Normal Values for Pentachlorophenol in Urine Samples Collected from a General Population

Transcription

1 Normal Values for Pentachlorophenol in Urine Samples Collected from a General Population Ronald G. Treble and Thomas S. Thompson Saskatchewan Health, Laboratory 8, Disease Control Services Branch, 3211 Albert Street, Regina, Saskatchewan, Canada S4S 5W6 I Abstract [ Aliquots of urine samples collected over a 24-h period from normal individuals were analyzed for pentachlorophenol (PCP). Urine samples were taken from subjects living in various regions (both rural and urban) throughout the province of Saskatchewan. Urinary PCP concentrations were determined with gas chromatography-mass spectrometry and stable isotope dilution. The normal PCP concentrations were found to range from 0.05 to 3.6 ng/ml. Because the aliquots analyzed were taken from 24-h sample collections, the normal range of PCP excreted on a daily basis was determined. A total of 69 samples taken from 26 males and 43 females who ranged in age from 6 to 87 years were analyzed. The average amount of excreted PCP was determined to be 4.3 nmol/day. Introduction Pentachlorophenol (PCP) is an example of a commonly used chemical that has become extensively distributed throughout the environment. PCP has been used for over 50 years in a variety of industrial, commercial, and domestic applications, although it has been primarily used for wood preservation and protection. The persistence of this chemical in the environment has resulted in its widespread existence throughout the food chain. The major route of human exposure to PCP (assuming that we are referring to the general population and not occupationally exposed individuals) is via the food chain. The second most significant means of exposure of the general population to PCP is through inhalation. According to Canada's Food and Drugs Act and Regulations, Health and Welfare Canada has established a maximum acceptable concentration of 0.1 ppm for all xenobiotics (presumably including PCP) in food (1). Although there is currently no Canadian guideline for ambient air quality with respect to PCP, the Ontario Ministry of the Environment has set an ambient air quality criterion of 20 g/m 3 air (1). There has been very little work involving the measurement of urinary PEP concentrations in samples collected from the general public. In the early 1980s, one group studied the presence of various organic pollutants, including PCP, in urine samples from a general population survey of 6000 people (2). With a detection limit of 2 ppb, 79% of the urine samples analyzed were found to contain quantitatable levels of PCP. In a case study involving children in Arkansas, urinary concentrations of selected herbicides and chlorinated phenols were compared between a group of children living near a chemical plant and a control group of children from another community (3,4). All 199 urine samples tested positive for PCP; however, there was no significant difference in the average levels found for the two groups of children. In a more recent study examining the differences in urinary chlorophenol levels between occupationally and non-occupationally exposed individuals, slightly elevated concentrations of PCP were found in the urine samples of the first group (5). In previous work carried out in our laboratory, we analyzed urine samples collected from residents from various locations throughout the province of Saskatchewan (6,7). PCP was found to be present in 0% of the 125 samples that were analyzed. If both studies are considered as one, samples were collected from non-occupationally exposed individuals ranging in age from 4 to 87 years with 67 males and 58 females comprising the subject group. The maximum urinary concentration of PCP was found to be 9.1 ng/ml, whereas the minimum detected was 0.1 ng/ml. The average PCP concentration for the combined studies was determined to be 1.4 ng/ml. In this study we will present urinary PCP data for 24-h collections from non-occupationally exposed individuals residing in various rural and urban areas throughout Saskatchewan. Because the urine samples were collected over a 24-h period, we were able to also present data regarding average levels of PCP excreted on a daily basis. This study was conducted in compliance with the standards established by the Laboratory & Disease Control Services Branch of Saskatchewan Health. Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. 313

2 Experimental Reagents Neat pentachlorophenol, which was of greater than 98% purity, was purchased from BDH Chemicals Canada (Toronto, Ontario, Canada). The surrogate standard, which consisted of a carbon-]3 isotopically labeled pentachlorophenol (13Cs-PCP) of 99% purity (i.e., 99% of all carbons are carbon-13) was purchased from Cambridge Isotope Laboratories (Woburn, MA). Petroleum ether, diethyl ether, and acetone were distilled in glass and suitable for residue analysis (BDH Chemicals Canada). Sulfuric acid (ultrapure reagent grade) was purchased from J.T. Baker (Phillipsburg, N J). The precursor used to produce diazomethane, 1-rnethyl-3-nitro-l-nitrosoguanidine (MNNG), was purchased from Aldrich Chemical (Milwaukee, WI). A standard solution of 2000 IJg/mL 2,3,5,6-tetrachloroxylene in methylene chloride was purchased from Supelco Canada (Oakville, Ontario, Canada). Acidified sodium sulfate, which was used for drying urine extracts prior to derivatization with diazomethane, was prepared by adding concentrated sulfuric acid to a slurry of anhydrous sodium sulfate (Fisher Scientific, Fair Lawn, NJ) in diethyl ether. The sulfuric acid was added at a ratio of 0.1 ml to 0 g of sodium sulfate. After the diethyl ether had been permitted to evaporate in a fume hood, the acidified sodium sulfate was stored in an oven at 1~ until required. Procedures Urine samples were collected from subjects over a 24-h period. Each urine donor was provided with a 4-L container made from high-density polyethylene (HDPE). The donors returned the samples to their respective medical clinics on the day which the collection period was completed. Staff at the medical clinics recorded the volume of the 24-h urine collection, and then transferred small aliquots (approximately 50 to ]00 ml) to smaller tfl)pe containers which were then shipped to our laboratory for analysis. A -ml aliquot of each urine subsample was pipetted into a 15-mL glass centrifuge tube. Using a micropipettor, 50 IJL of a solution containing 1 ng/ijl I:~C~-PCP in acetone was added to each sample. In order to ensure complete recovery of PCP, the samples were hydrolyzed with 200 IJL concentrated sulfuric acid prior to extraction (8). The urine samples were extracted with 3 ml petroleum ether by placing the capped tubes on a rocking mixer for 0.5 h. The samples were centrifuged for rain at 2500 rpm. to break up the emulsions that had formed. The upper organic layer was removed and dried by eluting it through a disposable glass pipette packed with a 2.5-cm layer of acidified sodium sulfate. The dried extract was collected in a clean centrifuge tube. The extraction procedure was repeated with two additional 3-mL aliquots of petroleum ether. Each extract volume was reduced to approximately 1 ml under a gentle stream of nitrogen gas. All extracts were derivatized with a freshly prepared solution of diazomethane in diethyl ether. After allowing the derivatized extracts to sit in a fume hood for 0.5 h (to permit excess diazomethane to dissipate), the solutions were gently evaporated to dryness with nitrogen gas. The residues were reconstituted with 0 IJL of a 314 solution containing 0.2 ng/tjl 2,3,5,6-tetrachloroxylene in toluene. The tetrachloroxylene was added to each extract in order to monitor the operational stability of the gas chromatographic-mass spectrometric (GC-MS) system. A series of laboratory reagent blanks and duplicate urine extracts were processed with the samples as a means of intralaboratory quality assurance. Also, the containers used for collection of the 24-h urine samples and those used for transportation of a subsarnple to our laboratory were examined to ensure that contamination did not result from the collection of the urine samples. This was accomplished by placing reagent water in representative containers and analyzing the water according to the same procedure used for the urine samples. The collection containers were also rinsed with organic solvent, which was then concentrated, derivatized, and analyzed by GC-MS to ensure that there was no contamination possible from leaching of the containers or caps. Instrumentation All GC-MS analyses were performed using a Fisons (Manchester, U.K.) MD800 system. The instrument consisted of a Carlo Erba (Milan, Italy) 8000 GC directly interfaced to a Fisons quadrupole MS via a heated capillary interface. The GC was configured with a splitless injection port and a DB-5MS fusedsilica capillary column coated with a Jm film of stationary phase (15 m x 0.25-mm i.d.; J&W Scientific, Folsom, CA). ~vo microliters of each extract was injected into the GC-MS using a Fisons AS800 GC autosampler. The GC oven temperature program consisted of an initial temperature of 120~ held for 2 min, ramped to 220~ at 8~ and to 300~ at 20~ The final oven temperature was held for min. A 1-min stabilization delay was employed to ensure that the system had reached proper temperature prior to the commencement of the next analytical run. The MS was operated in the electron impact ionization mode with an electron energy of 70 ev. The ion source temperature Table I. Summary of Subjects' Gender and Age* Number ot males 26 Number of females 43 Minimunl age of all subjects (years) 6 Maximum age of all subjects (years) 87 Average age of all subjects (years) 54.6 Median age of all subjects (years) 57 * Total number of subjects : 69. Table II. Summary of Urinary PCP Concentrations* Method detection limit Ing/mL) 0.05 Average PCP concentration plus or minus SD + (ng/ml) 0.75 _ Median PCP concentration (ng/ml) 0.5 Minimum PCP concentration detected (ng/ml) 0.05 Maximum PCP concentration detected (ng/ml) 3.6 * Total number of subje(ts : 69. f SD = standard deviation.

3 was maintained at 200~ for all analyses. In order to obtain the required instrumental sensitivity, the quadrupole mass filter was operated in the selected ion monitoring (SIM) mode. Four ions were monitored for the naturally incurred pentachlorophenol (m/z 263, 265, 278, and 280) and two ions were chosen for the isotopically labeled 13C6-PCP (m/z 288 and 290). Masses 242 and 244 were selected for the measurement of the GC-MS internal standard, tetrachloroxylene. A dwell time of 80 ms and a mass scan width of plus or minus 0.1 ainu was used for each ion monitored. Pentachlorophenol was positively identified as being present in any given urine extract when the following conditions occurred: 9 A peak appeared at the same retention time as the 13C6-PCP ( 0.02 min) in the reconstructed ion chromatograms of all four ions monitored for native PCP; 9 The reconstructed ion chromatogram peaks of all four ions monitored for native PCP had a signal-to-noise ratio of greater than 3:1; 9 The relative ratio of the peak areas for m/z 263, 265, 278, and 280 for the urine extract agreed within plus or minus 15% of the relative ratios obtained for a standard solution of PCP analyzed under identical conditions. The technique of stable isotope dilution was used to calculate the concentration of PCP in the urine samples. Isotopically labeled ]3C6-PCP has virtually identical physical and chemical properties as PCP, and therefore, it can be assumed that the 2s J recovery of ]3C6-PCP will be the same as I the recovery of PCP. Using known quantities of each compound, relative response factors based on the ratio of the peak areas for m/z 280 for native PCP and 288 for 13C 6-2o PCP were calculated. By comparing response factors for standard solutions and urine extracts, the concentration of PCP in "5 the original sample can be accurately determined, "~ 15 l Results A total of 69 urine samples collected from 26 males and 43 females were analyzed for PCP. Table I lists a summary of information regarding the subjects from whom urine samples were collected. Only 4 urine samples from the 69 subjects (< 6%) did not contain detectable levels of PCP (with an estimated method detection limit of 0.05 ng/ml). The remaining 65 urine samples were found to have PCP concentrations ranging from 0.05 to 3.6 ng/ml. Table II lists a summary of the analytical results obtained for the 69 urine samples analyzed E Z I in this study. The distribution of urinary PCP concentrations for the entire group of subjects is illustrated in Figure 1. The majority of the urine samples analyzed (56 of 69 or 81%) were found to have PCP concentrations less than I ng/ml In previous work performed in our laboratory, 0% of the urine samples were found to contain detectable levels of PCP (6,7). In the former study, the average concentration of PCP was calculated to be 1.6 ng/ml, whereas the average of the second study was slightly lower at 0.9 ng/ml. The average urinary PCP concentration in the current study was calculated to be 0.75 ng/ml, which is consistent with our previous results. None of the previous studies that examined PCP concentrations in human urine investigated the normal range of PCP excreted on a daily basis. This type of information is clinically more significant than taking a random urine sample and determining its PCP concentration. In order to diagnose an individual's exposure to PCP, it is best to examine the total amount of PCP excreted in the urine over a 24-h period. Because we have analyzed a measured portion of 24-h urine collections and the original total volumes are known, we can determine a normal range of urinary PCP for non-occupationally exposed individuals. Using the urinary PCP concentrations and 24-h excretion I I PCP concentration (ng/ml) Figure 1. Distribution of urinary PCP concentrations.! >

4 volumes, it is possible to determine the total amount of PCP excreted over the collection period. Table III summarizes the results of the determination of total PCP excreted during the 24-h collection based on the population surveyed in this study. Figure 2 illustrates the distribution of 24-h urinary PCP excretion. Although the highest concentration of total PCP excreted was found to be 20.2 nmol/day, the mean concentration, based on the samples analyzed, was determined to be 4.3 nmol/day with a standard deviation of 4.4 nmol/day. Approximately 90% (62 of 69) of the subjects in the study excreted less than nmol of PCP in a 24-h period, but about 70% (48 of 69) excreted less than 5 nmol. Table III. PCP Concentration in 24 h Urine Collections* Average level of PCP excreted plus or minus SD t (nmol) Median level of PCP excreted (nmol) 2.8 Minimum level of PCP excreted (nmol)* 0.5 Maximum level of PCP excreted (nmol) 20.2 * Total number of subjects = 69. SD = standard deviation. Based on a positive detection of PCP in the urine sample analyzed. (D 0..Q E z 3O o PCP excretion (nmol/day) Figure 2. Distribution of PCP concentrations excreted over 24 h Discussion When the data were examined to determine if any trends existed regarding the age of the subjects and their corresponding urinary PCP concentration, no direct relationship was apparent. Age also did not appear to influence the total amount of PCP eliminated in a 24-h urine collection. It must be noted that a high urinary PCP concentration (i.e., in terms of ng/ml) did not necessarily correspond to a large Mount of PCP excreted over a 24-h period. For example, one subject had a urinary PCP concentration of 3.1 ng/ml and a total 24 h urine volume of 460 ml, which yielded a total 24-h excretion of 5.3 nmol PCP. Another subject had a urinary PCP concentration of 1.0 ng/ml but had a total 24-h urine volume of 2800 ml, and therefore excreted.8 nmol of PCP over the same length of time. In order to more realistically compare the relative degree of exposure to PCP, it is advantageous to examine the total amount of PCP excreted over a 24-h period. As in many situations, the medical community sometimes finds it more valuable to have both timed (such as 24 h) collections, where a quantitated amount over a period of time is required, and random collections, where a concentration of the analyte in the random sample is required, for the purpose of being able to accurately diagnose and treat accordingly. As in the cases of renal failure or very young patients, it would be unreasonable to request a portion of a 24-h excretion for analysis. The rate of metabolism of PCP depends upon several factors, including the amount of PCP that one is exposed to and the route(s) of exposure. In general, however, PCP is fairly rapidly eliminated from the body via excretion, predominantly through urine and minor amounts removed with the feces. Unfortunately, what little information is known regarding rates of PCP metabolism and elimination has been obtained largely from animal studies. Very few studies involving occupationally exposed individuals or voluntary human dosage experiments have been reported. No work has been done examining the typical "background" levels of PCP to which the general population is exposed on a daily basis or the rate at which PCP is metabolized or eliminated from the body. For this type of work to be carried out, detailed analyses of PCP levels in food samples and air samples (assuming that dietary intake and inhalation are two most significant routes of 1 exposure) would have to be performed. This information would have to be coupled with data obtained through clinical analyses, specifically concentrations of PCP eliminated through urination and the rates of PCP elimination. 316

5 Given the widespread existence of PCP throughout the environment and the food chain, we feel that further work is necessary to examine human exposure to this chemical. By studying the amounts of PCP excreted in urine, it should be possible to estimate the levels of exposure of a particular population (e.g., the residents of Saskatchewan in our study). References 1. Health and Welfare Canada. Multimedia exposure analysis for pentachlorophenol. Report prepared for the Multi-media Guidelines Advisory Committee, R.S. Murphy, F.W. Kutz, and S.C. Strassman. Selected pesticide residues or metabolites in blood and urine specimens from a general population survey. Environ. Health Perspect. 48:81-86 (1983). 3. R.H. Hill, Jr., T. To, J.S. Holler, D.M. Fast, S.J. Smith, L.L. Needham, and S. Binder. Residues of chlorinated phenols and phenoxy acid herbicides in the urine of Arkansas children. Arch. Environ. Contarn. Toxicol. 18' (1989). 4. J.S. Holler, D.M. Fast, R.H. Hill, Jr., F.L. Cardinali, G.D. Todd, J.M. McCraw, S.L. Bailey, and L.L. Needham. Quantification of selected herbicides and chlorinated phenols in urine by using gas chromatography/mass spectrometry/mass spectrometry. J. Anal. Toxicol. 13" (1989). 5. M. Veningerova, V. Prachar, J. Uhnak, M. Lukacsova, and 1. Trnovec. Determination of chlorinated phenols and cresols in human urine using solid-phase extraction and gas chromatography. J. Chromatogr. B 657:3- (1994). 6. T.S. Thompson and R.G. Treble. Preliminary results of a survey of pentachlorophenol levels in human urine. BulL Environ. Contam. Toxicol. 53: (1994). 7. T.S. Thompson and R.G. Treble. Pentachlorophenol levels in human urine. Bull. Environ. Contain. Toxicol. 56: (1996). 8. T.R. Edgerton and R.F. Moseman. Determination of pentachlorophenol in urine: the importance of hydrolysis. J. Agric. Food Chem. 27: (1979). Manuscript received December 4, 1995; revision received March 8,