Recovery data from two different soil types (Sharkey clay loam and Plano loam) fortified with
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TABLE 3
Accuracy of PCB Immunoassay in Soila % recovery with extraction time soil Plano loam Sharkey clay loam average (SD)
PCB (ppm)
1 min
30 min
18 h
5 50 500 5 50 500
101 84 86 86 80 74 85 (9.0)
88 94 85 86 86 80 87 (4.5)
100 97 90 90 90 83 92 (6.0)
a Soils fortified with Aroclor 1254. Ten grams of each PCB-fortified soil was extracted with 20 mL of methanol. Soil extracts were diluted in the zero standard and analyzed in duplicate in the immunoassay. The unspiked soil assayed as less than the soil detection limit of 500 ppb.
FIGURE 3. Correlation between expected PCB concentrations in Aroclor 1254-fortified water samples and the magnetic particlebased immunoassay. n ) 30, r ) 0.992, y ) 0.848x + 0.082 ppm.
FIGURE 2. Correlation between PCB concentrations in Aroclor 1254fortified water samples as determined by the magnetic particlebased immunoassay and GC method 8080. n ) 30, r ) 0.909, y ) 0.819x + 0.887.
Aroclor 1254 at 5, 50, and 500 ppm and extracted for 1 min with 100% methanol is summarized in Table 3. To compare PCB extraction efficiency as a function of time, the soil samples were shaken for an additional 30 min and overnight (18 h) on a mechanical shaker. Average recoveries of PCB for the fortified soils were 85, 87, and 92% for a 1-min, 30-min, and 18-h extraction, respectively. A 1-min extraction was used for all further soil evaluations since most of the PCBs were extracted in this time period, and longer extractions did not significantly improve extraction efficiency. All unfortified soil samples assayed as less than the soil detection limit of 500 ppb. Method Comparison. Correlation of 30 Aroclor 1254fortified water samples (groundwater, surface water, and municipal water) using values obtained by the immunoassay method (y) and GC EPA Method 8080 (x) is illustrated in Figure 2. The regression analysis yields a correlation of 0.909 and a slope of 0.82 between methods. The difference between the two methods appears to be due to the variable recovery by GC analysis particularly at higher concentrations (>10 ppb). Figures 3 and 4 illustrate the recovery of the PCB-fortified water samples by comparing the immu-
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FIGURE 4. Correlation between expected PCB concentrations in Aroclor 1254-fortified water samples and GC method 8080. n ) 30, r ) 0.922, y ) 0.868x + 0.551 ppm.
noassay and GC results versus the analytically fortified concentrations. The source of the GC method variability was not investigated further. Seventy-four (74) soil samples, including Aroclors 1254 and 1260 field-contaminated soils and soils fortified with Aroclor 1254 from 0.5 ppm to 1000 ppm were analyzed by the immunoassay and GC Method 8080 (Figure 5). The regression analysis yields a correlation (r) of 0.960 and a slope of 0.971 between methods. The slope of the regression analysis (not significantly different from 1.00 at R ) 0.1) can be attributed to the immunoassay’s calibration to Aroclor 1254 and the fact that Aroclor 1254 was the major PCB soil contaminant measured. Comparison of the 15 weathered, field-contaminated soil samples in this study to Method 8080 yields a correlation (r) of 0.972 and a slope
TABLE 5
Effect of Possible Interfering Substances in Water compound
max concn 0 ppb of 2 ppb of of compd PCB sample PCBa sample (ppb) tested (ppm) (ppb) NDb ND ND ND ND ND ND ND ND ND ND ND ND ND ND
calcium (chloride) 500 copper (chloride) 250 iron (chloride) 100 magnesium (chloride) 250 manganese (chloride) 250 mercury (chloride) 250 nickel (sulfate) 250 zinc (chloride) 250 nitrate (sodium) 250 phosphate (sodium) 250 sulfate (sodium) 10000 sulfite (sodium) 250 silicates (sodium meta-) 1000 thiosulfate (sodium) 250 NaCl 1.0M a Samples fortified with Aroclor 1254. ppb).
FIGURE 5. Correlation between PCB concentrations in fieldcontaminated (0) and Aroclor 1254-fortified soil samples (O) as determined by magnetic particle-based immunoassay and GC method 8080. n ) 74, r ) 0.960, y ) 0.971x + 0.188 ppm. TABLE 4
Specificity (Cross-Reactivity) of Aroclors and Specific Congeners in PCB Immunoassay compound
LDDa (ppb)
I50b (ppb)
% cross reactivity
Aroclor 1254 Aroclor 1260 Aroclor 1248 Aroclor 1262 Aroclor 1242 Aroclor 1232 Aroclor 1268 Aroclor 1016 Aroclor 1221 2,2′,5,5′- c 3,3′,4,4′3,4,4′,53,3′4,4′,5- d 2,3,4,4',53,3′,4,4′,5,5′- e pentachlorophenol
0.10 0.10 0.11 0.18 0.17 0.42 0.46 0.47 6.77 0.24 0.28 0.69 0.09 0.10 1.96 184
1.80 1.15 2.11 2.37 4.40 9.38 10.9 12.8 81.3 18.2 47.0 100 2.04 61.4 >100 7260
100 156 85.3 75.9 40.9 19.2 16.5 14.1 2.2 9.9 3.8 1.8 88.2 2.9
