6
Chemical Assay of Specimens

INTRODUCTION

“Monitoring” implies routine measurement that is inherently closed-ended and based on established methods and practices. Monitoring programs vary with regard to requirements and approaches, but usually have as a base a list of analytes (“target chemicals”) and assay methods that have been validated for the sample type and concentration range of interest. A successful monitoring program maintains results over time for comparison and must therefore be technically adequate at the outset. Comparability is most easily achieved if assay methods are constant. A monitoring program often offers a good setting for other kinds of activities, and the National Human Monitoring Program includes aspects that are not monitoring, such as recognition of new agents of concern and detection of chemicals not previously included in monitoring protocols. The latter objectives are appropriate to a population-based biologic surveillance program, but they require an approach to chemical analysis different from that for monitoring. Design of a program of surveillance requires a plan for development and change in analytic methods, as well as a plan for maintaining stable methods and analytic quality control. Successful balancing of the routine and the innovative or exploratory components of such a program is a major challenge for the program’s management.

The following discussion of aspects of a program is based on several assumptions:

  • Present knowledge does not permit designation of all substances that might be detectable in tissues or that would be important if detected.

  • Present analytic technology is inadequate for surveillance of some sub-



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Monitoring Human Tissues for Toxic Substances 6 Chemical Assay of Specimens INTRODUCTION “Monitoring” implies routine measurement that is inherently closed-ended and based on established methods and practices. Monitoring programs vary with regard to requirements and approaches, but usually have as a base a list of analytes (“target chemicals”) and assay methods that have been validated for the sample type and concentration range of interest. A successful monitoring program maintains results over time for comparison and must therefore be technically adequate at the outset. Comparability is most easily achieved if assay methods are constant. A monitoring program often offers a good setting for other kinds of activities, and the National Human Monitoring Program includes aspects that are not monitoring, such as recognition of new agents of concern and detection of chemicals not previously included in monitoring protocols. The latter objectives are appropriate to a population-based biologic surveillance program, but they require an approach to chemical analysis different from that for monitoring. Design of a program of surveillance requires a plan for development and change in analytic methods, as well as a plan for maintaining stable methods and analytic quality control. Successful balancing of the routine and the innovative or exploratory components of such a program is a major challenge for the program’s management. The following discussion of aspects of a program is based on several assumptions: Present knowledge does not permit designation of all substances that might be detectable in tissues or that would be important if detected. Present analytic technology is inadequate for surveillance of some sub-

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Monitoring Human Tissues for Toxic Substances stances, because of limitations in sensitivity, in applicability to some chemicals of interest, and in cost. Individual target chemicals will increase or decrease in importance over time, but relatively slowly. The slowness of increases in importance reflects the slowness of increases in US population-wide exposures. Decreases in importance reflect similarly slow decreases in exposures and increases in understanding of the nature of the health implications of and reasons for some exposures. “Emergency” exposure assessments that might be required when a serious health hazard is discovered would be best addressed in focused special studies. Monitoring efforts will provide opportunities for exploration of tissue composition beyond a list of target chemicals. Those opportunities and other efforts parallel to the monitoring tasks should support continuing development of the monitoring program itself. MAJOR FEATURES OF A PROGRAM FOR CHEMICAL ANALYSIS OF TISSUES FOR POPULATION-BASED SURVEILLANCE OF EXPOSURES Monitoring-Program Development One must first establish goals, specify target chemicals and quantities, and identify analytic methods. The usual reason for establishing a monitoring program is recognition of a problem or potential problem related to known agents. Programs of environmental monitoring have used several kinds of information in formulating lists of target chemicals, such as case reports of environmental concentrations or industrial releases, volumes of chemicals produced or sold, toxicity or other hazard-ranking factors, and analytic feasibility. A chemical would be a good candidate for inclusion in a tissue monitoring program if it met the following requirements: The chemical is detectable with currently used or available methods. It would appear in the tissue at detectable concentrations in the event of exposure. Its presence in the tissue would indicate an exposure, i.e., would represent a marker of exposure or the actual substance or its metabolite would be present only in response to an exposure. There is reason to believe that exposure is possible or likely.

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Monitoring Human Tissues for Toxic Substances On the basis of those characteristics, several criteria and methods for selecting target chemicals are possible. A recent discussion of priorities for laboratory testing of chemicals (NRC, 1984a) presented many issues that are relevant here. They are now briefly described. Selection Based on Analytic Expediency and Constraints In formulating a new monitoring program, one might choose analytic methods to maximize the likelihood of detection of key target chemicals. Multi-agent analytic schemes that rely on gas chromatography and mass spectrometry (GC-MS) are common to many environmental monitoring laboratories. Within any such scheme, the ranges of applicability and sensitivity of detection are roughly fixed; analytical performance then dictates what goals (i.e., what list of target chemicals and detection limits) might be considered. For example, schemes currently used to detect EPA priority pollutants with detection limits of 1–10 ppb in water or wastewater might permit inclusion of additional target chemicals with similar analytic behavior (for example, alkylated polycyclic hydrocarbons in addition to the priority polycyclic aromatic hydrocarbons), but could not permit inclusion of polychlorinated dibenzodioxins (PCDDs) and dibenzofurans (PCDFs) at concentrations lower than parts per billion. Modification of analytic protocols might permit detection of PCDDs and PCDFs, but exclude detection of polycyclic hydrocarbons altogether. For monitoring purposes, established assay protocols represent analytic “windows” through which specific subsets of contaminant chemicals might be detected and measured. That is an advantage, because within such a window nontarget chemicals might be detected and new target chemicals added without extensive analytic-method development; but it is a disadvantage because “blind spots” outside established analytic windows might be neglected. The latter concern would be reinforced if analytic expediency were a main criterion for identifying new target chemicals. Selection Based on Regulatory Interest To the degree that a monitoring program is aimed at a specific regulatory issue, other criteria for the selection of target chemicals might be preempted. Analytic methods would then have to be devised to meet the requirements of the chemicals targeted by the regulation, and future additions to the list would probably require development of new methods. It is important to determine

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Monitoring Human Tissues for Toxic Substances what regulatory needs are to be served by a monitoring program, but it is equally important to determine whether the program is confined to those particular needs or has (or will have) a broader mission as well. Selection Based on Prior Detection in Human Tissues Reports of detection of contaminant chemicals in human tissue in the scientific literature or in regulatory-agency studies constitute convincing evidence that exposures can occur and that tissue concentrations are measurable, by at least some method. Compilations of such reports—as in the Chemicals Identified in Human Media data base (EPA, 1980) and the NIST Human Specimen Banking Reports (Wise and Zeisler, 1984)—not only are tools for locating candidate target chemicals that might conform to pre-existing analytic capabilities, but also might indicate a need for new analytic approaches to widen the range of chemicals detected. Selection Based on Indications of Health Relevance Testing programs conducted by NIH or by nongovernment researchers will continue to provide findings that increase or decrease health concerns related to possible exposures to individual agents. In lieu of case reports or other documented findings of agents in tissues to be monitored, results of toxicologic studies in animal models can indicate the likelihood that a candidate target chemical will be present and detectable in sample tissues. When such findings indicate a need for epidemiologic followup, inclusion of the agent in monitoring programs should be considered. Selection Based on Indications of Environmental Contamination Numerous environmental monitoring programs focus on drinking water (Wallace et al., 1986), air quality (Hunt et al., 1986; Wallace et al., 1986; EPA, 1987b), food (Reed, 1985; Reed, et al., 1987), biota (Lewis and Lewis, 1979; Becker et al., 1988; NOAA, 1988), and waste treatment and discharges (Hannah and Rossman, 1982). Findings that indicate widespread environmental contamination by specific chemicals would add to the importance of including those chemicals in a program of tissue monitoring.

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Monitoring Human Tissues for Toxic Substances Summary Clearly, those approaches are not independent. For example, regulatory priorities reflect toxicologic research, environmental monitoring, etc., and it is probable that some candidate target chemicals will satisfy several criteria. But, each approach can identify agents not currently being studied or considered in connection with the other approaches. Design of Chemical Assay Programs Determination of Requirements for Analytic Performance Precision, accuracy, and sensitivity must all be adequate to meet the specified goals of the program. Analytic goals are ideally based on knowledge of the relationships among tissue concentration, exposures, and health outcomes and on the variations in tissues concentrations that might be attributed to individual variations, to systematic variations in exposure, and to the time course of exposure. Such information is rarely available in practice, but analytic precision, sensitivity, and accuracy should be great enough to permit detection and measurement of exposures well below any plausible threshold of clinically evident effects. Decisions regarding type of detection method to be used or whether multiple target chemicals are to be included in a single assay (and if so, which ones) will depend on the analytic performance required. Specificity and sensitivity are closely related; when analytic needs are defined on the basis of high unit toxicity or a need to detect an agent in tissues of persons exposed at background concentrations, a dedicated assay may be required for the specific agent or a group of agents. Development and Validation of Method Ways of developing and validating specific analytic techniques should be specified in considerable detail and then applied to each chemical in the initial set or added later. At a minimum, method development will include modification of existing protocols and methods developed for use in other applications or taken from the scientific literature, so that one can demonstrate adequate performance for the specific target chemicals, tissue sample type, and concentration range desired. Some of the steps (not necessarily in order) are:

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Monitoring Human Tissues for Toxic Substances Demonstration that the method is adequately sensitive to detect each target chemical by assay of appropriate coded (blind) standards. Demonstration of adequate recovery of target chemicals in sample preparation and instrumental assay steps through analysis of standard mixtures in a blank (matrix-free) solution. Demonstration of adequate control of interference by chemicals in the sample background through analysis of both blank samples and spiked matrix samples. Demonstration of good overall recovery of target chemicals from representative samples. Actual (unfortified) samples that have been assayed thoroughly are needed for this step, because biologically incorporated target chemicals might not be as readily isolated from the sample as would a spiked contaminant. Several of those steps should be performed in replicates, so that assay variability can be assessed. More extensive method development will be required for analytic needs or target chemicals that do not conform closely to an existing protocol. If new or improved separation steps or methods of detection are needed, they must be developed before the steps outlined above. Once a method is demonstrated to meet stipulated analytical requirements, a validation study should establish the performance of the method with real samples over a realistic period and, if possible, with a range of operators, equipment, and laboratories. Precision would be characterized with replicate and coded (blind) analysis or assay of control materials, if available. Method accuracy would be established with reference samples or interlaboratory comparisons. Completion of this formalized method development and performance evaluation is necessary to support quantitative uses of monitoring results. Without such knowledge of system recoveries and performance, tissue concentrations cannot be defined and negative results cannot be properly interpreted. Design of Quality Assurance A detailed quality-assurance (QA) plan should formalize the analytic procedures for each assay. If results are to be acceptable it is necessary to specify calibration materials, acceptable levels for blanks, recovery samples, replicate analyses, other control samples, and the frequency with which procedures are applied. QA plans can also specify preventive-maintenance schedules for instruments, how data are to be validated, and what remedial procedures are to be used when QA procedures detect unacceptable results. The overall

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Monitoring Human Tissues for Toxic Substances intent is to build into the assay a system of diagnostic measurements that detect and correct degradation of analytic performance. Two tools of particular value in maintaining long-term analytic stability are the benchmark sample and the graphic control chart. The benchmark sample is of the same type as study samples and ideally has detectable concentrations of several or all of the target chemicals. It is of known stability and is prepared in many multiple replicate subsamples. The chemical homogeneity of these subsamples is established by assay of a statistical fraction of the total number of aliquots prepared. The sample might be composited from many individual samples, provided that the blending process achieves adequate homogeneity. Analysis of portions of the sample over time is used to detect and document changes in assay response. Correlation of assay results with the benchmark sample among methods and laboratories is used to establish reference values for the contaminants in the sample, which make it possible to verify assay accuracy and to detect assay bias. After statistical limits are established for subsample homogeneity and assay precision in the benchmark sample (usually as a part of validation experiments), a control chart is established for individual target chemicals. Results of reanalysis of the benchmark sample are posted on this chart, and control limits (usually reflecting 95% confidence values from the validation data set) are established. Maintenance of a correct control chart allows assay acceptability to be checked on a day by day or even batch by batch (Taylor, 1985). Identification of Additional Resources Needed Although it is possible for any monitoring program to prepare control materials by blending a large volume of samples, an alternative is to use standard reference materials provided by NIST and other organizations. Reference materials offer several advantages over blended samples: stability and homogeneity have been well established; reference values for certified contaminants are well established and documented; and a large community of users is sharing the same materials, so that evaluation of comparability of results is enhanced. The appropriateness of existing reference materials to the needs of any monitoring program will depend on the match between matrix type, analyte list, and concentration range in standard reference materials and monitoring-program samples and target chemicals. For a monitoring program that will continue for decades, development of reference materials should have high priority. Not only will such materials help in maintaining and documenting consistent monitoring results, but provision of portions of the materials to researchers or other monitoring programs

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Monitoring Human Tissues for Toxic Substances will help in establishing analytic comparability for related studies. Among the kinds of additional resource needs that might be identified at this point are alternative methods for specific analytes that show marginal or unacceptable analytic performance; identification of intermittent contamination sources, such as particular types of containers or sampling media; and data on the chemical stability of components that display high variability of recovery. Implementation of Method Development and Validation Studies Analytic-method development often involves complex choices, particularly when many kinds of performance must be optimized in a single procedure. If the choices are to be optimal, an analyst who focuses on specific needs or problems in the assay scheme must work closely with the program planner who is considering future needs and larger objectives. In general terms, the process of preparing a sample for assay can be viewed as a series of steps that exclude chemical constituents from the sample, simplify the sample composition, and increase the ability to detect and measure the target chemicals that remain. Collection of components from the sample by extraction or other phase-separation techniques is the first stage. In the optimization of a method, there are commonly many solutions to a given problem. Substitution of one chromatographic medium for another and modification of the mobile phases used to partition sample components in prefractionation steps are two examples. Given a choice between two or more procedures that accomplish the basic task of removing interfering (and presumably unimportant) sample constituents, the alternative that best meets other program needs (such as a high likelihood of including agents of possible future monitoring interest) should be selected. Such an alternative should, of course, be validated in practice before it becomes a part of routine operations. Pilot-Scale Monitoring Pilot-scale testing will identify and help to correct problems in the various elements of a monitoring program, including collection and transmission of samples, sample management before assay, chemical measurements, data management, and reporting. Each modification of the assay opens new possibilities for unforeseen technical problems. For example, addition of new target chemicals to an otherwise unchanged assay might require alteration of materials used for sample collection, to avoid contaminating a sample with a

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Monitoring Human Tissues for Toxic Substances new target chemical. In particular, when a new assay is added to a program, pilot testing of that assay might prevent large-scale waste of effort and resources. Continuing Monitoring: Review of Analytic Program and Goals The QA plan should contain a statement of what constitutes acceptable data and acceptable assay performance. A schedule of data review and validation, with periodic QA review, should be created. Outside review of the QA plan itself is desirable, as is periodic auditing of quality control-procedures and results by a group separate from the monitoring program. The standing committee that we recommend elsewhere in this report is a possibility. Monitoring of assay procedures will satisfy program objectives that are based on a specific monitoring need and a defined list of agents of concern. There will be incentives for further development within the narrowly defined mission to measure the same target chemicals more accurately, more quickly, and more cheaply. Not only can improved measurement technology permit dramatic increases in sensitivity and ease of measurement, but failure to keep pace with the state of practice will eventually weaken the credibility of monitoring-program results. Monitoring-program compromises imposed by analytic limitations, such as the number of assays supportable by program budgets or the requirement for compositing to ensure assay sensitivity, might be reduced or eliminated as technology improves. The desire to extend the monitoring program to new agents and to recognize otherwise unanticipated exposures is a second reason to encourage developmental tasks within a monitoring program. Reasons for incorporating new agents into a national monitoring program are to respond to needs and concerns broader than individual regulatory programs and to realize additional benefits from the considerable resources that a monitoring program requires. Exploratory activities within a monitoring program can provide an anticipatory approach to hazard recognition and provide for efficient application of new findings in the basic program. Incorporation of new agents into existing assay protocols and development of new protocols to address new monitoring goals can be planned as continuing activities. Exploratory Analyses Exploratory analyses are of two sorts: those that attempt to catalog nontar-

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Monitoring Human Tissues for Toxic Substances get contaminants as they occur in monitoring of sample preparations and those that seek to detect classes of chemicals that are not expected to occur in significant concentrations in assay-sample fractions but would be of great interest if they did. The latter kind might use sample-preparation procedures that differ from assay procedures or instrumental detection techniques other than the assay techniques used in monitoring. Results will be qualitative or at best semiquantitative, because method recoveries and other performance would not have been determined and instrument response calibration with valid standards might not have been possible. “Volunteer” Chemicals in Monitoring Assays Modern assay techniques, such as GC-MS, permit a degree of qualitative analysis of sample constituents other than target chemicals. Mass spectrometry as a detection and quantitation method typically involves collection and storage of sequential mass spectra representing the entire detectable portion of the sample or solution analyzed. Computerized comparisons with massspectrum libraries can tentatively identify some proportion of “unknown” components, if the library of standards contains mass spectra similar to the unknown spectrum from the sample. The reliability of a tentative identification of an unknown component will depend on the intensity and purity of the unknown spectrum and on the nature of the chemical detected. Confirmation of tentative identifications with standards to demonstrate matching mass spectral and gas chromatographic behavior is required. Alternatively, when sufficient quantities of the unknown can be isolated from a sample, the identities of the unknown might be determined by mass-spectrum interpretation combined with traditional techniques for the elucidation of chemical structure (infrared, and ultraviolet spectroscopy, nuclear magnetic resonance, elemental analysis, and high-resolution mass spectrometry). The undertaking is time-consuming and expensive, so a standardized approach might be desirable for deciding which unknowns should be identified. Some considerations might be apparent concentration (i.e., intensity of GC-MS peak); frequency of detection; mass-spectral features that suggest anthropogenic origins such as patterns indicative of chlorine or bromine atoms; or similarity of mass spectra to those of known toxicants. Alternative Detection Methods The versatility and specificity of GC-MS make it the most widely used tool

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Monitoring Human Tissues for Toxic Substances for environmental monitoring of organic contaminants in environmental samples. Under electron-impact ionization conditions, it is possible to generate similar mass spectra from a variety of instrument designs, so large publicdomain mass-spectrum libraries can be used. The adoption of GC-MS by EPA for many of its monitoring methods has led to further standardization of MS instrumental characteristics. However, several techniques are important complements to standard GC-MS. Chemical ionization improves detection of chemicals that show excessive fragmentation under electron-impact ionization conditions. Both positive and negative chemical ionization can also be used selectively to improve detection of chemicals with higher gas-phase proton or electron affinities, as well as providing molecular-weight information. Although those techniques are not widely used for routine monitoring, they are particularly useful for detecting traces of halogenated chemicals, such as PCDDs (Dougherty et al., 1980). Other emerging techniques can address those chemicals that are not detectable by any gas chromatographic technique because they are nonvolatile or thermally unstable. Among those methods are new interface designs to permit high-performance liquid chromatography and mass spectrometry (LC-MS) (Covey et al., 1986) and supercritical-fluid chromatography and mass spectrometry (SFC-MS) (Smith et al., 1986). Secondary-ion mass spectrometry with collisional activation, an MS-MS technique (Tondeur et al., 1987), is another approach that permits detection, identification, and ultimately quantitation of nonvolatile chemicals at low concentrations in biologic samples. Each of those techniques could play a useful role in exploratory analyses, and their incorporation into monitoring protocols will soon be feasible. The newer techniques open the way for detection of polar metabolites of toxic agents, as well as of “refractory” toxicants. Innovative uses of new approaches to develop new information for human-tissue monitoring should be encouraged. Alternative Sample-Preparation Schemes Exploration of alternative sample extraction and prefractionation techniques should occur as new analytic techniques are developed. “Throwaway” fractions obtained from monitoring protocols that are not amenable to conventional GC-MS analysis could be screened with LC-MS, SFC-MS, and MS-MS. Supercritical-fluid extraction methods (Kalinoski et al., 1986) and microcolumn liquid-chromatographic methods (Ozretich and Schroeder, 1986) are receiving wide attention for use in sample preparation.

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Monitoring Human Tissues for Toxic Substances   History or Sourcea Compound CAS No. A B C D E F G II. Target Compounds: PCDD, PCDF Protocol     X   Chlorinated dibenzodioxins and dibenzofurans   X   2,3,7,8-tetrachlorodibenzodioxin   X   1,2,3,7,8-pentachlorodibenzodioxin   X   1,2,3,4,7,8-hexachlorodibenzodioxin   X   1,2,3,6,7,8-hexachlorodibenzodioxin   X   1,2,3,7,8,9-hexachlorodibenzodioxin   X   1,2,3,4,6,7,8-heptachlorodibenzodioxin   X   Octachlorodibenzodioxin   X   2,3,7,8-tetrachlorodibenzofuran   X   1,2,3,7,8-pentachlorodibenzofuran   2,3,4,7,8-pentachlorodibenzofuran  

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Monitoring Human Tissues for Toxic Substances 1,2,3,4,7,8-hexachlorodibenzofuran   X   1,2,3,6,7,8-hexachlorodibenzofuran   X   1,2,3,7,8,9-hexachlorodibenzofuran   X   2,3,4,6,7,8-hexachlorodibenzofuran   X   1,2,3,4,6,7,8-heptachlorodibenzofuran   X   1,2,3,4,7,8,9-heptachlorodibenzofuran   X   Octachlorodibenzofuran   X   Polybrominated dibenzodioxins and dibenzofurans   2,3,7,8-tetrabromodibenzodioxind   1,2,3,7,8-pentabromodibenzodioxind  

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Monitoring Human Tissues for Toxic Substances   History or Sourcea Compound CAS No. A B C D E F G 1,2,3,4,7,8-hexabromodibenzodioxind   2,3,7,8-tetrabromodibenzofurand   1,2,3,7,8-pentabromodibenzofurand   1,2,3,4,7,8-hexabromodibenzofurand   a A: Historical NHATS pesticide analyte (1970–1981) B: SARA 313 chemical list C: SARA/ATSDR 110 chemical list D: EPA Method 1625 analyte E: Target analyte in analysis of fiscal year 1982 adipose tissue samples F: Bioaccumulative pollutant study target analyte, EPA contract 68–01–6951, WA7 and 13 G: Analytes previously qualitatively identified in broad scan analysis of fiscal year 1982 specimens bIndividual congeners used as standards; detected as mixtures in historical NHATS surveys cNonvalidated target analytes for qualitative analysis dNonvalidated target analytes for quantitative analysis

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Monitoring Human Tissues for Toxic Substances play an important short-term role in the toxicokinetic behavior of many common volatile solvents, but they are not known to accumulate in adipose tissue over long periods. The methods used for sample collection and storage had not been validated for volatile chemicals, and the committee questions the integrity of the samples for those applications. Little information exists on the concentrations of trace elements in adipose tissues from normal, abnormal, or overexposed persons, and the diagnostic value of such measurements is uncertain. The assays are not planned for incorporation into routine monitoring of adipose tissues. Selection of analytic methods was generally based on methods already used and reported in published applications to analysis of adipose tissues (Macleod et al., 1982), other tissues (Norstrom et al., 1986), and environmental samples (Lopez-Avila et al., 1981). The general NHATS approach consists of exhaustive extraction of tissue with a lipid solvent such as methylene chloride; prefractionation of the extract with size-exclusion chromatography to remove the bulk of biologic (predominantly lipid) background material from the extract; additional prefractionation with one or more conventional (adsorption-partition) column chromatographic steps to separate out the least polar (aliphatic) components and to separate interfering groups of target chemicals (e.g., toxaphene or other chemicals from PCBs); and final instrumental detection and quantitation with high-resolution GC/low-resolution MS. The scheme for sample analysis as presented generally in the 1989 “Program Strategy” document and more specifically in the reports resulting from the initial applications of those methods to adipose samples (Mack and Stanley, 1984) is shown in Table 6–2. The analysis of 1982 samples according to the new methods took place in 1984–1985 and was reported in 1986. Additional validation and method-development studies were undertaken concurrently with and after the 1982 tissue analyses. All four of the new analytic projects (semivolatile chemicals, dioxins and furans, trace elements, and volatile chemicals) are most properly regarded primarily as method-development exercises and only secondarily as tissue surveys. Because of the lack of prior method validation, the results of those analyses are referred to in the respective project reports as concentration estimates. An intermethod comparability study that used historical and 1984 samples was initiated to compare the historical pesticide-survey method with the pesticide results obtained under the 1982 protocol. That effort was carried out at a contracting laboratory other than the one that had performed the 1982 method development and analysis. Some problems in applying the newer protocols were reported, and results of the study are not yet released. Additional method development and application of the semi volatile-chemical

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Monitoring Human Tissues for Toxic Substances TABLE 6–2 NHATS Analytic Efforts Collection Year Activity Analytes Assay Method Completion Date or Status 1970–1981 Monitoring OCl pesticides PGC/ECDa Annual 1982 R&D, survey OCl pesticides, semivolatiles HRGC/MSb 1986   R&D, survey Volatiles HRCG/MSc 1986   R&D, survey Trace elements ICP, NAAd 1986   Validation PCDDs, PCDFs HRGC, MSe 1986   R&D, survey PCDDs, PCDFs HRCG/MSb 1986   Exploration Unknown peaks HRGC/MSf 1986 1983 Monitoring OCl pesticides PGC/ECDa Partially published 1984 Comparability study OCl pesticides, semivolatiles PGC/ECDa vs. HRCG/MSb Under review 1985 No analyses n/a n/a n/a 1986 Monitoring OCl pesticides, semivolatiles HRGC/MSb In progress   Exploratory Unknown peaks HRGC/MSf In progress 1987 Monitoring PCDDs, PCDFs HRGC/MSb Under review   R&D, survey PBDDs, PBDFs HRGC/MSb In progress 1988 Analyses being planned n/a n/a n/a aModified Mills-Gaither-Olney pesticide method with low-resolution gas chromatography with electron-capture detection.

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Monitoring Human Tissues for Toxic Substances bExtraction, size exclusion, and adsorption chromatographic sample prefractionation with high-resolution gas chromatography and mass-spectrometry detection. cPurge and trap collection of vapors from an aqueous slurry of tissue sample with high-resolution gas chromatography and mass spectrometry detection. dAcid sample digestion and quantitation with atomic-absorption spectrometry or inductively coupled plasma atomic-emission spectroscopy. eSolid-phase extraction and digestion of lipid matrix, adsorption chromatographic prefractionation on graphitized carbon, with high-resolution gas chromatography and mass-spectrometry detection and high-resolution mass-spectrometry confirmation of key analytes. fExtraction, size exclusion, and adsorption chromatographic sample prefractionation with high-resolution gas chromatography/mass spectrometry detection; unknown peak screening by mass spectral library comparison to reference spectra and to previously observed unknown components.

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Monitoring Human Tissues for Toxic Substances (“broad-scan”) protocol has occurred since then, but no formal results have been released. The target chemicals listed in part I of Table 6–1 were taken from the QA planning document for the most recent round of analysis (1986 sample year). Target chemicals designated with superscripts c and d are planned for inclusion as “qualitative” or “quantitative” analytes. Method validation has not been performed, but these chemicals are detectable with the GC-MS method to be used, and standards are available. The most successful of the method-development and validation projects, and one that resulted in the production of a well-documented protocol as a basis for future monitoring, was the dioxin-furan project. Several documents presented the accomplishments in PCDD and PCDF analysis. The first is a report of an analysis of 1982 tissue-survey samples that used developmental methods. The analytic work took place from the fall of 1984 through the winter of 1985 (EPA, 1986a). The report reflects an improvisational approach to the analytic method that is incompatible with monitoring goals, because EPA, through the NHATS, had attempted to implement methods that were not fully tested and validated. One must infer which analysis produced which results by examining the size of the tissue aliquot reported (Tables 5–14 in that report). For given target chemicals, different samples were assayed with different methods. Some samples were assayed with two methods, and that would presumably produce results for the complete list of target chemicals for each method, but the results are reported for one method for some analytes and for the other method for other analytes. Comparison of results of the two methods is limited to mean and standard deviation of recoveries for the two sets of data. No side-by-side comparison of results for each preparation method is shown, although such data were presumably generated. The consequences of that approach include the use of multiple methods of sample preparation when the method initially adopted was judged to be inadequate for some samples of target chemicals, lack of clarity about which methods produce which results, and uncertainty regarding comparability of results of the two methods. Method-related uncertainties, and a generally tentative level of confidence in the quality of the analysis are reflected in the following language of the report: “The data for a sample reported based on the original protocol may be considered suspect based on the possible differences in the recoveries of these chemicals according to the two methods used”. “A continued effort in following the trends of PCDD and PCDF will require that the analytical method…be fully validated through intra- and interlaboratory studies.” The second report describes method-development and validation work that was largely after the initial application of the methods to study samples (EPA,

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Monitoring Human Tissues for Toxic Substances 1986b). The effort addressed several of the problems of the previous results. The report is a good model for future method-development and validation projects. It discusses several prerequisites of the production of fully usable data: method precision, accuracy, sensitivity, and stability are characterized in a statistical manner; control materials and comparison results are developed and validated; and a step-by-step approach to quality control throughout the analytic protocol is developed. The revision of the latter project report contained in the third document indicates an intention to use the control material developed previously as a continuing quality control element (MRI, 1988). Additional planning for sample and data management and quality control are also reported. Preliminary efforts like those reported in the second and third documents provide a good basis for a monitoring effort. However inclusion of new but related chemicals in the overall framework of such a program introduces new risks. For example, the proposed inclusion of brominated analogues of PCDD and PCDFs as target chemicals without an explicit validation effort is a short cut that can lead to “messy” and unsatisfactory results. Overall confidence in the results of the program would be strengthened if results of “try it and see” assays of study samples were more clearly distinguished from results of well-validated analyses. The NHATS or its successor must document the rationale and specific analysis that result in program decisions during each year’s planning to add or delete target chemicals. SUMMARY AND RECOMMENDATIONS The NHATs project reports show several encouraging characteristics. Some of the weaknesses identified and discussed in the previous section have been recognized by EPA and its contractors, and efforts to remedy them are evident. Within the definition of each analytic task, the contractor has demonstrated a competent and sometimes innovative approach to analytic-method development. The most recent reports and planning documents show a good understanding of quality-assurance planning and quality-control techniques. In summary, there is no reason to doubt that the program can attain state-of-the-art analysis. The principal concerns in this regard would be with constraints imposed on the analytic effort by inadequacies of agency planning and direction, budgeting, or long-term commitment to a program of human-tissue monitoring. Several factors make the need for analysis of program goals and a sound planning process critical: the need to balance innovative method development

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Monitoring Human Tissues for Toxic Substances with the maintenance of stable assays for monitoring; the open-ended nature of the analytic effort and the cost per sample; the need for sufficient analyses to achieve sampling and statistical goals; and the inherently multiagency and multiuse nature of the program, which requires strong coordination among agencies and with other sources of technical information vital to program planning. Definition of Goals Efforts to date have been oriented toward those chemicals that previous reports suggested could be detected with present protocols. Once the agency has established the relative importance of various possible uses of the data, the rationales for selecting target chemicals should be incorporated into a systematic weighting scheme and ap plied as comprehensively as is feasible. Criteria for determining the relative importance of a candidate target chemical should be separated from issues of analytic feasibility until late in the planning. The identification of one or several analytes that might require a new assay protocol could be important in planning future method development. Problems are likely to occur when method-development projects are concurrent with tissue monitoring (as in the 1982 samples). Design of an adaptable monitoring program with mechanisms for selection of new analytes and for development and validation of collection, storage, and assay methods will permit the monitoring program to remain responsive to current needs and to take advantage of progress in analytic technology. Formalization of the Planning Process Present reports do not address the larger issues underlying selection of project goals, nor do they provide insight into the information and alternatives considered in defining those goals. The result is an appearance of arbitrary program decisions; in some cases, decisions regarding the choice of analytic method seem to reflect a “shotgun” approach (e.g., elemental analysis of adipose tissue and possibly the volatile-chemicals projects). The program would benefit from regular strategic planning by the agency, frequent agency consultation with program contractors, scien tific peer review, and advice from interested federal agencies.

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Monitoring Human Tissues for Toxic Substances Those efforts would maximize the likelihood of a clear decision-making process and one that is well documented and understood. Critical Evaluation of Program Results Evaluation of data should be a continuing part of program reporting. One limitation common to all the project reports produced in the 1980’s is that they report very little analysis of final results beyond analytic validity. Data sets are a prime product of a monitoring program, but interpretation of findings in relation to larger program goals (such as time trends, efficacy of interventions, relative importance of different environmental contaminants, and regional or demographic trends in exposures) is an important part of understanding and meeting additional data needs. It seems to the committee that only NHMP itself has broad responsibility for making certain that the program is productive in relation to its larger goals. Links to Analytic-Methods Research Analytic-methods research is conducted within EPA, in other government programs, and in academe and the private sector. The NHATS must be in a position to articulate and, within EPA, influence research priorities for development of new analytic applica tions of emerging technology and to benefit from new developments. Analytic-program managers must be specifically charged with the formulation of analytic needs and maintenance of awareness of potentially useful developments. The committee doubts that leadership in analysis can be effectively delegated to contracting organizations, and it believes that EPA must maintain substantially more activity and expertise in this regard. Regular Schedule of Analyses The analytic effort has been modified from year to year since 1981, and developmental activities have supplanted monitoring to some degree. The NHATS has released data from only 1 collection year for each of the new sets of analytes. Including current efforts, there are data from only 2 collection

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Monitoring Human Tissues for Toxic Substances years (3 years for the broad-scan pesticides, if the comparability study is included). Priority should be given to setting and maintaining a schedule for analysis of results of each assay type.