The Priority Toxicant Reference Range Study: Interim Report Larry L. Needham, Robert H. Hill, Jr., David L. Ashley, James L. Pirkle, and Eric J. Sampson Centers for Disease Control and Prevention, Atlanta, Georgia The relationship between human exposure to environmental toxicants and health effects is of utmost interest to public health scientists. To define this relationship, these scientists need accurate and precise methods for assessing human exposure and effects. One of the most accurate and pre- cise means of assessing exposure is to measure the level of the toxicant or its primary metabolite in a biologic specimen; this has been defined as measuring the internal dose. This measurement must be quantitative to best study the dose-response relationship. Pertinent questions asked dur- ing an exposure assessment include "How do the levels of a given toxicant in a particular population compare with the levels of that toxicant in other populations?" and "What is the prevalence of exposure to that toxicant in other populations?" To answer these questions for two chemical classes of environmental toxicants, we developed state-of-the-art analytic methods and then applied them to measure the levels of 44 environmen- tal toxicants in biologic specimens from 1000 United States residents who participated in the Third National Health and Nutrition Examination Survey (NHANES ll). These 1000 people are a cross-sectional subset of the NHANES IlIl population and were selected from urban and rural communities in four regions of the United States; all were between 20 and 59 years of age. This subset is not a probability-based sample. The 44 environmental tox- icants are 32 volatile organic compounds, which are measured at parts-per-trillion levels in whole blood, and 11 phenols and one phenoxy acid, which are measured at parts-per-billion levels in urine. We present statistical data for these toxicants in a large portion of our study's population. These analytic measurements have not been compared to any demographic characteristics, such as age and race, in this interim report. In addition, we also give examples of how the methods we developed and the reference range data we gathered have been used to assess exposure in other populations. - Environ Health Perspect 103(Suppl 3):89-94 (1995) Key words: priority toxicant reference range study, pesticides, urine, volatile organic compounds (VOCs), blood, NHANES IlIl Introduction Environmental public health scientists, including epidemiologists, clinicians, risk assessors, and risk managers, are interested in the relationship between human expo- sure and health effects. To accurately study this relationship, they must assess both exposure and health effects (including bio- logic changes). Exposure has been defined as "an event that occurs when there is con- tact at a boundary between a human and This paper was presented at the Conference on Human Tissue Monitoring and Specimen Banking: Opportunities for Exposure Assessment, Risk Assessment, and Epidemiologic Research held 30 March-1 April 1993 in Research Triangle Park, North Carolina. This work was supported in part by funds from the Comprehensive Environmental Response, Compensation, and Liability Act trust fund through an interagency agreement with the Agency for Toxic Substances and Disease Registry, Public Health Service, U.S. Department of Health and Human Services. We thank Marianne Gregg, Sandra Bailey, Samuel Baker, Cynthia Alley, Susan Head, Frederick Cardinali, Dr. Michael Bonin, Joan McCraw, and Joe Wooten for their technical assistance, and Dr. Robert Murphy and his staff for the collection of the specimens. Address correspondence to Dr. Larry L. Needham, Centers for Disease Control and Prevention, 4770 Buford Highway NE, Atlanta, GA 30341-3724. Telephone (404) 488-4111. Fax (404) 488-4546. the environment with a contaminant of a specific concentration for an interval of time" (1). Therefore, human exposure depends on two factors: the contaminant being present in the environment and a person coming in contact with that environment. Essentially, two means are used to assess total human exposure to environ- mental toxicants. One we shall call the "environmental approach" and the other, the "biologic approach." The environmental approach for assess- ing total human exposure is based on the above definition of exposure, that is a) the concentration of the toxicant is measured in one or more environmental media (air, water, soil, or food) to which the subjects may have been exposed; and b) the dura- tion of human contact with each environ- mental medium is estimated. Step one in this approach sometimes requires research- ers to conduct many analyses to character- ize the levels of the toxicants in certain matrices, such as soil. Other times fewer analyses may be necessary because their results can be placed on a geographic map, and the distribution of the pollutants mod- eled. Sometimes the toxicant may either no longer be in the media or no original media samples, such as food, may be available. Data for step two are normally gathered from responses to questionnaires or other historic information on human activity pat- terns (e.g., how many biscuits possibly con- taining the toxicant did one eat, or how far does one live from the source of the toxi- cant). These two steps, or these two steps combined with data on the route of expo- sure (ingestion, inhalation, or dermal absorption), absorption and distribution within the body, and human activity pat- terns, are then used to estimate total human exposure to a toxicant. Epidemiologists fre- quently develop an exposure index and clas- sify an individual's exposure on the basis of such data. However, as we have shown, use of the exposure index is frequently not an accurate means of assessing exposure (2). The biologic approach for assessing total human exposure is based on measur- ing the concentration of the toxicant, its metabolites, or reaction products (such as DNA and protein adducts) in a biologic specimen. This concentration is sometimes referred to as a measure of the body burden of a toxicant even though it usually does not reflect the total amount of the pollu- tant in the body. However, these measure- ments may be used in conjunction with physiologically based pharmacokinetic models to estimate the body burden. Environmental Health Perspectives 89 NEEDHAM ETAL. Even without such models, these measure- ments reflect a person's exposure to the toxicant and the absorption, metabolism, disposition, and elimination of the toxi- cant. To more accurately interpret the bio- logic measurements, however, researchers need biologic half-life and other pharmaco- kinetic data. One of the primary disadvan- tages of the biologic approach is that the use of a biologic matrix frequently entails an invasive procedure to collect the bio- logic material (e.g., blood). Another disad- vantage is that the analytic measurement is demanding because the concentration of the toxicant in a person's body is many times lower than in the environment; how- ever, with the continued evolution of ana- lytic equipment, the magnitude of this disadvantage is decreasing. Also, as with the environmental approach, the toxicant may no longer be in the matrix when it is sampled; the absence of the toxicant is both time and toxicant dependent. We believe that the biologic approach is the superior method for assessing human expo- sure, as we have previously indicated (2). Pertinent questions asked during a bio- logic approach are "Is a given toxicant nor- mally found? If so, at what levels?" These questions are the bases for our priority tox- icant reference range study. Obviously, before beginning the reference range study, we had to select the toxicants and the bio- logic matrix, develop the analytic method, establish a source for the specimens, and procure funding to support the study. The two chemical classes of interest for our initial reference range study were the volatile organic compounds (VOCs) and pesticides that are readily metabolized or eliminated. There was less human exposure information for these compounds than for the more persistent pesticides. Both classes of compounds have short half-lives in humans-on the order of minutes to hours for the first elimination phase. Nonetheless, by measuring these compounds in a large, diverse population (1000 people), one can establish a useful reference range. Exposure to both classes of compounds has been linked to health effects. Another reason we chose the VOCs is that more than 50% of the human exposure inquiries to the Centers for Disease Control and Prevention (CDC) and Agency for Toxic Substances and Disease Registry (ATSDR) relate to potential exposure to VOCs; hence, the public is concerned about exposure to these compounds. Finally, the Total Exposure Assessment Measurement (TEAM) studies showed that the indoor air concentrations of VOCs are much higher than the outdoor air concentrations, and since most people spend about 90% of their time indoors, the duration of indoor exposure is high (3). We chose the pesticides to be analyzed on the basis of need for reference data, the health effects associated with a pesticide, and the following four criteria: a) presence and ranking on the ATSDR priority toxi- cant list; b) the reported presence of the pesticide in indoor household air; c) the percentage of children with the pesticide present in their urine according to results of the Jacksonville, Arkansas, study (4); and d) how necessary it was to obtain human exposure data on the pesticide for regula- tory purposes (5). Once we decided on the classes of compounds, we had to complete other preliminary steps before analyzing the unknowns. Materials and Methods Select AnalyteMaic and Detection Limits The next step was to select the individual toxicants, the biologic matrix, and the lim- its of detection and quantification. A volatile organic compound has been defined as an organic compound that has limited solubility in water and boils at tem- peratures less than 260?C at atmospheric pressure (6). Our initial list of VOCs con- sisted of 48 toxicants; the final list that we have quantified in the reference range study consists of 32 chemicals (Table 1). Two of these chemicals, acetone and 2- butanone, do not meet the solubility por- tion of the definition for VOCs, but are accurately quantified by our method (7). We measured VOCs in whole blood because of the specificity of the analyses. By this, we mean that in the blood we measure the parent VOC, but in urine we frequently measure the metabolite, which Table 1. Thirty-two VOCs quantified in reference range study. Benzene Toluene Styrene Ethyl benzene 1,2-Xylene 1,3-Xylene 1,4-Xylene Chlorobenzene 1 ,2-Dichlorobenzene 1 ,3-Dichlorobenzene 1 ,4-Dichlorobenzene 1 ,1-Dichloroethane 1 ,2-Dichloroethane 1,1,-Dichloroethene cis-1 ,2-Dichloroethene trans- ,2-Dichloroethene 1,1,1 -Trichloroethane 1,1 ,2-Trichloroethane Trichloroethene 1,1 ,2,2-Tetrachloroethane Tetrachloroethene Hexachloroethane Methylene chloride Chloroform Carbon tetrachloride 1 ,2-Dichloropropane Bromoform Dibromomethane Bromodichloromethane Dibromochloromethane Acetone 2-Butanone may not be specific for only one VOC; for example, mandelic acid is a metabolite of both styrene and ethyl benzene. The disad- vantages of using blood are the invasiveness of the specimen collection procedure, and the fact that concentrations of VOCs in blood decrease more rapidly after exposure than do concentrations of VOC metabo- lites in urine. We decided that the limits of detection and quantification for the VOCs would have to be in the low parts-per-tril- lion range. We chose whole blood instead of serum because we did not know how the VOCs partitioned in blood, and pretreat- ing blood increases the likelihood of the matrix being contaminated and also increases the amounts of VOCs lost from the blood. Unlike the way we measured VOCs, we chose to measure the pesticides or their metabolites in urine at the low parts-per-billion levels. The list of the measured chemicals and their probable pesticide origin are given in Table 2. The list of the measured pesticides or their metabolites and why they were chosen are given in Table 3. We were particularly interested in 2,4-dichlorophenoxyacetic acid, which belongs to a class of com- pounds called peroxisome proliferators and which has been associated with non- Hodgkin's lymphoma in farmers (8). Because these pesticides or their metabo- lites are generally excreted in urine, we chose urine as the matrix of choice. We also measured creatinine in urine so that the data could be normalized. We selected the low parts-per-billion range as the neces- sary quantification limit for these toxicants. Analytical Method Some of the characteristics sought in an analytical method are: * Multianalyte * Compatible with matrix * Demonstrated sensitivity * Demonstrated specificity * Demonstrated precision * Demonstrated accuracy * Inexpensive * Fast These are demonstrated within and among studies by measurement of matrix-based pools. With the methods we developed, we were able to measure multiple toxicants in the biologic matrix of choice at the levels that we had earlier specified. By using a detailed quality assurance plan, which fea- tures the analyses and plotting of results of matrix-based quality control pools, we were able to demonstrate the necessary specificity, sensitivity, precision, and accu- racy-both for reference range study and Environmental Health Perspectives 90 PRIORITY TOXICANT REFERENCE RANGE STUDY Table 2. Selected chemicals measured in human urine and their pesticide origin. Chemical measured Pesticide origin 1 -Naphthol Naphthalene, carbaryl 2-Naphthol Naphthalene Isopropoxyphenol Propoxur Carbofuran phenol Carbofuran 3,5,6-Trichloro-2-pyridinol Chlorpyrifos 2,4-Dichlorophenoxyacetic acid (2,4-D) 2,4-D Pentachlorophenol PCP, HCB, y-BHC 2,4,5-Trichlorophenol (2,4,5-TCP) 2,4,5-TCP; 1,2,4-trichlorobenzene; y-BHC; HCB 2,4,6-Trichlorophenol (2,4,6-TCP) 2,4,6-TCP; 1,3,5-trichlorobenzene; y-BHC 2,5-Dichlorophenol (2,5-DCP) p-Dichlorobenzene 2,4-Dichlorophenol (2,4-DCP) m-Dichlorobenzene 4-Nitrophenol Methyl and ethyl parathion; nitrobenzene Table 3. Selected pesticides or metabolites and bases of choice. % Positives in Attempted or measured Analyte ATSDRa Household air Jacksonville (4) in NHANES 11(5) 2,4-Dichlorophenol X 27 2,5-Dichlorophenol X X 96 2,4,5-Trichlorophenol X 54 X 2,4,6-Trichlorophenol X Pentachlorophenol X X 100 X 4-Nitrophenol X X 1-Naphthol X X X 2-Naphthol X X 2-Isopropoxyphenol X X Carbofuranphenol X 3,5,6-Trichloro-2-pyridinol X 20 X 2,4-Dichlorophenoxyacetic acid X aAnalyte or parent compound on ATSDR's First List of 100 Substances (Federal Register, 17 April 1987) and Second List of 100 Substances (Federal Register, 20 October 1988). ATSDR's expanded list of 275 compounds includes all of these analytes or parent compounds except 2-isopropoxyphenol. bFrom newspaper articles citing unspecified U.S. EPA reports. for other exposure assessment studies. Although both procedures provide highly reliable results over the long term, they have the disadvantages of being time inten- sive and relatively expensive. Our analytic method for measuring VOCs in 10 ml of whole blood has been described (7). In summary, a known amount of the stable isotopically labeled VOC is added to the blood specimen; the blood is heated to 35'C and purged with helium, which drives the native and labeled VOCs onto a Tenax trap; the trap is then heated, which drives the VOCs to the head of the capillary gas chromatography col- umn, which is cooled to -150'C with liq- uid nitrogen; this column is then ballistically heated; and the VOCs are chro- matographed and analyzed at 3000 resolv- ing power (RP) by mass spectrometry. The mass spectrometer is run in the full-scan mode, which is not as sensitive as the selected ion-monitoring mode that is more often used in trace analysis. The full-scan mode, however, does allow researchers to acquire qualitative and semiquantitative data on additional VOCs. To meet the detection limits' specifications on a quadru- pole instrument, we used the selected ion- monitoring mode during our initial efforts to develop an analytic method; however, we have demonstrated that the accurate mass at this medium resolution is needed for accu- rate and automated quantification (9). Another problem we encountered was the contamination of the commercial vacu- tainers with the selected VOCs. We used several types of vacutainers before we set- tled on the gray-top vacutainers. Even those had to be disassembled, heated for 2 weeks to drive off the VOCs in the stop- pers, reassembled, sterilized, and then have a new vacuum established. Our method for measuring these pesti- cides or metabolites in urine consists of adding known amounts of stable isotopically labeled pesticides and conducting enzymatic hydrolysis, liquid/liquid extraction, derivati- zation, capillary column gas chromatogra- phy, and selective reaction monitoring by mass spectrometry/mass spectrometry. All steps prior to the concentration of the derivatized extract are performed by a robot. The robot relieves the laboratory workers of certain menial tasks and decreases their expo- sure to chemical solvents; the use of robotics may also improve laboratory precision (10). One of the difficulties associated with this method involves quantifying phenols and a carboxylic acid. Different conditions are gen- erally needed to extract and derivatize these two chemical dasses. Sources of Specimens We received the blood and urine speci- mens for the reference range study through the first cycle (1988-1991) and through the initial part of the second cycle (1992-1994) of the Third National Health and Nutrition Evaluation Survey (NHANES III), which is being conducted in collaboration with the National Center for Health Statistics (NCHS) of CDC. The urine specimens were received frozen by dry ice, and the blood specimens were received chilled by ice. The selected toxi- cants in the urine are stable as long as the urine is frozen; the selected VOCs are sta- ble for at least 7 weeks. Some VOCs, such as cis- and trans-1,3-dichloropropene, read- ily react with blood chemicals and thus have a very short "shelf life"; they were deleted from our original list. Whereas the population of the NHANES III is large, diverse, and chosen so as to be probability-based for the United States population, the 1000 individuals in our reference range study are not represen- tative of the entire United States popula- tion. They are, however, selected from a relatively broad spectrum of the population: * They are from all four regions of the contiguous United States. * They are from both urban and rural communities. * They are from 20 to 59 years old. * They include both men and women. * They are of different races. We have not decoded the samples to know the number of people in each cate- gory. The participants in the study answered a self-administered questionnaire designed to determine whether they had had recent exposures (including occupational exposure) to toxicants of interest. In addition, other results from the NHANES III, including cotinine levels, and other questionnaire data will be available for comparison. Results vOcs The data presented here are from the meas- urement of VOCs in about 600 specimens. Volume 103, Supplement 3, April 1995 91 NEEDHAM ETAL. Eleven of the VOCs (counting 1,3-xylene and 1,4-xylene as one because they are not separated by gas chromatography or mass spectrometry) were quantified in 75% or more of the reference population. The mean, median, and upper 95th percentile measurements of nine of these VOCs are represented in Figure 1. Nondetectable results were given a value of one-half the detection limit. All of the six nonchlori- nated aromatics (benzene, ethyl benzene, styrene, toluene, 1,2-xylene, and the non- separated combinations of 1,3 and 1,4- xylene) were found in more than 75% of the people. In general, human exposure to these compounds is from tobacco smoke and the exhaust from internal combustion engines. Previous blood measurements have shown an association between ele- vated blood benzene levels and tobacco smoking. For example, Brugnone et al. (11) reported that in Italy the mean blood benzene level of smokers (381 ppt) was sig- nificantly higher than that of nonsmokers (205 ppt); both of those levels are about 2 times or more higher than the mean blood benzene level reported herein. We also found lower benzene blood levels than those reported in Germany by Hajimiragha et al. (12) and Angerer et al. (13). Our mean benzene level is similar to that reported by Jermann et al. (14) for a popu- lation living in a European city with high traffic density. Of the chlorinated aromat- ics, which are the four compounds in the bottom of the first column of Figure 1, 1,4-dichlorobenzene was found in almost everyone. Its primary sources are bathroom deodorizing cakes and mothballs. Of the chlorinated ethanes and ethenes (shown in column 2 of Table 1), the widely used sol- vent, 1,1,1-trichloroethane, and the dry cleaning solvent, tetrachloroethene, were found in more than 75% of the people. These VOCs are essentially the same ones reported by the U.S. EPA's TEAM study as the primary VOCs in indoor air (3). Two other compounds, acetone and 2- butanone, were found in everyone at much higher levels because they are metabolic products. The relative concentration of these two compounds tended to track each other, which may be of clinical interest. Five other VOCs (chlorobenzene, trichloroethene, and the trihalomethanes- chlorodibromomethane, chloroform, bro- modichloromethane) were found in 10% or more of the reference population. The findings on the trihalomethanes may point to a need to more closely examine the effects of treating our water supply with halogenated compounds. All of the others Benzene Toluene Styrene o-Xylene m,p-Xylene Ethyl Benzene 1,1,1-Trichlorethane Tetrachloroethane 1,4-Dichlorobenzene 0 I @ I * i@. I-. I 0 I- * I * I * l I- p I * I I90 500 900 1700 Parts per trillion I1--I 1 2500 7500 12,500 Figure 1. Median (1), mean (@1, and 95th percentile (*) of VOCs in >75% of samples. were detected but in less than 10% of the samples. The entire data set with the vari- ous demographic data will be published separately by one of us (David L. Ashley). Pesticides More than 900 urine specimens have been analyzed for the selected pesticides or their metabolites. As was the case for the VOC data, data from these specimens have not yet been analyzed with respect to the demographic characteristics of the speci- men donors (i.e., age, race, gender, geo- graphic region of residence, urban versus rural) or matched with questionnaire data such as donor's occupation. They have not yet been normalized to the urinary creati- nine levels. These data will be published later by Robert H. Hill. In Figure 2, we present the prevalence percentages and the whole weight concentration ranges on a logarithmic scale for five chlorinated phe- nols and 4-nitrophenol. We calculated the first 95% range by using all of the data (and treating nondetectables as zero), and we calculated the second 95% range by using only the detectable values. The col- umn on the left gives the percentage that was detectable, which varies from 13% for 2,4,6-trichlorophenol to 97% for 2,5- 2-Naphthol 175%) * l 1-Naphthol 191 %) l4 Isopropoxyphenol 17%) Carbofuran phenol (1.5%)l 2,4-D 110%) _ 3,5,6-Trichloro-2-pyridinol r70% d 0 0 10 20 30 40 Parts per billion Figure 2. 95th percentile levels on a logarithmic scale (and percentage prevalence) for selected phenols. (-, using all data; o, using detectable data). dichlorophenol. The high prevalence of 2,5-dichlorophenol is consistent with the high prevalence of 1,4-dichlorobenzene found in the VOC portion of this study. Some of these chemicals have been mea- sured in other studies, results from which are presented in Table 4. In performing any comparisons, one should consider the differences in analytical methods used and the population studied; for example, only the NHANES II population is a probabil- ity-based sample. Nonetheless, these results suggest that the prevalence of 2,4,5- trichlorophenol and 4-nitrophenol is increasing while that of pentachlorophenol is decreasing; the decrease in the prevalence of pentachlorophenol is consistent with its decreased use since the middle 1980s. In Figure 3, we present data on five additional phenols and 2,4-diphenoxy- acetic acid in a similar manner (except not on a logarithmic scale) as the data pre- sented in Figure 2. The prevalence and 95th percentile are higher for 1-naphthol than 2-naphthol, differences that may be explained by the fact that although naph- thalene metabolizes to both compounds, carbaryl metabolizes only to 1-naphthol. Data for isoproxyphenol and carbofuran phenol indicate that although less than 2,4-DCP 161%) 1 0 0 2,4,6-TCP 113%1 2,4,5-TCP 118%) * 0 10 100 Parts per billion Figure 3. 95th percentile levels (and percentage preva- lence) for 2,4-D and selected pesticide metabolites (, using all data; o, using detectable data). Environmental Health Perspectives 1000 ) 92 PRIORITY TOXICANT REFERENCE RANGE STUDY Table 4. Percentage of selected phenols detected and the concentration (ppb, whole weight) of samples at various percentiles in urine (from previous studies). NHANES la U.S. population German population Arkansas children % Detected 90%e % Detected 100% % Detected 95% % Detected 95% 2,4-Dichlorophenol NR NR NR NR NR NR 27 7 2,5,-Dichlorophenol NR NR NR NR 88 33.6 96 280 2,4,5-Trichlorophenol 3.4 NR NR NR 54 4.5 54 7 2,4,6-Trichlorophenol NR NR NR NR 37 4.7 11 4 Pentachlorophenol 71.6 15.5 100 17 NR NR 100 110 4-Nitrophenol 2.4 NR NR NR NR NR NR NR NR, not reported. 8n= 13,980. From Kutz et al. (5). bn= 143. From Cline et al. (15). Cn= 258. From Angerer et al. (16). dn= 197. From Hill et al. (4). ?90th percentile of detectables. 10% of our population had been recently exposed to the carbamates (propoxur and carbofuran) more people had been recently exposed to propoxur. This difference may reflect the greater use of propoxur indoors (Table 3). The most striking statistic in Table 3 is the high prevalence that we found of 3,5,6-trichloro-2-pyridinol, a metabolite of chlorpyrifos; 3,5,6-trichloro- 2-pyridinol was reported in only 5.8% of the NHANES II population (5). The high exposure rate to chlorpyrifos is probably due to the large increase in the use of this termiticide since heptachlor and chlordane were banned. Consistent with these results are the findings from the U.S. EPA's Non- Occupational Personal Exposure Study (NOPES) that chlorpyrifos was the pesti- cide found at the highest prevalence rate; propoxur was found at the second highest rate in this 1986 to 1989 study (17). Discussion The primary objectives of this study were to better define background levels and prevalences of selected pesticides and VOCs. The results reported herein are pre- liminary and provisional, pending comple- tion of the study. Results from this study can also be used in monitoring trends if the levels of these compounds in humans are followed over time. Certainly the study results demonstrate that most Americans are continually exposed to a variety of known animal carcinogens and one known human carcinogen, benzene. This should be of interest to public health officials. One of the main uses of the reference ranges is as a basis of comparison in determining rel- ative levels of toxicants in potentially exposed populations. For example, the VOC reference ranges have been used to compare possible human exposures to oil well fires in Kuwait and Uzbekistan; occu- pational indoor air; drinking water con- taining selected VOCs; the environment around Superfund waste sites; and bathing water containing benzene. They have also been used to assess the exposure of people complaining of multiple chemical sensitiv- ity and of populations with an elevated rate of infants born with neural tube defects. Other data from this study are also use- ful. For example, although styrene is found at slightly lower levels than benzene in the reference population, human exposure to both of these compounds is likely to be from the same sources, as mentioned above. Evidence for this can be shown by plotting the concentrations of benzene and styrene. Thus, data on the internal dose are not only useful for linking exposure to health effects but also for linking exposure, back to the source of the pollutants. We mentioned previously that these VOCs were mass analyzed at a resolving power of 3000 under full-scan conditions. One of the advantages of such mass analyses is that not only will we be able to compare frequency distributions and other statistical data for the VOCs we targeted, we will also be able to compare such data for VOCs that were not targeted but that nonetheless were measured and for which the resulting data were stored. For example, we can compare human levels of benzene and methyl ter- tiary-butyl ether to see how the levels of these compounds differ between our refer- ence population and a population that is possibly exposed to the latter compound, which is used in oxygenated gasoline. Likewise, we hope to compare levels of ben- zene with those of cotinine, a metabolite of nicotine being measured in about 24,000 serum specimens from the NHANES III population (18). We will then compare their levels with other levels of VOCs that are known to originate from tobacco smoke, such as nicotine, 3-ethenylpyridine, mysomime, and pyridine (19). In addition to developing the reference ranges described here, we are developing ranges for other environmental toxicants of public health interest. The blood and urine for these studies are being acquired from the second cycle (1992-1994) of NHANES III. The classes of chemicals are polyaromatic hydrocarbons; polychlori- nated biphenyls, including the coplanar polychlorinated biphenyl congeners; very volatile organic compounds, such as vinyl chloride; and selected chlorinated hydro- carbon pesticides, such as dieldrin. REFERENCES 1. National Research Council. Principles of exposure assessment. In: Human Exposure Assessment for Airborne Pollutants: Advances and Opportunities. Washington:National Academy Press, 1991. 2. Needham LL, Pirkle JL, Burse VW, Patterson DG Jr, Holler JS. Case studies of relationship between external dose and internal dose. J Exp Anal Environ Epidem Suppl 1:209-221 (1992). 3. Wallace LA, Pellizzari ED, Hartwell TD, Sparacino C, Whitmore R, Sheldon L, Zelon H, Perritt R. The TEAM study: personal exposures to toxic substances in air, drinking water, and breath of 400 residents of New Jersey, North Carolina and North Dakota. Environ Res 43:290-307 (1987). 4. Hill RH Jr, To T, Holler JS, Fast DM, Smith SJ, Needham LL, Binder S. Residues of chlorinated phenols and phenoxy acid herbicides in the urine of Arkansas children. Arch Environ Contain Toxicol 18:469-474 (1989). 5. Kutz FW, Cook BT, Carter-Pokras OD, Brody D, Murphy RS. Selected pesticide residues and metabolites in urine from a survey of the U.S. general population. J. Toxicol Environ Health 37:277-291 (1992). 6. WHO. Indoor air quality: organic pollutants. In: EURO Reports and Studies, No 111. Copenhagen:World Health Organization Regional Office for Europe, 1989. 7. Ashley DL, Bonin MA, Cardinali FL, McCraw JM, Holler JS, Volume 103, Supplement 3, April 1995 93 NEEDHAM ETAL. Needham LL, Patterson DG Jr. Determining volatile organic compounds in human blood from a large sample population using purge-and-trap gas chromatography/mass spectrometry. Anal Chem 64:1021-1029 (1992). 8. Hoar SK, Blair A, Holmes FF, Boysen CD, Robel RJ, Hoover R, Fraumeni JF Jr. Agricultural herbicide use and risk of lym- phoma and soft-tissue sarcoma. JAMA 256:1141-1147 (1986). 9. Bonin MA, Ashley DL, Cardinali FL, McCraw JM, Patterson DG Jr. Importance of enhanced mass in removing interferences when measuring volative organic compounds in human blood by using purge-and-trap gas chromatography/mass spectrome- try. Am Soc Mass Spectrom 3:831-841 (1992). 10. Shealy DB, Hill RH Jr, Orti DL, Bailey SL, Miller BB, Turner WE. Automated sample preparation of serum cotinine for GCIMS analysis. In: Proceedings of the International Symposium on Laboratory Automation and Robotics, Vol. 7 (Strimaitis J, Little J, eds). Hopkinton, MA:Zymark Corporation, 1991 ;533-544. 11. Brugnone F, Perbellini L, Maranelli G, Romeo L, Guglielmi G, Lombardini F. Reference values for blood benzene in the occu- pationally unexposed general population. Int Arch Occup Environ Health 64:179-184 (1992). 12. Hajimiragha H, Ewers U, Brockhaus A, Bettger A. Levels of benzene and other volatile aromatic compounds in the blood of nonsmokers. Int Arch Occup Environ Health 61:513-518 (1989). 13. Angerer J, Scherer G, Schaller KH, Muller J. The determination of benzene in human blood as an indicator of environmental exposure to volatile organic compounds. Fresenius J Anal Chem 339:740-742 (1991). 14. Jermann E, Hajimiragha H, Brockhaus A, Freier I, Ewers U, Roscovanu A. Belatung von durch benzol und andere verkehrs- bedingte immissionen. Zbl Hyg 189:50-61 (1989). 15. Cline RE, Hill RH Jr, Philips DL, Needham LL. Pentachlorophenol measurements in body fluids of people in log homes and workplaces. Arch Environ Contam Toxicol 18:475-481 (1989). 16. Angerer J, Heinzow B, Schaller KH, Welte D, Lehnert G. Determination of environmentally caused chlorophenol levels in urine of the general population. Fresenius J Anal Chem 342:433-438 (1992). 17. U.S. EPA. The US Environmental Protection Agency Nonoccupational Pesticide Exposure Study (NOPES): Final Report. EPA/600/3-90/003, Washington:Environmental Protection Agency, January 1990. 18. Centers for Disease Control. Preliminary data:exposure of per- sons ages ?4 years to tobacco smoke in the United States 1988-1991. MMWR 22:37-39 (1993). 19. Eatough DJ, Benner CL, Bayona JM, Richards G, Lamb JD, Lee ML, Lewis EA, Hansen LD. Chemical composition of environmental tobacco smoke. Environ Sci Technol 23:679-687 (1989). 94 Environmental Health Perspectives