Environ. Sci. Technol. 1995, 29, 2754-2758
Determination of Pentackmphenol in Water and Soil by a Magnetic Particle-Based Enzyme Immunoassay CHARLES S. HOTTENSTEIN,* SCOTT W. JOURDAN, MARY C. HAYES, FERNANDO M. RUBIO, DAVID P. HERZOG, A N D T I M O T H Y S . LAWRUK Ohmicron Environmental Diagnostics, 375 Pheasant Run, Newtown, Pennsylvania 18940
A competitive enzyme immunoassay using specific pentachlorophenol (PCP) antiserum covalently coupled to a magnetic particle solid phase has been developed for the detection of PCP in water and soil. The immunoassay allows the quantification of PCP from 100 ppt (parts per trillion, ng/L) in water and 100 ppb (parts per billion, mg/L) in soil. Spike recovery from water samples with no sample pretreatment and from various soil types following a simple extraction technique averaged 105% and 94%, respectively. The method compares favorably with GC/MS and HPLC measurements in water ( r = 0.980) and soil ( r = 0.996) samples.
Introduction One of the most extensively used environmental chemicals in the world, 2,3,4,5,6-pentachlorophenol, commonly referred to as Penta or PCP, was introduced in 1936 as a timber preservative for the control of termites, fungal rots, and wood boring insects. Numerous applications since then, most of which are obsolete or prohibited, and the continued use as a wood preservative have led to the contamination of many environmental media. PCP residues have been detected at over 7% of the United States National Priorities List (NPL)hazardous waste sites (1).The United States Environmental Protection Agency (U.S.EPA) National Urban Run-off Program and National Organic Monitoring Survey have both reported frequent detections of PCP in stormwater runoff and public water supplies (2). Widespread public exposure has been documented with 71% of 6990 urine samples containing detectable levels of PCP with an average concentration of 6.3ppb (3).The U.S. EPA has classified PCP as a B2 probable human carcinogen based on sufficient evidence from animal toxicity studies and inadequate human clinical data ( 4 ) . The U.S. EPA has regulated PCP by setting a practical quantification limit * E-mail address:
[email protected]; telephone: (215) 8605115; fax: (215) 860-5213.
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ENVIRONMENTAL SCIENCE &TECHNOLOGY i VOL. 29. NO. 11, 1995
(PQL)and a maximum contaminant level (MCL)in drinking water at 1 ppb (ug/L) and a maximum contaminant level goal (MCLG) at 0 ppb (5). Government regulations, environmental persistence, and potential health concerns make efficient monitoring of PCP in soil and water necessary. Traditional analysis of PCP samples includes capillary column gas chromatography/mass spectrometry (GUMS)and high-pressure liquid chromatography (HPLC). Both require extensive sample cleanup, costly equipment, and highly trained personnel. The advantages of rapid, sensitive, inexpensive methods ideally suited for on-site analysis are desirable. Immunological methods provide a unique opportunity to screen large sample volumes quickly and cost effectively while detecting pesticide residues at ppb levels (6). Positive samples are then able to be confirmed by a complementary analytical method. The principles of immunoassay for the detection of pesticide residues have been previously described (7) and applied to PCP (8). These methods used polystyrene microtiter wells as the solid support for passively adsorbed antibody or hapten conjugate. This method of immobilization can compromise assay sensitivityand precision by desorption or leaching off of passively absorbed antibody or other proteins (9-11) causing well to well variability, which has been determined to be the largest source of overall assay imprecision within microtiter plates (12). Magnetic particle-based immunoassays have been developed and applied to the detection of pesticide residues (13-22). These immunoassays eliminate imprecision problems that are associated with coated beads, plates, and tubes through the covalent coupling of antibody to a superparamagnetic particle solid phase (23). The development and the performance of a competitive magnetic particle-based immunoassay that is rapid, sensitive, and accurate for the detection of PCP in water and soil (appropriate for both laboratory and field analyses) are described.
Experimental Section Reagents. Amine-terminated superparamagnetic particles were obtained from Perseptive Diagnostics (Cambridge, MA). Glutaraldehyde was purchased from Sigma Chemical Company (St.Louis, MO). Rabbit anti-PCP serum and PCPenzyme (HRP) conjugate are available from Ohmicron Corporation (Newtown, PA). PCP and related phenolic compounds as well as other nonrelated cross reactants were purchased from Riedel-de-Haen (Hanover, FRG) or Chem Service (west Chester,PA). Hydrogen peroxide and3,3’,5,5’tetramethylbenzidine (TMB)were obtained from Kirkegaard and Perry (Gaithersburg, MD). All other reagents were reagent grade or chemically pure. Apparatus. Soil collection and extraction were performed using a soil collection device (Ohmicron, Newtown, PA). The collection device is a self-contained unit with 30 mL capacity. The device includes screw caps, borosilicate glass fiber filter caps, and a detachable plunger rod that allows convenient sampling, extraction, filtration, and storage of samples. Gilson P-200 adjustable pipet (Rainin, Wobum, MA) and Eppendorf repeating pipet (Eppendorf, Hamburg, Germany) were used to dispense all reagents. A speciallydesigned 60-position test tube rack with a remov-
0013-936Xi95i0929-2754$09,0010
@ 1995 American Chemical Society
able magneticbase containingpermanently positioned rare earth magnets was used to separate antibody magnetic particles from unreacted reagents (21) (Ohmicron). Spectrophotometric measurements were determined using the RPA-IAnalyzer (Ohmicron)whose detailed operations have been previously described (22). Antibody Couplhg procedure. Rabbit anti-PCP coupled magnetic particles were prepared by glutaraldehyde activation of the magnetic particle solid phase (22). Immunoassay Procedure. All water samples and diluted soil extracts were tested by adding 200pL of sample, 250pL of enzyme conjugate, and 500pLof anti-PCP coupled magnetic particles to 12 x 75 mm polystyrene test tubes and incubating them at room temperature for 30 min. The reaction mixture was magnetically separated with the 60position test tube racklseparation base and decanted. The separated magnetic particles were washed twice with 1.0 mL of preserved Tris-buffered saline solution containing Tween 20. The amount of PCP-enzyme-labeled conjugate bound to the antibody during the competition step was measured by dispensing 500pL of hydrogen peroxidelTMB substrate (1:l) and color developed for 20 min at room temperature. The color formation was stopped and stabilized by the addition of 500pL of 2 N sulfuric acid, and the absorbance of each tube was measured at 450 nm using the RPA-I Analyzer. The RPA-I calculates sample concentrations from a linear regression line using a log-logit standard curve constructed from 0, 0.1, 2.0, and 10.0 ppb PCP calibrators (prepared in a buffered sodium acetate/ saline preserved solution). Samples greater than 10.0 ppb were diluted with the zero calibrator before analysis, and the actual sample concentrations were calculated by multiplying the results by the appropriate dilution factor. Water Analysis. Over 400 water samples from ground, surface, and municipal sources were collected fromvarious locations across the United States and analyzed by immunoassay as received. Twenty sampleswere fortified with known levels of PCP and evaluated by the immunoassay and U.S. EPA GUMS method 625 (241,which has a method detection limit of 10 ppb. The GClMS results were not corrected for procedural recoveries, and a correlation between methods was determined by a linear regression analysis of the data. The immunoassay performance characteristics were defined in detail by examining sensitivity, sample accuracy, precision, dilution linearity, metabolite specificity, and assay tolerance to ordinary groundwater compounds. Soil Exbraction and Analysis. Soils of known composition (sandy loam, silt loam, clay loam, muck, and loam) were fortified with PCP and air-dried (17) to determine the extraction efficiency, the precision, and the effect of soil interferences of the method. Ten grams of soil were weighed directly to the soil collection device and extracted by adding 20 mL of 100% methanol or methanollwater (75:25,v/v) basic solution (pH 11.5). The collection device was capped and shakenvigorouslyfor 1-30 min, after which the extraction mixture was allowed to sit for 5 min. The extract was filtered directly from the collection device by replacing the screw cap with a borosilicate glass filter cap and attaching a plunger rod to the base of the collection unit. The filtered extract was collected in a borosilicate glass vial and diluted 1:500 in the zero calibrator for analysis. The soil collection device was used for all soil extraction and analysis studies. Thirty soil samples collected from two lumber treatment facilities in Missouri and North
95 ,
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30
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1
2.0
0.1
10.0
-
Log Pentachlorophenol(ppb)
FIGURE 1. Pentachlorophenol calibration curve. Each point represents the mean of 79 runs with error bars indicating f 2 SD from the mean. BIB0 is the absorbance at 450 nm of a sample or standard divided by the absorbance of the zero standard.
Carolina were evaluatedby the immunoassay and EPA SW846 HPLC Method 8310 (25) to evaluate method comparison.
Results and Discussion Dose Response Curve and Sensitivity. A mean standard curve for the PCP calibrators was generated from 79 assays and transformed by a linear regression line with a logllogit curve fit (Figure 1). PCPvalues obtainedwithinthe standard range of the assay from 0.1 to 10.0 ppb were quantitatively reported. The error bar at each standard point represents the day to day variability from small differences in timing, temperature, or reagent age observed over 79 assays. To ensure accurate results, a standard curve was includedwith every assay to correct for this variability. From the mean standard curve, the assay detection limit (the lowest concentration that can be distinguished from zero) was estimated to be 60 ppt as the concentration corresponding to 90% BIBo (2@,where BIB. is the absorbance at 450 nm of a sample or standard divided by the absorbance of the zero standard. The quantification of the method from the first standard (0.1 ppb) is compatible with government regulatory levels for drinking water including the U.S. EPA MCL and PQL of 1 ppb. Accuracy. Method accuracy was evaluatedby analyzing two surface water samples obtained from a river and a pond and two drinkingwater samples obtained from a municipal source and a well before and after the addition of PCP at concentrations across the working range of the immunoassay. Each sample was evaluated three times in duplicate to verify reproducibility. The percent sample recovery was determined by subtractingthe neat value from the spiked value and comparing it to the known spiked concentration to calculate recovery. All samples recovered well (98-111%) with an average recovery of 105% (Table 1). Precision. To determine the reproducibility of the immunoassay, within and between day variation was examined by testing four individually fortified water samples from the above study as five singlicates per test over five consecutive days. The within and between day variation was determined by the method of Bookbinder and Panosian VOL. 29. NO. 11, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1
TABLE 3
Accuracy of Pentachlorophenol Immunoassay in Water
Linearity upon Santple DilutioP
amount of
water sample
pentachlorophenol recovered'
pentachlorophenol added (ppb)
mean Ippb)
0.50 1.50 3.00 8.00 av
0.49 1.63 3.34 8.43
SD
YO
n
(ppb)
recovery
12 12 12 12
0.09 0.17 0.29 0.71
98 108 111 105 105
a Four water samples individuallyfortified atthe above concentrations and assayed three times in duplicate in the immunoassay. All unfortified samples assayed less than the detection limit of 0.06 ppb pentachlorophenol.
TABLE 2
Precision of Pentachlorophenol Immunoassay in Water
river water obtained (ppb) expectedb (ppb) recovery (%) pond water obtained (ppb) expected (ppb) recovery (%) municipal water obtained (ppb) expected (ppb) recovery (%) groundwater obtained (ppb) expected (ppb) recovery (YO)
undiluted
1:2
1:4
1:8
8.10 8.10
3.60 4.05 89
1.90 2.02 94
0.92 1.01 91
8.32 8.32
4.22 4.16 101
2.07 2.08 100
0.96 1.04 92
7.24 7.24
4.14 3.62 114
1.94 1.81 107
0.95 0.91 104
3.44 3.44
1.52 1.72 88
0.71 0.86 83
0.37 0.43 86
a The water sampleswerediluted with thezerocalibrator. Expected concentrations were derived in a single assay from the pentachlorophenol concentration of each undiluted water sample.
samplea ~
replicates days n mean (ppb) % CV (within assay) % CV (between assay) % CV (total assay)
~~~
1
2
3
4
5 5 25 0.51 12.5 11.4 16.4
5 5 25 1.67 8.8 8.6 11.8
5 5 25 3.16 7.7 1.8 7.9
5 5 25 8.63 6.7 3.2 7.3
a Pentachlorophenol fortified groundwaters (sample 1 fortified at 0.5 ppb and sample 2 fortified at 1.5 ppb) and drinking waters (sample 3fortifiedat3.0ppbandsample4fortifiedat8.0ppb) wereeachassayed in five singlicates over 5 d.
(27) using SAS software (28) and is summarized in Table 2. Coefficients of variation (% CV) for within and between day values were less than 13% and 12%, respectively, demonstrating the reproducibility of the method. SampleDilution. Four PCP fortified water samples from a river, a pond, a municipal, and a groundwater source were diluted at 1:2, 1:4,and 1:8 in the zero calibrator and analyzed to determine concentration linearity. The expected values were derived from the PCP concentrations of the undiluted (neat) samples and agreed well with measured values (Table 3). If the results were affected by either specific or nonspecific interferences, sample dilution curves would not be parallel with the immunoassay standard curve (29),and the diluted sample values would not assay as expected. Specificity. Specificityof the rabbit anti-PCPserum was determined by testing a variety of phenolic analogs, including major PCP metabolites such as the tetra- and trichlorophenols, and many other nonstructurally related agricultural compounds. Percent cross-reactivity was defined as the amount of analog needed to displace 50% of the enzyme conjugate compared to the 50%displacement ( I d of PCP. The least detectable dose (LDD) was determined as the amount of analog required to achieve 90% BIBo in the immunoassay. The rabbit antiserum demonstrated low cross-reactivityto similar compounds (Table 4) but appeared to have the greatest affinity for phenolic compounds with the largest number of chlorine atoms in closest proximity to the hydroxyl moiety. Interferences. Assay tolerance to typical water matrices and a broad range of common groundwater components
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TABLE 4
Specificity of Various Pkenolic Compouads and Unrelated Agrochemicals in Pentachlorophenol Immunoassay compound
LDDa
Cb
(ppb)
(ppb)
PCP 0.06 2,3,5,6-tetrachlorophenol 0.21 2,3,4,6-tetrachlorophenol 0.91 2,3,6-trichlorophenoI 2.44 2,3,5-trichlorophenoI 1.52 2,3,5,6-tetrachloro8.7 1,4-benzenediol 2,4,6-trichlorophenoI 15.1 2,4,5-trichlorophenoI 21.5 2,3,4-trichlorophenoI 53.2 2,5-dichlorophenol 62.9 2,6-dichlorophenol 266 2,3-dichlorophenol 611 2,4-dichlorophenol 887 3,5-dichlorophenol 1670 hexachlorobenzene 1560 hexachlorocyclohexane 5790 (p isomer)
2.20 4.06 14.6 62.9 119 148 463 574 1730 7 830 5 990 >IO000 > 10 000 > I O 000 >IO000 > 10 000
cross-reactivity (YO)
100 54.1 15.1 3.4 1.8 1.4 0.5 0.4 0.1 0.03 0.04
