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APPLIED CHEMISTRY Microcontaminants in Pentachlorophenol Synthesis. 1. New Bioassay for Microcontaminant Quantification Jianli Yu, Terry J. Nestrick,† Randy Allen,‡ and Phillip E. Savage* Department of Chemical Engineering, UniVersity of Michigan, Ann Arbor, Michigan 48109-2136
We have refined and implemented a novel aryl hydrocarbon receptor capture bioassay for determining the toxic equivalent (TEQ) concentration of polychlorinated dibenzodioxins (PCDDs) and dibenzofurans (PCDFs) in pentachlorophenol samples. We calibrated the method for TEQ concentrations between 0.29 and 2.0 ppm. The standard deviation is typically about 20% of the reported value, but this value can be higher for samples with lower TEQ levels. Testing revealed that the single analysis of a sample is likely to be accurate to within (40% of the reported value, with the uncertainty being attributable to random error. Performing multiple analyses of the same sample reduces the error considerably, to only a few percent. Applying this bioassay method to pentachlorophenol samples synthesized previously via an isothermal reaction provided TEQ concentrations for these samples. The results showed that the TEQ concentration in the samples increased as the synthesis temperature increased. Very high TEQ concentrations (9 ppm) were present in samples from synthesis at 188 °C, which points to the importance of keeping the temperature as low as possible during pentachlorophenol synthesis. Introduction Pentachlorophenol finds use as a wood preservative, for example, in utility poles. The preservative is synthesized commercially via the Lewis acid catalyzed chlorination of phenol in a molten liquid phase. During this chlorination reaction, some phenoxyphenols form, which then react further to form chlorinated dibenzodioxins (but no 2,3,7,8-TCDD) and dibenzofurans. Typically, the octachlorinated compounds are the most abundant, and the abundance of the other compounds decreases as the degree of chlorination decreases. These microcontaminants, though present in low parts per million (ppm) levels, are undesired from an environmental perspective because of their toxicity. There is interest in developing strategies to reduce their concentrations in the pentachlorophenol product. In the past, efforts to reduce microcontaminants have been hampered by the expensive (a few thousand dollars per sample) and time-consuming (days) procedures required for conventional analysis of dioxins and furans in pentachlorophenol by highresolution gas chromatography with mass spectrometric detection (HRGC-MS). As a result, investigators have opted for less precise but more convenient indicators of the microcontaminant level in pentachlorophenol. For example, in our earlier work1,2 we used liquid-liquid extraction and the total area of all peaks in the extract identified by gas chromatography with flame ionization detection (GC-FID) as an indicator of the total microcontaminant level. This indicator is convenient and useful, but it lacks a direct connection to the toxicity of the microcontaminants, which is the issue of central concern. In this article, we describe the development and application of a new method for analysis of microcontaminants in technical* To whom correspondence should be addressed. Tel.: (734) 7643386. Fax: (734) 763-0459. E-mail:
[email protected]. † Present address: 4520 Washington St., Midland, MI 48642. ‡ Present address: Hybrizyme Corp., Raleigh, NC 27607.
grade pentachlorophenol. The target matrix to be analyzed in this work is pentachlorophenol as it is manufactured, not contaminated soils where the microcontaminant level would be orders of magnitude lower. This new method is based on a novel bioassay,3 and it measures directly the toxicity via the toxic equivalent (TEQ) concentration. Bioassays for dioxin quantification is an active area of research and development.4-7 The TEQ concentration can be calculated as the sum of the products of a toxic equivalency factor (TEF) for a given polychlorinated dibenzodioxin (PCDD) or dibenzofuran (PCDF) and the absolute amount of that dioxin or furan present in the sample (e.g., from HRGC-MS analysis). The TEF compares the toxicity of a particular PCDD or PCDF to the toxicity of 2,3,7,8-TCDD, which has a TEF value of unity.8 Only dioxins with 2,3,7,8substitution are toxic, so the TEF for congeners lacking chlorine atoms at these positions is zero. We show herein that reliable estimates for TEQ in pentachlorophenol samples can be obtained from a bioassay that employs real-time PCR (polymerase chain reaction) for analysis. We also report results for the TEQ concentrations in samples obtained in previous work.2 Experimental Section The materials, reactor system, and GC analyses have been described in detail in earlier articles.1,2 Here we focus on the new work done to develop and implement the novel bioassay for TEQ analysis. Hybrizyme Corp. developed and Eichrom Technologies now markets an aryl hydrocarbon receptor capture (AhRC) assay for the detection of PCDDs and PCDFs in a sample.3 These compounds activate the Ah receptor (AhR) to a form that binds the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. The transformed AhR/ARNT complex is then able to bind a DNA response element (DRE) specific for dioxins. The DRE probe includes both a DRE consensus sequence for
10.1021/ie0602106 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/27/2006
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Figure 1. Sample growth curve from PCR.
AhR/ARNT binding and a primer recognition sequence for polymerase chain reaction (PCR) amplification. The complex is next trapped onto a microwell and the cellular material and unbound DRE probe are washed away. The receptor-bound DRE probe is then amplified and measured by real-time polymerase chain reaction (PCR). In real-time PCR, the quantification occurs at each cycle, yielding a primary growth curve (fluorescence vs cycle number). Fluorescence correlates with the amount of input DNA and thus with the concentration of TEQ in the test sample. Having provided an overview of this analysis method, we now provide a more detailed discussion of the experimental procedure. Procedure. We first dissolve 0.1 g of a pentachlorphenol sample in 30 mL of methanol. A 300 µL volume of this solution is then transferred to a 30 mL vial that also contains 20 mL of 0.1 N NaOH (Fisher) and 3 mL of cyclohexane (HPLC grade from Aldrich). The contents of this vial are shaken by hand for about 30 s and then allowed to settle. After the aqueous and organic phases separate, we transfer 50 µL of the cyclohexane layer (which contains the microcontaminants) to a 15 × 85 mm glass tube and evaporate to dryness. The material remaining in the tube is then redissolved in 500 µL of methanol for the AhRC PCR assay. The AhRC PCR test kit used in these analyses was purchased from Hybrizme Corp. (now marketed by Eichrom Technologies as Procept), and it contains enough material to run up to 96 samples. The test kit includes activation solution (12 vials, 0.5 mL), which is the source of the Ah Receptor and DNA Probe (stored at -80 °C prior to use), capture strips (1 plate, 8 × 12 wells) and strip rack, assay buffer (1 bottle, 20 mL, stored at 4 °C prior to use), capture reagent (1 vial, 0.6 mL), primer/probe (1 vial, 0.6 mL, stored at -20 °C prior to use), PCR wash solution (2 bottles, 40 mL each, stored at 4 °C prior to use), and glass vials (rack with 96 vials). Preparing the microwell capture strips for the real-time PCR analysis involves several steps. First, we reconstitute the PCR wash solution for use with the automated platewasher (Wallac Columbus Washer) by mixing the entire contents of one bottle (40 mL) with 960 mL of distilled or deionized water. We then prepare the capture reagent by diluting 40 µL of the stock reagent in 600 µL of the assay buffer for each strip that is to be used. Next, the desired number of strips is placed in the strip frame and washed using the “3XWash” program of the platewasher. Using a multichannel pipet (Wheaton), we dispense 50 µL of the capture-reagent-
containing buffer solution to each well in the strips and then shake the strips for 60-90 min on a plate shaker (Heidolph Titramax 100). While the strips are being shaken, we dispense 50 µL of the assay buffer (at room temperature) into each glass vial using a multichannel pipet. We next add 5 µL of a pentachlorophenol sample solution to each glass vial by using a P-10 Pipetman (Gilson). After adding samples to an entire row of glass vials, we tap them gently to mix the contents and then repeat the process for the next row. We thaw the activation solution immediately prior to use and mix the contents of the individual vials together when analyzing more than one strip. We add 50 µL of activation solution to each glass vial using a multichannel pipet, and shake the vials for 1 h at room temperature for incubation. At this point, we wash the strips again using the “3XWash” program of the platewasher to remove unwanted material and leave behind capture reagent on the walls of the well. Using a multichannel pipet, we transfer 30 µL from each glass vial into wells on the strips and shake the strips for 30 min. We then wash the strips using the “PCRWash” program of the platewasher. This series of soaks and washes takes about 15 min. This step removes unbound DNA and leaves behind only the material bound to the walls. At this time, we thaw the primer/probe solution and make a 1 mL solution using 500 µL of Taqman PCR Master Mix with UNG (uracil-N-glycosylase) (Applied Biosystems), 400 µL of water, and 100 µL of Primer/Probe. We add 40 µL of this solution into each well, seal the wells with adhesive tape and cover them with two compression pads (Applied Biosystems), insert the strips into a thermocycler (ABI Prism 7700 Sequence Detector), and run the real-time “PCR” template. The CCD camera exposure time was set to 10 ms. An MGB-FAM (6carboxyfluorescein) probe was selected. The default cycle conditions for the ABI 7700 were used, which were 50 °C for 2 min (stage 1), 95 °C for 10 min (stage 2), and then 40 cycles of 95 °C for 15 s and 60 °C for 60 s (stage 3). The sample volume was set to 40 µL. Output from the ABI 7700 is in the form of growth curves or amplification plots, as shown in Figure 1. These curves show the amount of fluorescence as a function of the number of PCR cycles completed. The threshold cycle (Ct) value is the one for which the fluorescence first exceeds some user-determined threshold value. The Ct value for a particular well thus reflects the point during the reaction at which a sufficient number of
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amplifications have accumulated in that well, to be at a statistically significant point above the baseline. Samples with higher TEQ concentrations require fewer cycles to reach the threshold value, so the TEQ concentration can be correlated with the threshold cycle value, Ct. Quantitative results were obtained from the AhRC PCR analysis by always analyzing a set of secondary reference standard pentachlorophenol samples along with the samples generated from reaction experiments. The TEQ concentration in these standard samples had been determined in advance at a contract analytical laboratory (Alta Analytical Perspectives) by multiplying the absolute concentration of each isomer by its TEF, as established by the World Health Organization. This analysis involved a state-of-the-art, two-stage, HRGC-HRMS procedure capable or measuring all 17 chlorinated PCDD/PCDF isomers that contribute to TEQ. These samples of commercially produced technical-grade pentachlorophenol provided a TEQ calibration range from 0.3 to 2.0 ppm. This range was selected because it was thought to represent the range we were likely to encounter when analyzing pentachlorophenol samples synthesized in our laboratory. The method is not limited to this range, however. We used commercially produced pentachlorophenols as secondary reference standards (rather than use pure PCDDs/ PCDFs) because the microcontaminants in these standards resided in the same matrix present in the pentachlorophenol samples we were synthesizing and interested in analyzing. We typically ran three pentachlorophenol standards to generate a linear calibration (on a semilogarithmic plot), and then ran a fourth standard as an independent assessment of the quality of the calibration. Results and Discussion
Figure 2. Calibration using pentachlorophenol standard reference samples. (a) Analysis at University of Michigan. (b) Analysis at Hybrizyme Corp. Table 1. Results for Individual Pentachlorophenol Standards low: 0.294 TEQ TEQ
In this section, we first present results that show the accuracy and precision of the AhRC PCR analysis. We then apply this new bioassay to determine the TEQ concentrations in pentachlorophenol samples synthesized previously and compare these results with the total microcontaminant peak areas as determined previously by GC-FID. Accuracy and Precision of AhRC PCR Analysis. To test the accuracy and precision of the AhRC PCR analysis for the present application, we worked with commercial pentachlorophenol samples with known TEQ concentrations. The TEQ concentrations in these materials, which serve as calibration standards, were determined in advance by HRGC-MS. We had five standards available. We used three of these standards, which spanned the largest possible range of TEQ concentrations, to develop a calibration. These standards had TEQ concentrations of 2.03, 1.20, and 0.294 ppm. We refer to these samples as the high, medium no. 1, and low standards. We also had two other standards, medium no. 2 with 1.44 ppm TEQ and medium no. 3 with 1.43 ppm TEQ. To check the reproducibility of the method, we made two sets of samples for each of the three standards to be used in the calibration. We did all of the sample preparation and analysis in our laboratories for one set, but did only the extraction and evaporation to dryness steps for the other set. This latter set was sent to Hybrizyme Corp., where they prepared different concentrations by using different amounts of methanol (2, 1, and 0.5 mL) to redissolve the samples. Figure 2 presents the calibrations for the set done entirely at Michigan and the set analyzed at Hybrizyme. The pentachlorophenol standards and the experimental protocol led to good calibrations (linear on semilogarithmic plot) in both cases.
0.213 0.180 0.306 0.218 0.415 0.577 0.346 mean 0.322 std dev 0.140
medium no. 1: 1.20 TEQ
high: 2.030 TEQ
% error
TEQ
% error
TEQ
% error
-28 -39 4 -26 41 96 18
0.897 0.897 1.563 1.349 1.246 1.411 0.918
-25 -25 30 12 4 18 -24
1.982 2.051 3.415 2.486 0.928 2.914 2.005
-2 1 68 22 -54 44 -1
10
1.183
2.254
11
0.277
-1.4
0.793
We next used the calibration generated from the runs done entirely in our laboratory (Figure 2a) to calculate the TEQ concentrations in each of the standards used to generate the calibration. This analysis provides an indication of the sampleto-sample variation one can expect from this method. Table 1 provides the calculated TEQ concentrations for seven separate runs of the low, medium no. 1, and high TEQ pentachlorophenol standards. Sixteen of the 21 individual samples show less than 40% error. This error is random and not systematic, because the mean values for each pentachlorophenol standard exhibit only about 10% error. These results show that a single analysis of a sample should routinely be reliable to within about (40% of the true TEQ value, but that higher errors could occasionally be encountered. Better accuracy can be obtained by performing multiple analyses of the same sample. Having determined that the reproducibility and precision of the AhRC PCR analysis were adequate for the needs of this research project, we next proceeded to assess the accuracy by doing additional analyses of the reference standards. Three of the reference samples (TEQ ) 0.294, 1.20, 2.03 ppm) were used to develop the calibration for each set of samples analyzed,
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Table 2. Results for Multiple Analyses of Pentachlorophenol Samples bioassay used in calibration treated as “unknowns”
lab-generated penta samples
sample
GC-MS TEQ
mean TEQ
std dev
av error, %
n
low medium no. 1 high medium no. 2 low medium no. 3 high A B C
0.29 1.2 2.03 1.44 0.29 1.43 2.03 0.66 0.69 0.80
0.29 1.24 2.02 1.41 0.39 1.59 2.14 0.50 0.51 0.65
0.01 0.22 0.24 0.29 0.16 0.26 0.38 0.19 0.14 0.16
1 3 -1 -2 32 11 6 -25 -26 -19
20 20 20 15 4 4 4 3 3 3
and all but the medium no. 1 standard were used as “unknowns” to assess the accuracy of the method. In addition, three pentachlorophenol samples generated in our lab were analyzed at Alta by HRGC-MS to determine the TEQ concentration. We used the calibration to calculate the TEQ concentration both in the standards used for calibration and in the samples that were included in analyses as an accuracy check. Table 2 provides the results from these analyses. The first three rows show the results obtained for the standards used in the calibration. The accuracy is very good (as expected for samples used to make the calibration), as evidenced by the mean value departing from the actual value by only a few percent. The precision is reasonably good as well, with the standard deviation for these calibration standards being no more than 18% of the mean. The next four rows of Table 2 contain data for the analysis of standards that were not used in the calibration, and the following three rows show data for the pentachlorophenol samples synthesized in our lab. These results provide an important glimpse into the accuracy of the AhRC PCR analysis method as we have implemented it. The medium no. 2 standard with TEQ ) 1.44 ppm was run on 15 different strips (n ) 15), and the average error is -2%, showing good accuracy. The other three standards were run fewer times, and the largest average error is 32%. This largest error is for the reference standard with the lowest TEQ (0.29 ppm). This sample also had the poorest precision, as the standard deviation for the four analyses was 57% of the true value. The other three reference standards had standard deviations of less than 20% of the true value. Figure 3 shows a parity plot that compares the TEQ concentrations determined by HRGC-MS with those determined by the new bioassay for the seven different pentachlorophenol samples. The bioassay gives accurate results over the
Figure 3. Parity plot for TEQ analysis.
entire TEQ range examined, and there is no indication of any systematic errors. Recognizing that we can get adequate accuracy from this method, we proceeded to analyze numerous samples from synthesis experiments in our laboratory. During the course of these analyses, we routinely used the medium no. 2 standard as an internal check on the accuracy of the calibration we obtain from each set of PCR analyses. Examining the data obtained for this standard revealed that it had been analyzed 108 times. The mean value of the TEQ concentration so determined was 1.48 ppm, which compares very well with the known value of 1.44 ppm determined by HRGC-MS. The range of values for individual determinations is 1.00-1.99, however, showing that a single analysis can be trusted to only about (40%. The standard deviation for these 108 analyses is 0.26 ppm, and the 95% confidence interval is (0.05 ppm. Analysis of Reaction Samples for TEQ. In this section we report results for the TEQ concentration in selected samples obtained from isothermal synthesis experiments completed previously.2 Figure 4 shows results from pentachlorophenol synthesis runs done isothermally at five different temperatures. For synthesis at 105 °C, the lowest temperature investigated, the TEQ concentration was about the same (around 0.5 ppm) in all samples examined. In contrast, the TEQ concentrations in the samples produced at 188 °C show an increase with time. The trends of TEQ concentration with time are difficult to determine because the differences are small (comparable to the 40% uncertainty we estimate for a TEQ concentration determination by a single analysis). The trends with temperature are much easier to discern because these differences are more substantial. The TEQ concentrations increased steadily with increasing temperature. For example, the TEQ concentrations in the samples with the highest tetrachlorophenol yield in each experiment were 0.56, 0.44, 1.16, 1.82, and 6.85 ppm, respectively, for synthesis at 105, 125, 145, 165, and 188 °C, respectively. A similar increase in TEQ concentration with temperature is apparent if one considers the final sample in each experiment. Here the TEQ concentration increased from 0.48 to 9.61 ppm as the synthesis temperature increased from 105 to 188 °C. Figure 5 presents the TEQ concentrations for synthesis at each temperature as a function of the pentachlorophenol yield (rather than reaction time). These data reveal that the TEQ concentration at a given pentachlorophenol yield generally increases with temperature. Thus, keeping temperature no higher than absolutely necessary to maintain a liquid phase in the reactor is important for keeping TEQ concentrations as low as possible. Correlation between Total Peak Area and TEQ. In addition to determining the TEQ concentration for some reaction samples using the AhRC PCR method, we also have analyzed these samples for microcontaminants by determining the total
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Figure 4. Temporal variation of chlorophenol yields and TEQ concentrations for synthesis at different isothermal temperatures: (a) 105 °C; (b) 125 °C; (c) 145 °C; (d) 165 °C; (e) 188 °C.
peak area using GC-FID.2 Thus, we have the opportunity to compare a convenient approximate measure for the microcontaminant quantity (total peak area) with the actual TEQ concentrations. Figure 6 presents the correlation of these two measures of microcontaminants for the samples for which we have data for both measurements. The samples with the two highest total peak areas also were among the three highest in TEQ concentration. Overall, though, there does not appear to be much of a correlation between these two measures. Significant scatter exists. For example, samples with roughly the same total peak area of about 5000 had very different TEQ concentra-
tions (0.7-3.6 ppm). Likewise, separate samples with roughly the same TEQ concentration of about 3 ppm had total peak areas spanning a very wide range. That there is little correlation between total peak area and TEQ is not surprising. The compounds most likely to be the most abundant microcontaminants (octachlorinated compounds) possess very small TEF values, so large changes in the total peak area need not be accompanied by significant changes in TEQ. In addition to the reactor samples discussed above, we also analyzed four of the five pentachlorophenol standards (low, medium no. 1, medium no. 2, and high) by the extraction and
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GC-MS analysis. As such, this method should be useful to manufacturers (to monitor product quality) and to researchers working with pentachlorophenol. 2. There is little correlation between the TEQ concentration and the total peak area of microcontaminants determined by GC-FID. Samples with similar total peak areas have very different TEQ concentrations, and samples with similar TEQ concentrations gave very different total peak areas. Therefore, the GC measurement of microcontaminants by total area, though convenient, is not a very reliable substitute for the TEQ concentration. 3. The TEQ concentrations were highest in pentachlorophenol synthesized isothermally at 188 °C and lowest in the samples synthesized at 105 °C. For a given phenol yield, the TEQ concentration generally increased as the isothermal synthesis temperature increased. Thus, it is important to keep the synthesis temperature as low as possible to minimize the production of PCDDs and PCDFs during pentachlorophenol production. Figure 5. TEQ concentrations from synthesis at different temperatures as a function of pentachlorophenol yield.
Acknowledgment We are grateful for the assistance and expertise provided by Dr. Yves Tondeur of Alta Analytical Perspectives and Dr. Taocong Jin at the University of Michigan. This work was supported under a research contract with the Microcontaminant Reduction Venture, an industrial consortium of pentachlorophenol producers. Literature Cited
Figure 6. Correlation between total peak area and TEQ concentrations.
GC-FID method. The total peak areas for these standards were 241, 1207, 2366, and 2630, respectively, which were not quantitatively consistent with the TEQ content in each standard, as the two “medium” standards had similar TEQ concentrations but total peak areas that differed by a factor of 2. These results indicate that the correlation between total peak area and TEQ is not sufficiently good to justify the use of total peak area as a surrogate for the TEQ concentration in a sample. Summary and Conclusions 1. The AhRC PCR bioassay, as implemented herein, provides reliable quantification of the TEQ concentration in pentachlorophenol samples within the range of 0.3-2.0 ppm. Accuracy to within a few percent of the true value can be obtained through multiple analyses of the same sample. A single analysis, on the other hand, is likely to be accurate to within (40% of the true value. This analysis method is much less costly and much more rapid than the conventional method involving high-resolution
(1) Yu, J.; Savage, P. E. Reaction Pathways in Pentachlorophenol Synthesis. 1. Temperature Programmed Reaction. Ind. Eng. Chem. Res. 2004, 43, 5021-5026. (2) Yu, J.; Savage, P. E. Reaction Pathways in Pentachlorophenol Synthesis. 2. Isothermal Reaction. Ind. Eng. Chem. Res. 2004, 43, 62926298. (3) InnoVatiVe Technology Verification Report. Technologies for Monitoring and Measurement of Dioxin and Dioxin-like Compounds in Soil and Sediment; Hybrizyme Corporation AhRC PCR Kit; EPA/540/R-05/005, March 2005. Available at http://www.epa.gov/ord/SITE/. (4) Behnisch, P. A.; Hosoe, K.; Sakai, S. Bioanalytical Screening Methods for Dioxins and Dioxin-like CompoundssA Review of Bioassay/ Biomarker Technology. EnViron. Int. 2001, 27, 413-419. (5) Safe, S. Development of Bioassays and Approaches for the Risk Assessment of 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Related Compounds. EnViron. Health Perspect. 1993, 101, 317-325. (6) Schwirzer, S. M. G.; Hofmaier, A. M.; Kettrup, A.; Nerdinger, P. E.; Schramm, K. W.; Thoma, H.; Wegenke, M.; Weibel, F. J. Establishment of a Simple Cleanup Procedure and Bioassay for Determining 2,3,7,8Tetrachlorodibenzo-p-dioxin toxicity Equivalents of Environmental Samples. Ecotoxicol. EnViron. Saf. 1998, 41, 77-82. (7) Vanderperren, H.; Van Wouwe, N.; Behets, S.; Windal, I.; Van Overmeire, I.; Fontaine, A. TEQ-value Determinations of Animal Feed; Emphasis on the CALUX Bioassay Validation. Talanta 2004, 63, 12771280. (8) Van den Berg, M.; et al. Toxic Equivalency Factors (TEFs) for PCBs, PCDDs, PCDFs for Humans and Wildlife. EnViron. Health Perspect. 1998, 106, 775-792.
ReceiVed for reView February 20, 2006 ReVised manuscript receiVed May 9, 2006 Accepted May 13, 2006 IE0602106
