Chapter 3
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The Use of Reference Range Concentrations in Environmental Health Investigations Robert H. Hill, Jr., Susan L. Head, Sam Baker, Carol Rubin, Emilio Esteban, Sandra L. Bailey, Dana B. Shealy, and Larry L. Needham National Center for Environmental Health, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, 4770 Buford Highway, Atlanta, GA 30341-3724 Reference range concentrations are obtained by measuring xenobiotics, their residues, or their metabolites in human specimens from the general population. These reference range concentrations provide a foundation for assessments of exposure to xenobiotics in specific exposure situations and also provide information about the extent and magnitude of xenobiotic exposure in the general population. Reference range concentrations for p-nitrophenol served as a basis for comparison for residents exposed to methyl parathion following inappropriate residential exposure. Reference range concentrations for 2,5-dichlorophenol and 3,5,6-trichloro-2-pyridinol also provide information about the magnitude and extent of exposure to environmental contaminants, p-dichlorobenzene, and chlorpyrifos, respectively. Biological monitoring, sometimes called biomonitoring, evaluates exposure to xenobiotics by measuring the concentration of that xenobiotic in a biological sample, usually urine, serum, blood, or tissue. The xenobiotic measured may be the toxicant itself, a metabolite, a product of the toxicant and a bio-molecule such as DNA, hemoglobin, or other proteins, or a change in a naturally occurring biochemical within the body, such as cholinesterase depression. These are known as biomarkers of exposure. The concentration of the xenobiotic or its metabolite(s) is known as the internal dose and reflects toxicant exposurefromall routes and sources, providing a summary or integrated index of exposure. Air monitoring, wipe sampling, and food and water analyses are typically used to estimate exposurefroma single source, and these environmental monitoring methods are useful inriskmanagement to locate and prevent exposurefroma source or primary route of exposure. The objective of measuring internal dose is to assess actual human exposure and to correlate this internal dose with biomedical changes or adverse health effects. We have extensive experience in biological monitoring, particularly in This chapter not subject to U.S. copyright Published 1996 American Chemical Society In Biomarkers for Agrochemicals and Toxic Substances; Blancato, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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BIOMARKERS FOR AGROCHEMICALS AND TOXIC SUBSTANCES
support of various epidemiologic investigations involving known or possible exposures to toxic compounds. One important finding is that often a measurement of internal dose provides a much better assessment of exposure than all exposure indices (1,2). Exposure indices are estimates of exposure based upon information collected from questionnaires, medical history, and environmental measures. However, the paucity of biomarker data makes interpreting biological monitoring difficult. Detection of a xenobiotic in urine, blood, or serum indicates exposure; nevertheless, the magnitude and extent of exposure is at the heart of exposure assessment and risk analysis. In an effort to improve exposure assessments, we conducted a program to establish reference range concentrations for 32 volatile organic compounds in blood and 12 pesticide metabolites or residues in urine (5). Reference range concentrations are those concentrations of a toxicant, its residue, or its metabolite that are found in the general population. We assume that the general population is not occupationally exposed to the parent compound and is not otherwise overtly exposed. Of course, most people in the general population are exposed to many xenobiotics - for instance, to pesticide residues in foods; generally however, these residue concentrations are usually low. Reference range concentrations can be used as baseline or normal concentrations for the "unexposed," general population. These values are analogous to normal values of clinical laboratory measurements, but they are not normal biochemical constituents of the body. Reference range concentrations provide a foundation for assessments of exposure to xenobiotics by serving as a basis of comparison for possibly exposed populations. Reference range concentrations also provide information about the extent and magnitude of exposure of the general population to xenobiotics (e.g. pesticide residues), and they can suggest areas for future research. The uses for reference range concentrations for pesticide residues or metabolites will be illustrated below. Methods and Materials Reference range concentrations were determined in samples collected from approximately 1000 people - a subset of the National Health and Nutrition Examination Survey III [NHANES III], a national survey of the general population of the United States (4). Twelve pesticide residues were measured in urine samples by using an isotope dilution technique, enzyme hydrolysis and extraction, derivatization and concentration, and finally capillary gas chromatography combined with tandem mass spectrometry (5). This method was also used to measure p-nitrophenol in urine samples collectedfromLorain County residents. Non-Occupational Exposure to Methyl Parathion Methyl parathion is a highly toxic pesticide and its use, which is principally to spray cotton, is restricted by the Environmental Protection Agency (EPA) (6). Furthermore, methyl parathion's highly toxic properties require that field re-entry standards be observed with this pesticide (that is, workers may not re-enter fields that have been sprayed with methyl parathion for at least 48 hours). In late 1994, state officials from Ohio discovered that this pesticide had been used to exterminate pests
In Biomarkers for Agrochemicals and Toxic Substances; Blancato, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
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3. HILL ET AL.
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Use of Reference Range Concentrations
from a home- a use not permitted by federal or state regulations and laws. The ensuing investigation showed that an unlicensed pesticide operator had illegally used this pesticide in hundreds of homes, thus exposing several hundred people, including children, expectant mothers, and the elderly. Local, state, and federal health officials working together quickly went to selected homes to assess whether the people in those homes had suffered exposure or adverse health effects from methyl parathion. This population not only had exposure that could cause acute illness but also had experienced chronic, long term exposure to methyl parathion. Exposure was determined by measuring methyl parathion in air and wipe samplesfromthese homes and by measuring /7-nitrophenol (PNP), a metabolite of methyl parathion, in urine. Figure 1 shows an ion chromatogram (top) of a urine extractfromone of the residents selected for evaluation; the ion chromatogram (bottom) of a urine extractfromthe reference range population represents a PNP concentration of 16 //g/L- the 99th percentile of the PNP reference range concentrations. The PNP urinary concentration (4000 //g/L) from the resident was 250 times greater than the 99th percentile concentration. To accurately measure such a high concentration, the sample had to be diluted by a factor of 100. All residents of this household had PNP concentrations greater than or equal to 4000 //g/L; a child had a concentration of4800 yUg/L - almost 1000 times greater than the 95th percentile concentration of the reference range. Table I shows a comparison of PNP concentrations from 131 Lorain County residents to PNP reference range concentrations. For every category of comparison, these residents had concentrations 75 to 250 times greater than the reference concentrations. Comparisons with reference range data can be made using frequencies of detection, mean, and various percentile concentrations. Usually we consider concentrations less than or equal to the 95th percentile concentration of the
Table I. Comparison between p-Nitrophenol Urinary Concentrations Found in Lorain County Residents Exposed to Methyl Parathion and in the Reference Range Subjects. Measures of Comparison
Lorain County Reference Range (N=131) (N = 974)
Frequency of detection
86%
Mean concentration, Mg/L
240
1.6
28
