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Complex Mixtures: Methods for In Vivo Toxicity Testing (1988)

Chapter: 4. Sampling and Chemical Characterization

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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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Suggested Citation:"4. Sampling and Chemical Characterization." National Research Council. 1988. Complex Mixtures: Methods for In Vivo Toxicity Testing. Washington, DC: The National Academies Press. doi: 10.17226/1014.
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4 Sampling and Chemical Chamctenzation This chapter presents a set of general guidelines and strategies to consider when designing a scheme for sampling and analyzing complex mixtures. No protocol or group of protocols for sampling or analysis will be applicable or appropriate for all types of mixtures. What follows is not intended to constitute a handbook of sampling and analytic methods for complex mixtures. Rather, researchers should design or modify methods as appropriate in each particular instance. However, some general rules can be followed. All of them are based on the approach in which sampling of a complex mixture has three components: the relevance of the material collected to the human situation, the use to which the material will be put, and the potential for human exposure, including routes and extent of exposure and bioavailability. CLASSIFICATION It is important to know as much as possible about the chemical composition of a mixture before any useful toxicologic prediction can be undertaken (toxi- cologic testing can be performed on a mixture in the absence of chemical knowledge if prediction is not the objective). Information on the chemical and physical properties of a mixture can have a direct impact on the selection of testing strategies. Once the mixture has been partially defined, sampling tech- niques, bioavailability, and chemical characterization can be considered. In light of the understanding that complex mixtures can vary widely from combustion and distillation products of fossil or synthetic fuels, to pyrolysis products of tobacco or synthetic materials in buildings, to components leaking from waste-dump sites some criteria must be identified for the mixture, re- 65

66 COMPLEX MIXTURES gardless of origin. The first criterion, physical state, must be determined. One can encounter such complexity as a mixed-state situation, a slurp of particu- late matter with a liquid or a gas or vapors and gases, which differs substan- tially from each fraction and is not toxicologically comparable with the "mix." The second, chemical-class diversity and component multiplicity, will have to be evaluated; this is difficult, in that a continuum of complexity can be encoun- tered, and whether the components are known or unknown will influence the predictability of the composition and the biohazard. The third criterion, stabil- ity of the mixture, will be important, because chemical instability (i.e., the potential for chemical interactions) will seriously affect the reliability of the sample and the reproducibility of the toxicologic experiment to evaluate the mixture. Complex mixtures can be divided according to whether they are derived from combustion or distillation products or from noncombusted materials. Ex- amples of the first case are fossil and synthetic fuels and vegetable and syn- thetic materials. Examples of the second are food, water, and drugs; hazardous waste; municipal and sewage waste. If one knows the origin of a sample, one can deduce qualitative and quantitative information about the mixture in ques- tion, even if physical characterization and chemical characterization are mini- mal. Extensive information on chemical characterization of a series of fossil- fuel-related materials has been published (e.g., Wright and Dauble, 19861. That information makes it possible to predict chemical classes to be found in mixtures derived from these sources. A detailed discussion of the various ori- gins of complex mixtures is found in Appendix A. SAMPLING Consideration of bioavailability (relevant to human exposure) and of ulti- mate sample use must be incorporated into the sampling strategy, if it is to be effective in producing materials for assay that will provide data relevant to human health. It is important also to consider the preservation of sample integ- rity; sample alterations must be minimized to ensure that the substance assayed is the substance presented to the human environment. COLLECTION STRATEGY Exposure In designing a sampling protocol, input from several kinds of specialists is necessary. Toxicologists, industrial hygienists, or other experts in human health effects should be consulted regarding the most likely routes of exposure to the mixture in question. Exposure of humans via inhalation or ingestion of water or food is most common. In occupational settings, absorption through

SAMPLING AND CHEMICAL CHARACTERIZATION 67 the skin can also be important. Exposure to a mixture via a combination of routes is not unusual. For example, particles might be inhaled and later swal- lowed after being cleared from the lungs by mucocilia~y action. It is usually practical to consider only the presumed primary route of exposure in designing a sampling protocol. Meteorology Seasonal or meteorologic considerations are particularly germane to the de- sign of protocols for sampling environmental mixtures. Increases in air tem- perature can result in loss of benzoLa~pyrene (BaP), a carcinogenic polycyclic aromatic hydrocarbon (PAH), from particles collected during high-volume at- mospheric sampling (De Wiest and Rondia, 19761. Seasonal variation can also affect surface areas and density of atmospheric particles (Corn et al., 1971; Flessel et al., 19841. Wastewater, soil, and waste-dump sample composition can be expected to vain seasonally. Changes in temperature, freezing condi- tions, and rainfall can all modify the water table and influence environmental sample composition. Temporal Factors Industrial hygienists and process engineers should be consulted as to tempo- ral considerations of sampling. For industrial samples, the time course of pro- duction is important. One must consider whether to sample only at the end of a particular batch process, at the middle of a run, or throughout the production of the material in question. For example, it has been shown that the composition of coal-liquefaction recycle oils changes markedly during the first few days after startup of a process, but is relatively stable after that (Burke et al., 19841. In the case of air sampling at a manufacturing site, both the time of day and the point in the workweek are important. The variability of samples during a work- week has been illustrated in the monitoring of oncology nurses handling cyto- static drugs; the mutagenicity of material extracted from their urine increased toward the end of the week and decreased during the weekend (Nguyen et al., 1982). The duration of sampling is important, particularly for emission materials. If the substances of interest are in low concentration, the sampling period must be long enough to collect material required for the assays proposed. This state- ment of the obvious must be balanced by two other considerations. First, it can take some preliminary investigation to ascertain the likely concentration and to determine whether the concentration of the material is relatively stable or likely to fluctuate. Second, the nature of the collected material might change as the capacity ofthe collection device is approached. Gorse et al. (1982) showed that the biologic activity of diesel exhaust particles changed as the collecting filter

68 COMPLEX MIXTURES became loaded: the percentage of extractable material, the fluorescence of high-pressure liquid chromatography (HPLC) fractions, and the mutagenicity with respect to Salmonella typhimurium all increased linearly, presumably be- cause the loaded filter became a more efficient collector of mutagenic vapors. Spatial Considerations Inclusion of spatial considerations in the sampling design requires the advice of experts, including industrial hygienists, engineers, and biostatisticians. The primary consideration should be the relevance of the sample site to potential human exposure. If the material is from a manufacturing or refining process, the design should ignore materials in closed systems in favor of open points of maintenance, ingredient addition, product removal, and so forth. Waste sites should be studied as to the availability of deposited materials. Personnel access to the site and the potential for materials to enter water supplies, soil, and food chains are important. In the case of emission, the nature of the research or regulatory questions asked will define whether sampling should focus on the point of discharge, some distant site, or both. A testing program will probably require consideration of multiple testing sites for comparative studies. Whether sampling is to be purposive or of a random statistical design will be determined largely by the nature of the poten- tial for human exposure. Purposive sampling requires selection of a set of sites for generating samples of a particular type. In a study of coal-liquefaction biohazard potential, sam- pling sites would be at various process points: the coal-slur~ying step, the reac- tion vessel, points of refining and separation, discharge of wastewater, final products, recycle materials, and waste. Similarly, sampling could be done in a waste-dump site with a history of material escape or near an emission source and at designated distances from it. Random statistical designs seek to provide an overall picture of an area by sampling at a statistically determined number of places within that area. In a series of publications prepared for the Department of Energy, Gilbert (1983, 1984) described a number of sampling designs, generally termed "probability sampling" (see Table 4-1~. In simple random sampling (see Figure 4-1), eveIy unit of a target sample population (or geographic area) has an equal probability of being collected for assay. The units are numbered from 1 to N. and samples are chosen by drawing n numbers (n < N) from a random-number table or by use of a computer. The units designated by the selected numbers are sampled. This mode of sampling is best applied to a homogeneous population with no dominant spatial or temporal trends. Gilbert noted that most statistical inferen- tial procedures assume that data were collected through simple random sam- pling. Sites of likely sampling of complex mixtures might not be homogeneous with regard to composition or bioavailability of components (e.g., waste

SAMPLING AND CHEMICAL CHARACTERIZATION TABLE 4-1 Summary of Sampling Designs and When They Are Most Useful 69 Sampling Design Haphazard sampling Most Useful When Population homogeneous over time and space essential; method not recommended, because of difficulty in verifying assumption of homogeneity Target population well defined and homogeneous, so sample-selection bias is not a problem; or specific environmental samples selected for unique value and interest, rather than for making inferences to wider population Judgment sampling Probability sampling Simple random sampling Stratified random sampling Systematic sampling Multistage sampling Cluster sampling Double sampling Homogeneous, i.e., no dominant trends or patterns Homogeneous within strata (subregions); might want to consider strata as domains of study Trends over time or space must be quantified or strictly random methods are impractical Target population large and homogeneous; simple random sampling used to select contiguous groups of population units Population units cluster (schools of fish, clumps of plants, etc.); ideally, cluster means are similar in value, but concentrations within clusters should vary widely Must be strong linear relation between variable of interest and less expensive or more easily measured variable dumps). In addition, practical considerations in the collection of field samples can limit the use of simple random samples. Another type of random sampling is stratified sampling. This assumes a number of nonoverlapping populations that differ from each other, but are internally homogeneous. The population or study site is divided into as many strata as appropriate, and simple random sampling is applied to each stratum. Another technique is multistage subsampling, which involves division of the site or population into primary units (Cochran, 19771. A set of primary units is chosen by simple random sampling, and samples are collected within each of these, under the direction of simple random sampling. This type of design is particularly applicable to geographic areas with variations in terrain, habitat, or other factors that require comparison. It is also useful for a relatively homo- geneous, but large, study site, if the number of samples that can be collected is limited. In cluster sampling, which is related, population units are grouped into clusters, a number of clusters are randomly selected, and all units within the selected clusters are sampled. Distinct from the random designs are the methods of systematic sampling. Gilbert (1984) indicated that these methods often find use in environmental monitoring, because they are generally easier to implement under field condi- tions. In addition, data from statistical investigations have indicated that sys-

70 SIMPLE RANDOM (a) (c) (e) (g) COMPLEX MIXTURES STRATIFIED RANDOM ~ - (b) · · ~ - · /- ~ TWO-STAGE SAMPLING I A. I . I . | . . PRIMARY UNITS — (d) · | · | · | \m -- 1 1 _ . . _ SYSTEMATIC GRID RANDOM WITHIN BLOCKS .. 1 . .. ... . I 1. 1- (flu .~.~.~-e 1. 1.1- .-- ·. A. SYSTEMATIC UNALIGNED _ AI B I C I D ·E I F |.G |.H _~1 J it ; (h) SYSTEMATIC RADII CLUSTER SAMPLING 6~9 CLUSTERS FIGURE 4-1 Some probability field sampling designs. Reprinted with permission from Gilbert (1983). tematic sampling can be superior to simple or stratified random sampling for some types of environmental variables. In general, systematic sampling begins with random selection of one unit of the study population. This serves as the starting point of a sampling pattern according to some established spatial or temporal frequency. A problem common to systematic designs is that, if the variable being measured is subject to periodicity or cycles, misleading results

SAMPLING AND CHEMICAL CHARACTERIZATION 71 will be obtained. Gilbert cited another difficulty: the accuracy of estimates of sampling errors and other statistics depends on the study population's being random. In the aligned-square grid design, a location (or other unit) is chosen at random. The location of each sample is then chosen by applying a grid of fixed dimensions over the chosen area, and two random coordinate numbers are drawn to fix the location of the original point. Each sample site is then fixed in relation to this original point. Variations include the use of a triangular grid and the unaligned-grid-pattern design. In the latter (Figure 4-2), a point A is chosen randomly, and X and Y coordinates are established. To set points B. C, and D, one uses the X coordinate of A and three new randomly chosen Y coordinates. To set points E through I, the point A coordinate Y and random X coordinates are used. One can also design systematic sampling methods based on lines. Figure 4-1 illustrates periodic sampling of radii from a central point, such as a known discharge source. Aligned Square Grid · I ~ I I J I -HA my- ~ -- T.-- (a) (c) Central Aligned Square Grid _ —_ - - · — 1_ - - I1 1 1 2- it- 1~- r. (b) : l 1 , 1 1 1 1 - . - 1 _ - _ _ — 1 1 t_~- _ ~ ~ ~ I , 1 1 1 Unaligned Grid ~ .BI I .D| ~—Hi— Hi'—- '—~1 ·E | I G I ·H 1 (d) ) \\ >/ \ A\ ,~ \ I ~ I ~ K .~ | ~ / \|\ / ~ / ~ / \ Triangular Grid FIGURE 4-2 Some systematic designs for sampling in space. Reprinted with permission from Gilbert (1984).

72 COMPLEX MIXTURES COLLECTION PROCEDURE Designing the collection process requires the input of a toxicologist to pro- vide information on the nature of the assays and of a chemist and an engineer to produce a protocol that will efficiently provide a relevant sample material. One type of information to be supplied by a toxicologist is the amount of material that needs to be collected. Testing protocols geared to chemical analysis gener- ally require smaller samples than more biologically oriented approaches. In vitro tests are more sparing of sample than are whole-animal studies. The collection procedure depends largely on the source of the material and its physical state. Environmental samples are likely to be mixtures of materials in various physical states. They might include mixtures of gases, aerosolized liquids, and suspended particles; combinations of liquid and solid waste; bio- logic tissues, which are largely water; and so forth. We present here examples of sample types and methods deemed appropriate for their collection. Many of these were described at greater length in a recent EPA document (U. S. EPA, 1985), and some useful techniques were also reviewed by Alfheim et al. (19841. Gases and Aerosols Vapor-phase organic chemicals have been collected from ambient air, com- bustion exhaust gases, cigarette smoke, and indoor spaces for chemical charac- terization and bioassay (Hanson et al., 1981 , 1984; Griest et al., 1982; Higgins et al., 1983; Pellizzari et al., 1976; Pellizzari, 1982; Hughes et al., 1980; Krost et al., 1982~. Chemical transformations and perturbations of the original chemical mixture can occur during the sampling step (Berkley and Pellizzari, 1978; Pellizzari et al., 1984; Pellizzari and Krost, 19841. Air particles are a common source of mixtures that have environmental or health significance. Alfheim et al. (1984) recommended that particle-size frac- tionation be included in the collection scheme to increase the biologic rele- vance of a particulate sample. The International Standards Organization (ISO, 1983) and the American Conference of Governmental Industrial Hygienists (Lippmann, 1985) have established cut sizes appropriate to fractionation. They depend on the toxicity at deposition sites in the airways or at sites along the clearance pathways or storage sites. Inspirable particles are those which can be aspirated by the nose or mouth. Thoracic particles are those which can enter the thorax. Respirable particles are those which penetrate the lung's conductive airways. Another reason for size separation is that the chemical composition and biologic activities are related to particle size. Among urban air particles, small size is associated with the presence of acidic compounds, and larger sizes with basic groups (Miller et al., 19791. In comparative assays, most mutagenic activity has been associated with smaller

SAMPLING AND CHEMICAL CHARACTERIZATION 73 particles, less than 2.5,um in diameter (Talcott and Harger, 1980; Preidecker, 1980; Chrisp and Fisher, 19801. Most of the total PAH content of air particles is found in particles less than 5 ,um in diameter. It has been recommended, however, that results of assays of particles collected with size fractionation be compared with results of assays of particles collected without fractionation. Artifacts of size separation reportedly have reduced activity in mutagenicity assays (Alfl~eim et al., 1983), whereas other chemical transformations report- edly have increased mutagenicity (Clark et al., 19811. Point-source sampling for air particles includes use of impactors (Cheng and Yeh, 1979), cyclones, electrostatic precipitators, and filters (U.S. EPA, 1985~. Sampling methods have been described by Brusick and Young (1982) and Lentzen et al. (19781. Collection of particles from ambient air generally uses high-volume samplers (U.S. EPA, 1971), so-called massive-volume samplers (Henry et al., 1978; Cheng et al., 1984), and medium-, low-, or ultrahigh- volume samplers (Fitz et al., 19831. It has been recommended (U.S. EPA, 1985) that preparation of particulate samples for bacterial-mutagenicity moni- toring begin with collection of standard high-volume samples with inert filter- collection media. For collection of larger amounts of material or when circum- stances dictate a shorter collection period, use of an ultrahigh-volume sampler would be appropriate (U.S. EPA, 19851. If gaseous emission is collected at high temperature, there will be less oppor- tunity for condensation of materials on particles. To collect a gaseous mixture that adequately reflects the material being produced or emitted to the human environment, the possible presence of volatile materials must be considered in the collection scheme. When it is feasible, a gas can be cooled before introduc- tion to an adsorbent material. Alfheim et al. (1984) indicated that XAD-2 resin was most commonly used for this purpose. Gaseous samples can thus be re- duced to collected particles, extracts from absorbents, and condensates. The testing of volatile materials not captured that way is a subject for research and is not generally applicable to routine environmental monitoring or widely used in bioassays. Aqueous Materials Aqueous samples may be collected in a relatively homogeneous state or may contain suspended solids. The most common method of sampling water is manual-grab collection of the required volume. Water generally has low con- centrations of biologically active materials, which must be concentrated for application in most tests. Concentration protocols are therefore identified as the major part of the collection procedure. Methods for concentration of water with less than 5 % associated solids, particularly drinking water, have been well described (Kopfler, 1980; Jolley, 1981; NRC, 19821. Aqueous solutions can be concentrated by removing the water via freeze concentration, lyophiliza-

74 COMPLEX MIXTURES lion, vacuum evaporation, and reverse osmosis (Shapiro, 1961; Baker, 1970; Dawson and Mopper, 1978; Crathorne et al., 1979; Jolley et al., 1975; Kopfler et al., 19771. These methods concentrate organic and inorganic materials to- gether, a procedure that Kopfler (1980) claimed was a disadvantage if the bio- assay system to be used could not tolerate high concentrations of inorganic materials. Another scheme involves selective concentration of organic materials from drinking water by adsorption of the contaminants on activated carbon or XAD resin columns (Middleton et al., 1956; Kopfler, 1980; McGuire and Suffet, 1983; U.S. EPA, 1985~. A variety of solvents can be used for elusion of or- ganic substances from the activated-carbon columns. Supercritical liquid car- bon dioxide is an efficient elusion solvent (Modell et al., 1978~. This method can be used for large-scale processing of water samples and results in the re- cove~y of gram quantities of contaminants. The limitations of these techniques include the nonextraction or nonrecove~y of inorganic materials and highly polar materials, ionic organic species, or volatile low-molecular-weight or- ganic compounds. Methods that might be suitable for collection of these sub- stances were cited by EPA (19851. Kopfler (1980) noted that organic acids and bases might be amenable to collection on resin columns after adjustment of the water pH to suppress ionization. Altering the pH of a mixture of materials, however, also results in changing its chemical composition or biologic activity. Aqueous samples with large amounts of suspended solids are generally best prepared by separation ofthe phases. Gravity partitioning is done by storing the sample at 4°C for 24 hours. The solid phase can then be removed and pro- cessed separately for assay. Alternatively, high-pressure filtration or high- speed centrifugation can be used for phase separation. The liquid portion can be separated into aqueous and nonaqueous components, concentrated if neces- saty, and assayed. Some samples with suspended solids are amenable to pro- cessing by liquid-liquid extraction methods. Details of some of these protocols were described by EPA (19851. Nonaqueous Materials Many environmental and indust~y-related samples are nonaqueous liquids, including organic liquids, light and heavy oils, and some tars. Samples can contain mixtures of volatile (b.p., 36-100°C), moderately volatile (b.p., 100- 300°C), and nonvolatile (b.p., over 300°C) materials, and there might be associated solids. Generally, the composition of such materials is poorly char- acterized and subject to variation after sampling. Much of the information on samples of this sort has been derived from research on fossil-fuel materials (Wright and Dauble, 19861. Sampling itself is usually not difficult; grab sam- pling often suffices. The samples are generally concentrated enough for assay of undiluted or unprocessed material. The preparatory issue with these materi-

SAMPLING AND CHEMICAL CHARACTERIZATION 75 als therefore is not sampling, but rasher treatment to make the sampled material compatible with the bioassay in question. Some of these issues are discussed later. Solids and Sediments Solid materials appear to be amenable to simple sampling techniques. How- ever, the entire mass of material collected might not constitute the sample of interest. Soils and sediments are examples of materials wherein an unknown or small concentration of a substance of biologic interest is associated with a large quantity of matrix. The protocol for these materials must therefore include extraction and concentration. Specific recommendations for treatment of soils were made by EPA (19851. Efforts should first be made to provide a homogeneous sample. Aggregates can be broken by crushing or cutting. To avoid a particle-size bias, the material can be quartered. This is done by spreading out the material on a clean surface and mechanically dividing the sample into four parts; three parts are returned to the storage vessel, and the remainder is quartered until the desired amount is obtained. Sediments might contain water, which can be removed as described previ- ously. The next step should be extraction in a Soxhlet apparatus with highly pure organic solvents. The nature of material to be recovered will depend largely on the solvents used. Solvents used for extracting materials from soil matrices for chemical analysis have included the following: benzene, ethyl acetate, and benzene-methanol-acetone (2: 1: 1) for humic substances (Ogner and Schnitzer, 1970; Cifrulak, 19691; n-pentane, carbon tetrachloride, and methylene chloride for oil (Jobson et al., 1974; Jensen, 1975; McGill and Rowell, 1980~; and hexane-isopropanol (3:1) for polychlorinated biphenyls (Carey and Gowen, 19781. Other extracting solvents have been used specifically to produce samples that would be amenable to biologic testing. Mutagenic materials were obtained from agricultural soils extracted with hexane-acetone (2:1) and dichloro- methane (Goggelmann and Spitzauer, 1982; Brown et al., 19851. Likewise, mutagenic sediment extracts have been obtained through sequential extraction with diethyl ether and methanol (Kinae et al., 198 11. Ether has also been used on sediments (Sato et al., 19831. The choice of solvents can likely be dictated solely by the nature of the material to be recovered, if the solvent can be evapo- rated or otherwise removed before bioassay. EPA (1985) has recommended rotary evaporation. The pH of the soil or sediment will be a major influence on the types of compounds extracted. For most other types of solid samples, the primary preparative consideration will be to constitute a material compatible with the bioassay selected. The sample must be representative of the environmental or human exposure poten-

76 COMPLEX MIXTURES PABLE 4-2 Samplers Recommended for Various Types of Solid Wastea Waste Type Sluny Dry solid Waste Location or Container Sampling Devices Weighted bottle, dipper Thief, scoop, shovel Sticly or moist solid and sludge Hard or packed waste aData from U.S. EPA (1982). Tank, bin, pit, pond, lagoon Dmm, sack, pile, truck, tank, pit, pond, lagoon Drum, tank, tmck, sack, pile, pit, pond, lagoon Drum, sack, truck Auger tial. This can be ensured by taking multiple samples (multiple sites on repeated occasions) and combining them. EPA (1985) has suggested that heating the material or conducting the sampling procedure at a high temperature might increase the homogeneity of some solids. Mixing is to be done before taking the sample, if that is feasible. EPA (1985) also cautioned that heating might increase volatilization of some sample components and alter characteristics of others. Table 4-2 lists types of sampling devices recommended for solid wastes. MAINTENANCE OF SAMPLE INTEGRITY When a sample has been collected, further safeguards must be in place if it is to be assumed that the data from the bioassay are appropriate for evaluating the sample's hazard potential. Mechanisms must be instituted at each step of sam- ple handling to ensure that the material collected is the material tested. Bal- anced against this need to maintain sample integrity is the need to deliver the sample to the bioassay appropriately. The delivery must be done so that there is even distribution of the test item. It is also important that the test organisms not be killed and that they not be affected in any way that would impinge on the biologic end point to be measured. It is likely that all these conditions could be met only in an ideal world. Nonetheless, intelligent consideration of factors bearing on sample integrity is necessary. Storage A suitable means of sample storage is the first consideration. In all but the rarest of instances, there is a period between collection of the sample and its application in a biologic test. The researcher will want to preserve an aliquot for future reference, retesting, and other possible characterization. Storage conditions must be tailored to particular samples. Some general suggestions have been made for various classes of mixtures (U.S. EPA, 19851. Factors that must be considered for storage conditions include the following:

SAMPLING AND CHEMICAL CHARACTERIZATION 77 · Chemical transformations. Potentially biohazardous substances are likely to be chemically reactive themselves or readily converted to reactive forms. In some instances (as with fossil-fuel mixtures), biologic activity has been shown to be lost with time after sampling (Schoeny, 19851. Temperature plays an important role; storage under liquid nitrogen or at—70°C enhances preservation. · Photochemical reactions. Components of complex mixtures might be subject to photoactivation or photodegradation by near-UV light that would alter the biologic activity (Larson et al., 1977; Barnhart and Cox, 1980; Selby et al., 1983; Kalkwarf et al., 19841. It would be prudent to store samples in dark brown or actinic glass containers. · Microbial transformations. Various species of bacteria and fungi can me- tabolizeorganic compounds found in wafer end soil samples (Cerniglia, 19811. Preservation might require autoclaving, although heat Idly or steam steriliza- tion) can alter the contaminants. Liquid samples, such as water without sus- pended sediments, can be filter-sterilized. · Physical state. It must be recognized that heterogeneous materials can be expected to separate into phases during storage. Particles will settle out of liquids; volatile materials can escape; and aqueous and organic phases might separate, as might organic phases from other organic phases. _ _ Preparation for Assay In preparing test materials for assay, it is of the greatest importance to con- sider their mode of administration to the bioassay. The sample of interest might require modification of physical state for compatibility with the bioassay de- sign. The most widely applied modification is solubilization of the sample in a substance that can be safely used for its delivery. The choice of solvents de- pends on the chemical properties of the mixture. For less complex mixtures of similar substances, such solvents as dimethyl sulfoxide, ethanol, methanol, acetonitrile, or methylene chloride might be adequate for most chemical or toxicologic studies. More complex mixtures (coal tar or liquids derived from coal liquefaction) might require a mixed solvent or a combination of solvents with increasing polarity. The failure of these to solubilize the entire mixture might affect the overall results. An estimate of the nonsolubilized (and ulti- mately nontested) material should be included in the sample evaluation. For all chemical and toxicologic studies, solvent controls are necessary to gauge the effects of the delivery system on the assay outcome or test-organism viability. The solvent must be of the highest grade practicable (e.g., spectro- scopic- or pesticide-grade). Sample preparation methods, like sample collection and maintenance, must be tailored to specific sample types. Solubilization and extraction procedures should be undertaken in glass containers to minimize contamination. Collected

78 COMPLEX MIXTURES particles and gases can also be extracted and the adsorbed materials assayed independently of the matrix. One recommended method entails the extraction of 1.0 g of a sample twice with dichloromethane either by Soxhlet extraction or by sonication followed by filtration and concentration. When these types of extractions are conducted, solvent incompatibility between chemical fraction- ation and bioassay systems can occur and might require solvent exchange (Bur- ton et al., 1979; Springer et al., 1982, 1984; Hackett et al., 1983; Kelman and Springer, 1981; Mahlum, 1983a,b; Mahlum and Springer, 19861. Quality Assurance Quality assurance quantifies and documents the extent to which measures taken to maintain sample integrity have been successful. Information on sam- ple source, collection, separation, and analysis should be recorded in a coher- ent and consistent fashion in a language devoid of jargon to facilitate informa- tion interchange among the members of the research team. The precision of the analytic method is assessed to evaluate the variability in the technique and requires the assay of independent replicate samples collected in the same man- ner at the same place and time. EPA has recommended that, for large sample lots, a fixed frequency of replicate measurements (e.g., 1 in 10 or 1 in 20) be chosen. For small sample lots, a greater frequency of repetition will be neces- sa~y (U.S. EPA, 19841. Quality assurance also extends to the preparation of a sample for chemical or toxicologic evaluation, because, in addition to the mixture's being complex, numerous steps in separation, extraction, and fractionation could give rise to variable results. The addition of a known surrogate or reference material to the original complex mixture, to act as an internal standard for later extraction and evaluation, is useful. Distinctive chemicals, having physical and chemical properties similar to those found in the complex mixture, should be introduced at the collection site, thereby allowing an assessment of sample change during handling, transportation, and storage, as well as recovery during sample prepa- ration. However, these internal standards should be carefully selected, because they might interfere with the anticipated bioassay. ANALYSIS The following discussion on chemical characterization of complex mixtures presents highlights of past and current analytic strategies and techniques. It introduces analytic tools of the future that can play an important role in more comprehensive characterization of mixtures and identification of components. This section should be regarded as a conceptual guide to mixture classification.

SAMPLING AND CHEMICAL CHARACTERIZATION CHEMICAL CLASSIFICATION 79 To make toxicologic predictions, analytic chemists and toxicologists need to know which chemical classes are present in a mixture. Toxicologists would be interested in major chemical classes such as PAHs, aromatic amines, nitro- samines, halogenated hydrocarbons, direct-acting alkylating agents, and metals—because they can indicate the types of causative agents that might be present in the mixture. Analytic chemists would be interested in the similarities between compounds within a chemical class, because they can provide clues as to appropriate analytic procedures and strategies that could be used for sample fractionation, component separation, and identification. Organic compounds can be divided into aliphatic, aromatic, cyclic aliphatic, and organometallic compounds, and inorganic compounds can be divided into metallic and nonmetallic elements and compounds. (The presence of heteroatom-containing compounds in each class is also important.) Aliphatic compounds can be further divided into compounds with one car- bon atom such as methane, methanol, methylamine, methylnitrosamine, and methylene chloride and compounds with two or more carbon atoms. The reactivity, volatility, stability, and solubility of a mixture can depend heavily on the numbers of carbon atoms in its aliphatic constituents. Aromatic com- pounds can be divided into one-ring compounds such as benzene, toluene, anisole, thiophene, pyrrole, and aniline and compounds with two or more rings. Many cyclic aliphatic compounds are solvents, such as cyclopentane, cyclohexane, decalin, and pyrrolidine. In most schemata, these compounds are not included, although they are present in some complex mixtures. In or- ganometallic compounds, a metal atom is bound directly to two or more carbon atoms (e.g., organotin). This chemical classification for the aliphatic and aro- matic compounds, and so on, is important when considering the strategies to be used in the chemical and toxicologic analysis of the mixture. Although this type of chemical classification limits the number of classes, irrespective of the degree of complexity of the mixture in question, it permits the chemical and toxicologic aspects of the research to be interactive, in that only one chemical nomenclature needs to be used. GENERAL CONSIDERATIONS IN FRACTIONATION OF COMPLEX MIXTURES Many approaches have been used for analyzing complex mixtures. For de- veloping data bases on toxicity, standardized strategies on separation and chemical analysis might be beneficial, especially if comparisons are antici- pated (Guerin et al., 1983; Haugen and Stamoudis, 1986; Guerin, 1981; Wilson et al., 1981; Wright et al., 1985; Benson et al., 1984; Hanson et al.,

80 COMPLEX MIXTURES 1980, 1982, 1985; Stamoudis et al., 1983; Later et al., 1981, 1983a,b; Smith, 1983; Weimer and Wilson, 19831. Requirements for Analysis As part ot selecting a strategy for chemical fractionation and characteriza- tion of complex mixtures, the in vitro and in viva bioassay requirements must be clearly delineated. Toxicologists and chemists should collaborate to address the needs of the testing strategies and logistics discussed here. Whether the purpose is to identify the causative agents in a mixture (Clax- ton, 1982; Bridbord and French, 1978; Hite and Skeggs, 1979; Florin et al., 1980) or to predict toxicity, some fundamental information is needed in design- ing chemical characterization experiments. Consideration should be given first to the total quantity of material (mass) and the mode of sample administration (fractions and subfractions). This preliminary information is necessary to set up the analytic and preparative procedures to support toxicity testing. For ex- ample, the analyte concentrations in the extracts, the quantity of material needed for each assay, and the total amount of material to be administered should be calculated. Generally, much more material is needed for a bioassay than for chemical analysis. The route of administration must be considered, because it defines the deliv- e~y system that can be used. Is the route of administration to be inhalation, ingestion, topical application, or injection? The route of exposure for in viva tests should simulate the exposure of humans. Thus, the mode of administra- tion can be inhalation for in viva experiments, but exposure can be to either gases, aerosols, or particles for in vitro tests. The physical state of the mixture, including particle or droplet size for inhalation exposure, must be defined; it will have a bearing not only on the form of administration, but on the predictive power of the test. Engineering factors, particularly in inhalation experiments for chamber design, must be considered, so that the exposure will simulate environmentally and biologically important conditions. In general, extraction, fractionation, and subfractionation of mixtures for chemical analysis are con- ducted on a relatively small scale, compared with those for bioassays. An analyst must be aware of the bioassay and chemical-analysis requirements dur- ing the application of isolation, fractionation, and subfractionation schemes. Analysts have a repertoire of techniques available for elucidating chemical structures and quantifying analyses in mixtures. They consider mass require- ments for successful analysis, the physical state of materials to be analyzed, the mode required to introduce an analyte or mixture into an analytic system, and the degree of complexity in a mixture (e.g., how much purification is needed). Some analytic techniques can resolve simple mixtures, such as pesticides, to the extent that qualitative and quantitative analysis can be performed on indi- vidual analyses. The extent to which this can be achieved with an analytic

SAMPLING AND CHEMICAL CHARACTERIZATION 81 technique should be recognized, so that sufficient fractionation and subfrac- tionation are conducted in advance. Some spectroscopic techniques are ultrasensitive and have low mass re- quirements (e.g., emission spectroscopy), whereas other techniques need rela- tively large quantities (e.g., nuclear magnetic resonance). The selectivity and specificity of a spectroscopic technique also determine the extent of purifica- tion required before analysis. All four factors quantity, physical state, mode of introduction, and purifi- cation requirements- should be thoroughly examined before approaches are chosen for creating fractions and subfractions. All the issues referred to here should be noted in concert with the bioassay requirements, to ensure a compatible bioassay and chemical analysis ap- proach. For information from a bioassay to correlate with information from a chemical analysis, the mixtures, fractions, and subfractions must have a com- mon denominator; that is, the same chemical separation procedures should be used for both bioassay and chemical analysis (Claxton, 1982; Later et al., 1981; Guerin et al., 1980, 1983; Ho et al., 1980; Pellizzari et al., 19781. Separation and Preliminary Examination The identification of analyses in a mixture involves separation into individ- ual components and structural elucidation with spectroscopic and spectromet- ric techniques. Separation and purification of the constituents are necessary because it is rarely possible to identify the constituents of a mixture directly. Separation of the constituents of a mixture should be quantitative for both bioassay and chemical characterization. It is also important that the compo- nents be in as pure a state as possible to avoid erroneous structural assignments. Methods for separating and purifying components of a mixture should not chemically alter any components. As indicated elsewhere, the history or origin of a mixture (see Appendix A) can reveal important information that aids in selecting separation processes and, to some extent, spectroscopic or spectro- metric techniques. Preliminary review of the origins of a chemical mixture, its sampling characteristics, and its sample history will help in selecting a strategy for chemical separation and analysis. Extraction and Concentration Solvents and extraction methods used for various complex mixtures have recently been reviewed by several investigators (Epler, 1980; Chrisp and Fisher, 19801. Liquid and gaseous samples can be chemically analyzed as is; sometimes a concentrated sample is required (Hughes et al., 1980; Higgins et al., 1984; Flotard, 19801. Liquid-liquid extraction is suitable when small sam- ple volumes are needed (Alfheim et al., 1984; Boparai et al., 19831. Dichloro-

82 COMPLEX MIXTURES methane and diethyl ether are commonly used for complex mixtures in aqueous samples (Maruoka and Yamanaka, 1980; van Hoof and Verheyden, 1981; Kringstad et al., 19811. Impunties or preservatives in the solvent should be noted, lest Key be concentrated with the sample components and cause inap- propnate interpretations of sample components. For example, peroxides and cyclohexene are present in ether and dichloromethane, respectively. Diethyl ether extraction of aqueous samples has been extensively used. Many solvents (methanol, dichloromethane, acetone, and dimethyl sulfoxide) have been used to extract and elute adsorbed material from particulate matter and analyses adsorbed on sorbent materials (Kool et al., 1981a,b; Epler, 1980; Chnsp and Fisher, 19801. Mixtures of solvents or sequential solvents with increasing po- lanty have also been used to elute analyses. The matrix of the sample limits both the choice of solvent and the method of extraction for all mixtures. SEPARATION Two principal approaches liquid-liquid partitioning and chromatogra- phy—have been used to separate complex mixtures into chemically distinct fractions that are suitable for chemical testing or toxicity testing (Alfheim et al., 19841. These approaches are also used in fractionation for bioassay. Con- ventional methods are listed in Table4-3. Each method has a different principle of action. Fractionation of complex extracts might require a combination of these methods. Two important characteristics of fractionation methods are the accuracy and reproducibility of analyte recovery. When the recovery of mass is poor, the TABLE 4-3 Conventional Separation Methods Type Variations Materials - Liquid-liquid partitioning Chromatography Column (open), thin-layer (TLC) Adsorption Solvent-solvent; solvent-acid/base Partition Molecular exclusion Ion-exchange High-performance liquid (HPLC) Adsorption Partition Molecular exclusion Ion-exchange Organic solvents of different polarity; aqueous acid/base solutions Aluminum, silica gel, polyamide, Florisil Cellulose Porous polymers, gels Ion-exchange resins (anionic, cationic) Aluminum, silica gel, polyamide, Florisil Cellulose, bonded phases Porous polymers,.gels Ion-exchange resins (anionic, cationic)

SAMPLING AND CHEMICAL CHARACTERIZATION 83 method should obviously be abandoned. The recovery of toxic activity, how- ever, is more complicated to interpret (Alfheim et al., 19841. There might tee a lack of additivity in a fractionated complex mixture, because of the loss of toxic analyses, because chemical reactions transform toxic analyses to nontoxic sub- stances (or vice versa), or because of the nature of the assay itself. The toxic activity of a reconstituted mixture (a sample made by combining the individual fractions) is often compared with that of the original complex mixture (Schoeny et al., 19861. The sum of the toxic activities of the individual frac- tions might reveal effects of interactions, such as additivity and antagonism, between substances (Alfheim et al., 19841. Thus, it is important that both reproducibility and precision tee established for the fractionation scheme. Spill- over of an analyte into various fractions must be minimal, so that toxicity-test results are not obscured. Liquid-Liquid Partitioning This method has been applied to particulate organic matter (Daisey et al., 1980; Pellizzari et al., 1978), fly ash (Chrisp and Fisher, 1980), coal gasifica- tion and liquefaction products (Schoeny et al., 1980), and water samples (Kopfleret al., 19771. Acid-base fractionation has been applied to many different types of complex mixtures (Hughes et al., 1980; Pellizzari et al., 1978; Swain et al., 1969; Bock et al., 1969; Wynder and Wright, 1957; Kieret al., 19741. It has the following advantages: · It generally yields a good separation of chemical classes. · It yields knowledge of chemical classes expected in various fractions, because the principles of the method are well understood and can be correlated with the physical and chemical properties of compounds. · It is applicable to small and large samples. · It can be applied to a wide variety of sample matrices and to organic extracts, whether lipophilic or hydrophilic. Liquid-liquid partitioning is very useful for making preliminary assessments of toxicity data (Alfheim et al., 1984) . Because some chemical knowledge can be inferred from the fractions derived by liquid-liquid partitioning, toxic activ- ity in a fraction can be correlated with general chemical-class functionalities (Ho et al., 1980; Later et al., 1981; Later and Lee, 1983; Toste et al., 1982; Wright et al., 1985). The disadvantage of liquid-liquid partitioning is that the process does not clearly define the chemical classes in each solvent used; instead, spillover often occurs between the individual fractions (i.e., an analyte appears in more than one fraction). Several investigators have observed this problem (Moller and Alfheim, 1983; Teranishi et al., 19781. In addition, the analyst should be

84 COMPLEX MIXTURES cognizant of potential chemical reactions (e.g., hydrolysis, dehydrohalogena- tion, elimination, and oxidation) when a complex mixture is in contact with acids and bases. Such reactions might alter the bioactive compounds present in the original sample or even create new toxic compounds. Reports on extracts of particles from various sources have indicated that such reactions can occur (Teranishi et al., 1978) . Practical disadvantages are that the procedure is gener- ally laborious and that material can easily be lost by adsorption or volatilization when solutions are transferred from one container to another. Several liquid-liquid partitioning schemes have been used in combination with toxicity testing to avoid the use of acids and bases (Dehnen et al., 1977; Hoffmann et al., 1980; Guerin et al., 1978; Chan et al., 19811. This reduces the potential for many undesired chemical reactions. Chromatographic Separation The general benefits of chromatographic separation methods lie in the vast choice available for application in the presence of different chemical and physi- cal properties. The methods should be chosen to lead to only minimal irrevers- ible chemical reactions and analyte losses. Chromatographic separations can be divided into adsorption, partitioning, molecular exclusion, and ion exchange, as shown in Table 4-3, for open- column chromatography (Browning, 1969~. In adsorption, there is competi- tion between solid and gas (gas-solid chromatography, GSC) or between solid and liquid (column, thin-layer). Partitioning involves competition between liq- uid and gas (gas-liquid chromatography, GLC) or between liquid and liquid (column, paper, thin-layer). Gel-permeation chromatography in its purest form is based on size exclusion; however, it rarely is a homogeneous process, and instead can be a mixture of size exclusion, adsorption, and partitioning. Modern forms of chromatography—such as high-performance liquid chroma- tography (HPLC), high-performance thin-layer chromatography (HPTLC), and supercritical-fluid chromatography (SFC) are improved variations of either adsorption or partitioning methods. Column Chromatography Open-bed columns of various materials have been used for over40 years. Silica gel has been used for the fractionation of air particles (Wynder and Hoffmann, 1965) and cigarette-smoke condensates (Kieret al., 1974; Lee et al., 19771. Airborne particles have also been fraction- ated on alumina (Dehnen et al., 1977; Tokiwa et al., 19771. Solvent partition- ing, Sephadex chromatography, and alumina chromatography (in the case of separations of fractions from synfuels) have been reported (Guerin et al., 1980; Hsie et al., 19801. Chromatography with alumina, Florisil, XAD-2, Ambersorb, and silica gel has been evaluated for separating organic substances from drinking water

SAMPLING AND CHEMICAL CHARACTERIZATION 85 (Chriswell et al., 19781. Because of poor recovery and the production of chem- ical-reaction artifacts of organic compounds during fractionation with silica or alumina, Florisil might tee preferred in some circumstances. Artifacts can often be minimized and better reproducibility attained when the adsorptive activity of the sorbent is controlled. It is important to define the state of activation ofthe medium. Gel-Permeation Chromatography Because of the potential for artifacts of susceptible compounds in acid-base fractionation schemes, alternate schemes with gel-permeation chromatography (GPC) have been developed (Guerin et al., 1978; Epler et al., 19781. This technique has been very beneficial for separating PAHs. Molecular size, shape, and polarity are major influences on the separation of PAHs (Wilk et al., 1966; Edstrom and Petro, 1968; Oelert, 1969; Vithayathil et al., 19781. GPC methods are believed to involve hydrophobic-hydrophilic partitioning, molecular size separation, and aliphatic-aromatic separation. Basic fractions can also be separated with Sephadex chromatography (Guerin et al., 19781. Serum extracts of fly ash have been subjected to GPC to separate protein- bound mutagens from low-molecular-weight mutagens (Chrisp et al., 19781. Thin-Layer Chromatography Thin-layer chromatography (TLC) is gener- ally considered a mild method and can be used for especially sensitive com- pounds. TLC has been applied not only to air particles, but to coal-derived heavy distillates (Wilson et al., 1980), diesel particulate extracts (Pederson and Siak, 1981; Pederson, 1983), and other complex mixtures. High-Performance Liquid Chromatography Because HPLC has the ad- vantages of higher reproducibility and shorter analysis time, it has for the most part supplanted open-column chromatography and TLC. However, HPLC and TLC are complementary techniques; HPLC can be used to measure accurately the components identified by TLC with its impressive array of specific colori- metric sprays. Both normal-phase chromatography and reverse-phase chroma- tography are widely used (Eisenberg, 1978; Kaiser et al., 1981; Rappaport et al., 1980; Later and Lee, 1983; Later et al., 1981; Toste et al., 19821. Because the mobile phase can interact with the solute at mild operating temperatures, HPLC is suitable for the analysis of nonvolatile and thermally labile com- pounds (Ahuja, 19841. Nonetheless, achieving good resolution of these types of polar analyses with HPLC is still a problem. This is a concern, because the polar fraction is often also toxic. High-Resolution Techniques High- and ultrahigh-resolution techniques, currently under vigorous development, are the analytic tools of the future for resolving complex mixtures, particularly those with many components in the

86 COMPLEX MIXTURES same chemical class. High-resolution techniques are not practical for generat- ing fractions for bioassay (the quantities obtained are insufficient for most assays), so a search for causative agents generally stops with subfractions that might still be relatively complex. If toxicity is associated with subfractions that are prepared by the above methods, then high-resolution chromatography is applied in concert with chemical analysis. If all or most of the analyses in a subfraction are identified, then a predictive strategy can be applied to the indi- vidual components found to be present. High-resolution chromatography evolved with capillary gas chromatogra- phy—a technique that remained dormant for a long period, because it was difficult to prepare suitable glass capillary columns, but changed abruptly with the introduction of fused-silica capillaries (Ahuja, 19841. High-resolution gas- liquid chromatography is often used in combination with a detector that yields molecular information (e.g., mass spectrometer and Fourier-transform infra- red spectroscopy). Even though excellent separating power is attainable with capillary gas- liquid chromatography, only 10% of the millions of known compounds are amenable to this technique (Gouw et al., 19791. The unequivocal separation of all constituents in very complex chemical mixtures, such as synfuels, is rarely achieved (Later and Wright, 19841. The limitations stem from two factors analyte volatility and polarity. Chemicals with low volatility (e.g., boiling point over 350°C) or high polarity will not go through a gas-liquid chromato- graphic column. That is a serious limitation, because toxic chemicals do not have the same polarity and volatility boundaries. Clearly, the separation of analyses in complex mixtures requires exquisite resolution, if the specific chemicals responsible for toxicity in mixtures are to be assessed comprehen- sively. Two-dimensional systems, in which a portion ofthe eluent is rechroma- tographic under different conditions, have also shown promise (C. W. Wright, 1984~. Research progress has been steady in high-resolution separation techniques, and complementary methods, including computerized data analysis, have now been developed that allow the remaining chemicals to be analyzed (Demirgian, 1984; Stamoudis and Demirgian, 1985; Stamoudis, 19821. HPLC has made substantial advances in the last decade (Ishii and Takeuchi, 19841. New avenues for achieving ultrahigh resolution have been developed, and micropacked fused silica for microbore columns and long capillary columns can provide an enormous number of theoretical plates (that is, a num- ber of degrees of separation equivalent to many simple distillations) and high resolution that was not possible a few years ago (Ishii and Takeuchi, 19841. Analytes with widely differing polarity can be separated on high-resolution micropacked fused-silica columns in combination with gradient elusion. Mi- crodetectors have also been designed—a requisite in combination with high

SAMPLING AND CHEMICAL CHARACTERIZATION 87 resolution. HPLC is more versatile than GLC, but has been difficult to use with molecular detection systems. Supercritical-fluid chromatography (SFC) takes advantage of the special properties of substances that have been compressed beyond and heated above their critical pressures and temperatures (Eckert et al., 19861. This emerging technique provides yet another dimension to ultrahigh-resolution chromatogra- phy and permits resolution of polar chemicals that is not possible with GLC or, in some cases, even with HPLC (Randall, 1984; Novotny, 19851. Because of high solute diffusitivity, lower viscosity, and excellent solvating properties, high resolution can be attained. Because the supercritical fluid used as the mobile phase is a hybrid with some characteristics of both gases and liquids (n- pentane, carbon dioxide, and nitrous oxide), it can be considered as a combina- tion of and complementary to gas chromatography (GC) and HPLC (Randall, 19841. High chromatographic efficiencies and fast analysis, compared with those of HPLC, are predominant characteristics. Nonvolatile and high-molec- ular-weight chemicals can be separated at relatively low temperatures with efficiencies approaching those of GLC. Relatively polar analyses can be sepa- rated effectively (Randall, 19841. Current results indicate its great potential for separation of complex mixtures (Randall, 1984; Chess and Smith, 1984; Smith et al., 1984; Wright et al., 19841. The high resolution obtainable and the ability to couple capillary SFC to most of the conventional HPLC and GO detectors make this technique very versatile. Detectors that have been successfully cou- pled to SFC include ultraviolet-absorption, fluorescence, flame-ionization, and nitrogen thermoionic detectors, as well as mass spectrometry. The avail- able methods of separating complex mixtures are HPTLC, SFC, field-flow fractionation, electrophoresis, electroosmosis, and isoelectric focusing. When choosing a chromatographic technique for separating complex mixtures, the investigator must consider analyte volatility and thermal stability, chromato- graphic efficiency, and speed of analysis. Development of a strategy, however, has not been explicitly standardized. CHEMICAL ANALYSES An extensive repertoire of analytic techniques (Table 4-4) are available for structural elucidation of organic molecules. The terms "spectroscopy" and "spectrometry" encompass a group of techniques that differ widely in their mode of application and in the information they reveal about chemical struc- ture (Buchanan et al., 1983~. A comprehensive strategy has not been devised for applying spectroscopic techniques to the analysis of organic substances and complex mixtures. The approach selected generally is targeted to obtain infor- mation about a specific portion of a mixture, but a comprehensive strategy would be useful in extracting the relevant molecular information that can be

88 COMPLEX MIXTURES TABLE 4-4 Conventional Instrumental Techniques for Chemical Analysis Instrumental Technique Ultraviolet and visible spectroscopy Vibrational spectroscopy (infrared and Raman) Information Content Conjugation Selected References Brown, 1980; Silverstein and Bassler, 1963 Borwn, 1980; Strommen and Nakamoto, 1984; Gans, 1980; Case and Fately, 1980; Clerc et al., 1981 Penzer, 1980 Chemical functionalities and assembly of atoms Molecular emission spectroscopy Mass spectrometer Nuclear magnetic resonance spectrometer Atomic absorption spectroscopy and inductively coupled argon . . . plasma emission Electron paramagnetic spin resonance Selective detection Molecular weight, chemical functionalities, overall structure, and assembly of atoms Anangement of atoms in molecule Elements Free-radical formation Brown, 1980 Watson and Throck, 1985; Wilson et al., 1981; Smith and Udseth, 1983 Jones, 1980; Jackrnan and Sternhell, 1969 Wood, 1980; Cantle, 1982 used to identify the chemicals responsible for toxicity. Extreme caution should be exercised when one is comparing chemical data bases derived from different complex mixtures, because the separation techniques introduce uncertainty as to whether structural elucidation is unequivocal or merely postulative or tentative. For structural analysis, these modern methods are used most efficiently if they are combined, because they can provide complementary information that increases their overall effectiveness. Guidelines for the combined application of spectroscopic methods are available (Clerc et al., 1981; Silverstein and Bassler, 19631. The most generally applied methods for the characterization of organic substances are ultraviolet spectroscopy, infrared spectroscopy, proton and carbon-13 nuclear magnetic resonance, and mass spectrometer (Clerc et al., 19811. Specialized techniques such as magnetic resonance of other nu- clei, Raman spectroscopy, and optical rotary dispersion are used less often. Gas chromatography/Fourier-transform infrared (GC/FT-IR) spectroscopy is now being developed for routine use in identifying analyses in complex mix- tures with the aid of software programs for the automatic interpretation of spectra (Growths, 1980; Gurka, 1985~. Infrared, Raman, visible, ultraviolet, electron-spin resonance, and magnetic circular dichroism spectroscopies have all been used in conjunction with matrix isolation (Barnes and Orville-Thomas, 19801. Matrix isolation traps isolated molecules of the species to be studied in a

SAMPLING AND CHEMICAL CHARACTERIZATION 89 large excess of an inert material by rapid condensation at a low temperature, so that the diluent forms a rigid cage or matrix (Barnes and Orville-Thomas, 1980~. The noble gases (primarily argon) and nitrogen are most widely used for matrix materials. More recent uses of matrix isolation have been in combina- tion with GC effluents in which high-resolution gas chromatography (HRGC) has been coupled with FT-IR spectroscopy. Several HPLC/FT-IR systems have been described (Growths, 19801. The exquisite sensitivity of GC/FT-IR with matrix isolation rivals that of mass spectrometer; these two methods can also be combined to provide simultaneous and complementary information during chromatography of a complex mixture. The gas chromatograph is the most commonly used inlet system for analysis of complex mixtures with mass spectrometry (Watson and Throck, 19851. It is rarely possible to isolate in pure form each of the hundreds of individual sub- stances present. However, HRGC combined with mass spectrometer (GC/ MS) can often serve as the final purification step by resolving the various components and presenting them one at a time to the mass spectrometer. The mass spectrometer can also serve as a universal detector for SFC and HPLC (Smith and Udseth, 19831. Several types of devices are under develop- ment (e.g., moving wire or moving belt, direct inlet, and thermospray) to introduce relatively nonvolatile materials after separation into the ion source of the mass spectrometer. A fast-atom-bombardment (FAB) ionization technique combined with a moving belt exploits the attributes of FAB ionization of non- volatile analyses and allows analysis in a manner very similar to that of GC/MS (Smith and Udseth, 19831. An alternative to resolving components chromatographically is separating them by mass spectrometric methods the so-called MS/MS methods (Watson and Throck, 1985; Smith and Udseth, 1983; Henderson et al., 1982, 1983, 19841. The complex mixture is ionized, usually gently, to produce charactens- tic ions from each component. The ions suspected to arise from the component of interest are selected by mass analysis in the first stage of a double mass spectrometer, and their identity is confirmed by mass analysis in the second stage on the basis of the fragment ions arising from collision-induced associa- tion of the selected ions. REFERENCES Ahuja, S. 1984. Overview: Multiple pathways to ultrahigh resolution chromatography, pp. 1-8. In S. Ahuja (ed.). Ultrahigh Resolution Chromatography. ACS Symposium Series 250. American Chemical Society, Washington, D.C. Alfheim, I., G. Lofroth, and M. Molter. 1983. Bioassay of extracts of ambient particulate matter. Environ. Health Perspect. 47:227-238. Alfheim, I., A. Bjorseth, and M. Molter. 1984. Charactenzation of microbial mutagens in complex samples—methodology and application. CRC Crit. Rev. Environ. Control 14:91-150.

9o COMPLEX MIXTURES Baker, R. A. 1970. Trace organic contaminant concentration by freezing. IV: Ionic effects. Water Res. 4:559-573. Barnes, A. J., and W. J. Orville-Thomas. 1980. FT-IR matrix isolation studies, pp. 157-170. In J. R. Durig (ed.). Analytical Applications of FT-IR to Molecular and Biological Systems. D. Reidal, Dordrecht, Holland. (607 pp.) Barnhart, B. J., and S. H. Cox. 1980. Mutation of Chinese hamster cells by near-UV activation of promutagens. Mutat. Res. 72:135-142. Benson, J. M., R. L. Hanson, R. E. Royer, C. R. Clark, and R. F. Henderson. 1984. Toxicological and chemical characterization of the process stream materials and gas combustion products of an experi- mental low-Btu coal gasifies. Environ. Res. 33:396-412. Berkley, R. E., and E. D. Pellizzari. 1978. Evaluation of Tenax GC sorbent for in situ formation of N-nitrosodimethylamine. Anal. Lett. 4:327-346. Bock, P. G., A. P. Swain, and R. L. Stedman. 1969. Bioassay of major fractions of cigarette smoke condensate by an accelerated technic. Cancer Res. 29:584-587. Boparai, A. S., D. A. Haugen, K. M, Suhrbier, and J. F. Schneider. 1983. An improved procedure for extraction of aromatic bases from synfuel materials, pp. 3-11. In C. W. Wright, W. C. Weimer, and W. D. Felix (eds.). Advanced Techniques in Synthetic Fuels Analysis. PNL-SA-11552. CONF- 811160. U.S. Department of Energy, Technical Information Center, Oak Ridge, Tenn. (Available from NTIS as DE83015528.) Bridbord, K., and J. G. French. 1978. Carcinogenic and mutagenic risks associated with fossil fuels, pp. 451-463. In P. W. Jones and R. I. Freudenthal (eds.). Carcinogenesis—A Comprehensive Sur- vey, Vol. 3. Polynuclear Aromatic Hydrocarbons. Raven Press, New York. Brown, K. W., K. C. Donnelly, J. C. Thomas, P. Davol, and B. R. Scott. 1985. Mutagenicity of three agricultural soils. Sci. Total Environ. 35:799-807. Brown, S. B. 1980. Introduction to spectroscopy, pp. 1-13. In S. B. Brown (ed.). An Introduction to Spectroscopy for Biochemists. Academic Press, New York. Browning, D. R. 1969. Chromatography. McGraw-Hill, New York. (151 pp.) Bn~sick, D. J., and R. R. Young. 1982. IERL-RTP Procedures Manual: Level 1 Environmental As- sessment, Biological Tests. EPA 600/S8-81-024. Office of Research and Development, U.S. Envi- ronmental Protection Agency, Washington, D.C. (8 pp.) Buchanan, M. V., M. R. Guerin, G. L. Kao, I. B. Rubin, and J. E. Caton. 1983. Comparative spectro- scopic characterization of synthetic fuels, pp. 286-298. In C. W. Wright, W. C. Weimer, and W. D. Pelix (eds.). Advanced Techniques in Synthetic Fuels Analysis. PNL-SA-11552. CONF-811160. U.S. Department of Energy, Technical Information Center, Oak Ridge, Tenn. (Available from NTIS as DE83015528.) Burke, F. P., R. A. Winschel, and G. A. Robbins. 1984. Recycle Slurry Oil Characterization: Third Annual Report. DOE/PC 30027-56. Conoco Inc., Coal Research Division, Library, Pa. Burton, F. G., R. E. Schirmer, D. D. Mahlum, and F. D. Andrew. 1979. Disposition of process solvent from solvent refined coal in tissues of the rat after oral dosing. Abstract No. 54. Toxicol. Appl. Pharmacol. 48:A27. Cantle, J. E. 1982. Atomic Absorption Spectrometry. Elsevier, New York. (448 pp.) Carey, A. E., and J. A. Gowen. 1978. PCB's in Agricultural and Urban Soil. U.S. Environmental Protection Agency, Washington, D.C. (Available from NTIS as PB-276 315t9.) (4 pp.) Case, J. C., and W. G. Fately. 1980. One view of the advantages of infrared interferometry, pp. 3-10. In J. R. Durig (ed.). Analytical Applications of FT-IR to Molecular and Biological System. D. Reidel, Dordrecht, Holland. (607 pp.) Cerniglia, C. E. 1981. Aromatic hydrocarbons: Metabolism by bacteria, fungi, and algae, pp. 321- 361. In E. Hodgson, J. R. Bend, and R. M. Philpot (eds.). Reviews in Biochemical Toxicology. Vol. 3. Elsevier, New York. Chan, T. L., P. S. Lee, and J.-S. Siak. 1981. Diesel-particulate collection for biological testing: Comparison of electrostatic precipitation and filtration. Environ. Sci. Technol. 15: 89-93.

SAMPLING AND CHEMICAL CHARACTERIZATION 91 Cheng, Y.-S., and H.-C. Yeh. 1979. Particle bounce in cascade impactors. Environ. Sci. Technol. 13: 1392-1396. Cheng, Y.-S., R. L. Hanson, R. L. Carpenter, C. H. Hobbs. 1984. Use of a massive volume air sampler to collect fly ash for biological characterization. J. Air Pollut. Control Assoc. 34:671-674. Chess, E. K., and R. D. Smith. 1984. Development and Evaluation of Supercritical Fluid Chromatog- raphy/Mass Spectrometry for Polar and High Molecular Weight Coal Components. PNL-SA-12298. CONF-840694-9. Battelle Pacific Northwest Labs, Richland, Wash. (Available from NTIS as DE850008271XAB.) (8 pp.) Chrisp, C. E., and G. L. Fisher. 1980. Mutagenicity of airborne particles. Mutat. Res. 76: 143-164. Chrisp, C. E., G. L. Fisher, and J. E. Lammert. 1978. Mutagenicity of filtrates from respirable coal fly ash. Science 199:73-75. Chriswell, C. D., B. A. Glatz, J. S. Fritz, and H. J. Svec. 1978. Mutagenic analysis of drinking water, pp. 477-494. In M. D. Waters, S. Nesnow, J. F. Huisingh, S. S. Sandhu, and L. Claxton (eds.). Application of Short-Term Bioassays in the Fractionation and Analysis of Complex Environmental Mixtures. Plenum, New York. Cifrulak, S. D. 1969. Spectroscopic evidence of phthalates in soil organic matter. Soil Sci. 107:63-69. Clark, C. R., T. J. Truex, F. S. C. Lee, and I. T. Salmeen. 1981. Influence of sampling filter type on the mutagenicity of diesel exhaust particulate extracts. Atmos. Environ. 15:397-402. Claxton, L. D. 1982. Review of fractionation and bioassay characterization techniques for the evalua- tion of organics associated with ambient air particles, pp. 19-34. In R. R. Tice, D. L. Costa, and K. M. Schaich (eds.). Genotoxic Effects of Airborne Agents. Environmental Science Research. Vol. 25. Plenum, New York. Clerc, J. T., E. Pretsch, and J. Seibl. 1981. Structural Analysis of Organic Compounds by Combined Application of Spectroscopic Methods. Elsevier, New York. (288 pp.) Cochran, W. G. 1977. Sampling Techniques, 3rd ed. John Wiley & Sons, New York. (274 pp.) Corn, M., T. L. Montgomery, and N. A. Esmen. 1971. Suspended particulate matter: Seasonal vari- ation in specific surface areas and densities. Environ. Sci. Technol. 5: 155-158. Crathorne, B., C. D. Watts, and M. Fielding. 1979. Analysis of non-volatile organic compounds in water by high-performance liquid chromatography. J. Chromatography 185:571 -690. Daisey, J. M., T. J. Kneip, I. Hawryluk, and F. Mukai. 1980. Seasonal variations in the bacterial mutagenicity of airborne particulate organic matter in New York City. Environ. Sci. Technol. 14: 1487-1490. Dawson, R., and K. Mopper. 1978. A note on the losses of monosaccharides, amino sugars, and amino acids from extracts during concentration procedures. Anal. Biochem. 84: 186-190. Dehnen, W., N. Pitz, and R. Tomingas. 1977. The mutagenicity of airborne particulate pollutants. Cancer Lett. 4:5-12. Demirgian, J. 1984. Computerized rapid analysis of complex mixtures by gas chromatography. J. Chromatogr. Sci. 22:153-160. De Wiest, F., and D. Rondia. 1976. Sur la validite des determinations du Benzo (a) pyrene atmosphe- rique pendant les mods d~ete. Atmos. Environ. 10:487~89. (English abstract.) Eckert, C. A., J. G. Van Alsten, and T. Stoicos. 1986. Supercritical fluid processing. Environ. Sci. Technol. 20:319-325. Edstrom, T., and B. A. Petrol 1968. Gel permeation chromatographic studies of polynuclear aromatic hydrocarbon materials. J. Polym. Sci. Part C (21): 171-182. Eisenberg, W. C. 1978. Fractionation of organic material extracted from suspended air particulate matter using high pressure liquid chromatography. J. Chromatogr. Sci. 16: 145- 151. Epler, J. L. 1980. The use of short-term tests in the isolation and identification of chemical mutagens in complex mixtures, pp. 239-270. In P. J. de Serres and A. Hollaender (eds.). Chemical Mutagens: Principles and Methods for Their Detection, Vol. 6. Plenum, New York. Epler, J. L., B. R. Clark, C.-H. Ho, M. R. Guerin, and T. K. Rao. 1978. Short-term bioassay of complex organic mixtures: Part II, mutagenicity testing, pp. 269-289. In M. D. Waters, S. Nesnow,

92 COMPLEX MIXTURES J. L. Huisingh, S. S. San&u, and L. Claxton (eds.). Application of Short-Term Bioassays in the Fractionation and Analysis of Complex Environmental Mixtures. Plenum, New York. Fitz, D. R., G. J. Doyle, and J. N. Pitts, Jr. 1983. An ultrahigh volume sampler for the multiple filter collection of respirable particulate matter. J. Air Pollut. Control Assoc. 33: 877-879. Flessel, P., G. Guirguis, J. Cheng, K. Chang, and E. Hahn. 1984. Monitoring of Mutagens and Carcinogens in Community Air. ARB-R-84/223. California State Air Resources Board, Sacramento, Calif. (Available from NTIS as PB85-173763/XAB.) (134 pp.) Florin, I., L. Rutberg, M. Curvall, and C. R. Enzell. 1980. Screening of tobacco smoke constituents for mutagenicity using the Ames' test. Toxicology 15:219-232. Flotard, R. D. 1980. Sampling and Analysis of Trace-Organic Constituents in Ambient and Workplace Air at Coal-Conversion Facilities. Argonne National Laboratory, Argonne, Ill. (Available from NTIS as ANL/PAG-3.) (51 pp.) Gans, P. 1980. Vibrational spectroscopy, pp. 115-147. In S. B. Brown (ed.). An Introduction to Spectroscopy for Biochemists. Academic Press, New York. Gilbert, R. O. 1983. Field sampling designs, simple random and stratified random sampling. TRANS- STAT Statistics for Environmental Studies, No. 24. PNL-SA-11551. Battelle Pacific Northwest Labs, Richland, Wash. (Available from NTIS as DE83016826.) (38 pp.) Gilbert, R. O. 1984. Field sampling designs: Systematic sampling. TRANS-STAT Statistics for Environmental Studies. No. 26. PNL-SA-12180. Battelle Pacific Northwest Labs, Richland, Wash. (32 pp ) Goggelmann, W., and P. Spitzauer. 1982. Mutagenic activity, content of polycyclic aromatic hydro- cargons (PAM) and humus in agricultural soils. Abstract No. 50. Mutat. Res. 97: 189-190. Gorse, R. A., Jr., I. T. Salmeen, and C. R. Clark. 1982. Effects of filter loading and filter type on the mutagenicity and composition of diesel exhaust particulate extracts. Atmos. Environ. 16: 1523- 1528. Gouw, T. H., R. E. Jentoft, and E. J. Gallegos. 1979. Some recent advances in supercritical fluid chromatography, pp. 583-592. In K. D. Timmerhaus and M. S. Barber (eds.). High-Pressure Sci- ence and Technology, Vol. 1: Physical Properties and Material Synthesis. Plenum, New York. (583 pp.) Griest, W. H., C. E. Higgins, R. W. Holmberg, J. H. Moneyhun, J. E. Caton, J. S. Wike, and R. R. Reagen. 1982. Characterization of vapor- and particulate-phase organics from ambient air sampling at the Kosovo gasifier, pp. 395-410. In L. H. Keith (ed.). Energy and Environmental Chemistry, Vol. 1. Ann Arbor Science, Ann Arbor, Mich. Griff~ths, P. R. 1980. Chromatography and PT-IR spectrometry, pp. 149-155. In J. R. Durig (ed.). Analytical Applications of FT-IR to Molecular and Biological Systems. D. Reidal, Dordrecht, Hol- land. (607 pp.) Guerin, M. R. 1981. The integrated approach to chemical-biological analysis, pp. 1-16. In J. C. Harris, P. L. Levins, and K. D. Drewitz (eds.). Proceedings, 2nd Symposium on Process Measure- ments for environmental Assessment, February 25-27, Atlanta, Gal, 1980. Industrial Environmen- tal Research Lab., Research Triangle Park, N.C. (Available from NTIS as PB82-211574.) Guerin, M. R., B. R. Clark, C.-H. Ho, J. L. Epler, and T. K. Rao. 1978. Short-term bioassay of complex organic mixtures: Part I, chemistIy, pp. 247-268. In M. D. Waters, S. Nesnow, J. L. Huisingh, S. S. San&u, and L. Claxton (eds.). Application of Short-Term Bioassays in the Fraction- ation and Analysis of Complex Environmental Mixtures. Plenum, New York. Guerin, M. R., C.-H. Ho, T. K. Rao, B. R. Clark, and J. L. Epler. 1980. Separation and identification of mutagenic constituents of petroleum substitutes. Int. J. Environ. Anal. Chem. 8:217-225. Guerin, M. R., J. Dutcher, E. S. Olson, E. J. Peterson, V. C. Stamoudis, D. H. Stuermer, and B. W. Wilson. 1983. Summary of chemical characterization methodologies with future directives, pp. 263-266. In C. W. Wright, W. C. Weimer, and W. D. Felix (eds.). Advanced Techniques in Syn- thetic Fuels Analysis. PNL-SA-11552. CONF-811160. U.S. Department of Energy, Technical In- formation Center, Oak Ridge, Tenn. (Available from NTIS as DE83015528.)

SAMPLING AND CHEMICAL CHARACTERIZATION 93 Gurka, D. F. 1985. Interim protocol for the automated analysis of semivolatile organic compounds by gas chromatography/Fourier transform infrared (GC/FT-IR) spectrometry. Appl. Spectrosc. 39:827-833. Hackett, P. L., R. L. Music, D. D. Mahlum, and M. R. Sikov. 1983. Developmental effects of oral administration of solvent refined coal materials to rats. (Abstract.) Teratology 27:47A. Hanson, R. L., R. L. Carpenter, and G. J. Newton. 1980. Chemical characterization of polynuclear aromatic hydrocarbons in airborne effluents from an experimental fluidized bed combustor, pp. 599- 616. In A. Bjorseth and A. J. Dennis (eds.). Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects. Battelle Press, Columbus, Ohio. Hanson, R. L., C. R. Clark, R. L. Carpenter, and C. H. Hobbs. 1981. Evaluation of Tenax-GC and XAD-2 as polymer absorbents for sampling fossil fuel combustion products containing nitrogen oxides. Environ. Sci. Technol. 15:701-705. Hanson, R. L., R. E. Royer, J. M. Benson, R. L. Carpenter, G. J. Newton and R. F. Henderson. 1982. Chemical fractionation and analysis of organic compounds in process streams of low Btu gasifies effluents, pp. 205-223. In E. L. Fuller, Jr. (ed.). Coal and Coal Products: Analytical Characteriza- tion Techniques. Polynuclear Aromatic Hydrocarbons. ACS Symposium Series 205. American Chemical Society, Washington, D.C. Hanson, R. L., C. R. Clark, R. L. Carpenter, and C. H. Hobbs. 1984. Comparison of Tenax-GC and XAD-2 as polymer absorbents for sampling combustion exhaust gases, pp. 79-93. In L. H. Keith (ed.). Identification and Analysis of Organic Pollutants in Air. Butterworth, Boston, Mass. Hanson, R. L., A. R. Dahl, S. J. Rothenberg, J. M. Benson, A. L. Brooks, and J. S. Dutcher. 1985. Chemical and biological characterization of volatile components of environmental samples after fractionation by vacuum line cryogenic distillation. Arch. Environ. Contam. Toxicol. 14:289-297. Haugen, D. A., and V. C. Stamoudis. 1986. Isolation and identification of mutagenic polycyclic aromatic hydrocarbons from a coal gasifies condensate. Environ. Res. 41 :400-419. Henderson, T. R., R. E. Royer, C. R. Clark, T. M. Harvey, and D. F. Hunt. 1982. MS/MS of diesel emissions and fuels treated with NO2. J. Appl. Toxicol. 2:231-237. Henderson, T. R., J. D. Sun, R. E. Royer, C. R. Clark, A. P. Li, T. M. Harvey, D. H. Hunt, J. E. Fulford, A. M. Lovette, and W. R. Davidson. 1983. Triple quadrupole mass spectrometry studies of nitroaromatic emissions from different diesel engines. Environ. Sci. Technol. 17:443-449. Henderson, T. R., J. D. Sun, A. P. Li, R. L. Hanson, W. E. Bechtold, T. M. Harvey, J. Shabanowitz, and D. F. Hunt. 1984. GC/MS and MS/MS studies of diesel exhaust mutagenecity and emissions from chemically defined fuels. Environ. Sci. Technol. 18:428-434. Henry, W. M., R. I. Mitchell, and R. J. Thompson. 1978. Development of a Large Sampler Collector of Respirable Particulate Matter. EPA-600/4-78/009. Battelle Columbus Labs, Columbus, Ohio. (Available from NTIS as PB-281 528/0.) (54 pp.) Higgins, C. E., W. H. Griest, and G. Olerich. 1983. Application of Tenax trapping to analysis of gas phase organic compounds in ultra-low tar cigarette smoke. J. Assoc. Off. Anal. Chem. 66: 1074- 1083. Higgins, C. E., W. H. Griest, and M. R. Guerin. 1984. Sampling and Analysis of Cigarette Smoke Using the Solid Adsorbent Tenax. ORNL/TM-9167. Oak Ridge National Lab, Oak Ridge, Tenn. (Available from NTIS as DE 84012025.) (25 pp.) Hite, M., and H. Skeggs. 1979. Mutagenic evaluation of nitroparaffins in Salmonella typhimurium/ mammalian-microsome test and the micronucleus test. Environ. Mutagen. 1 :383-389. Ho, C.-H., C. Y. Ma, B. R. Clark, M. R. Guerin, T. K. Rao, and J. L. Epler. 1980. Separation of neutral nitrogen compounds from synthetic crude oils for biological testing. Environ. Res. 22: 412-422. Hoffmann, D., K. No~poth, R. H. Wickramasinghe, and G. Muller. 1980. The detection of mutagenic air pollutants from filter samples by the salmonella/mammalian S-9 mutagenicity test^ (Ames test) with S. typhimurium TA98 (Part 1). Zentralbl. Bakteriol. Mikrobiol. Hyg. 1 Abt., Orig. B 171: 388-407.

94 COMPLEX MIXTURES Hsie, A. W., P. A. Brimer, J. P. O'Neill, J. L. Epler, M. R. Guerin, and M. H. Hsie. 1980. Mutagenic- ity of alkaline constituents of a coal-liquefied crude oil in mammalian cells. Mutat. Res. 78:79-84. Hughes, T. J., E. Pellizzari, L. Little, C. Sparacino, and A. Kolber. 1980. Ambient air pollutants: Collection, chemical characterization and mutagenicity testing. Mutat. Res. 76:51-83. ISO (International Organization for Standardization). 1983. Air Quality—Particle Size Fraction Defi- nitions for Health-Related Sampling. ISO/TR 7708-1983. International Organization for Standard- ization, Geneva. (13 pp.) Ishii, D., and T. Takeuchi. 1984. Application of micro high performance liquid chromatography to the separation of complex mixtures, pp. 109-120. In S. Ahuja (ed.). Ultrahigh Resolution Chromatog- raphy. ACS Symposium Series 250. American Chemical Society, Washington, D.C. Jackman, L. M., and S. Sternhell. 1969. Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry. 2nd ed. Pergamon, New York. (456 pp.) Jensen, V. 1975. Bacterial flora of soil after application of oily waste. Oikos 26: 152-158. Jobson, A., M. McLaughlin, F. D. Cook, and D. W. S. Westlake. 1974. Effect of amendments on the microbial utilization of oil applied to soil. Appl. Microbiol. 27: 166-171. Jolley, R. L. 1981. Concentrating organics in water for biological testing. Environ. Sci. Technol. 15:874-880. Jolley, R. L., S. Katz, and J. E. Mrochek. 1975. Analyzing organics in complex, dilute aqueous solutions. Chem. Technol. 5:312-318. Jones, R. 1980. Nuclear magnetic resonance, pp. 235-278. In S. B. Brown (ed.). An Introduction to Spectroscopy for Biochemists. Academic Press, New York. Kaiser, C., A. Kerr, D. R. McCalla, J. N. Lockington, and E. S. Gibson. 1981. Use of bacterial mutagenicity assays to probe steel foundry lung cancer hazard, pp. 583-592. In M. Cooke and A. J. Dennis (eds.). Chemical Analysis and Biological Fate: Polynuclear Aromatic Hydrocarbons. Bat- telle Press, Columbus, Ohio. Kalkwarf, D. R., D. L. Steward, R. A. Pelroy, and W. C. Weimer. 1984. Photodegradation of Muta- gens in Solvent-Refined Coal Liquids. PNL-4982. Battelle Pacific Northwest Labs, Richland, Wash. (Available from NTIS as DE84010760.) (46 pp.) Kelman, B. J., and D. L. Springer. 1981. Fetal exposure to benzo[a]pyrene across the hemochorial placenta, pp.387-397. In D. D. Mahlum, R. H. Gray, and W. D. Felix (eds.). Coal Conversion and the Environment: Chemical, Biomedical, and Ecological Considerations. Proceedings of the Twen- tieth Hanford Life Sciences Symposium at Richland, Wash., October 19-23, 1980. CONF-801039. U.S. Department of Energy, Technical Information Center, Oak Ridge, Tenn. (Available from Na- tional Technical Information Sen~ice as DE82000105.) Kier, L. D., E. Yamasaki, and B. N. Ames. 1974. Detection of mutagenic activity in cigarette smoke condensates. Proc. Nat. Acad. Sci. U.S.A. 71:4159~163. Kinae, N., T. Hashizume, T. Makita, I. Tomita, and I. Kimura. 1981. Kraft pulp mill effluent and sediment can retard development and lyse sea urchin eggs. Bull. Environ. Contam. Toxicol. 27: 616-623. Kool, H. J., C. F. van Keijl, H. J. van Kranen, andE. deGreef. 1981a. The use ofXAD-resins for the detection of mutagenic activity in water. I. Studies with surface water. Chemosphere 10:85-98. Kool, H. J., C. F. van Keijl, H. J. van Kranen, and E. de Greef. 198 lb. The use of XAD-resins for the detection of mutagenic activity in water. II. Studies with drinking water. Chemosphere 10:99-108. Kopfler, F. C. 1980. Alternative strategies and methods for concentrating chemicals from water, pp. 141-153. In M. D. Waters, S. S. Sandhu, J. L. Huisingh, L. Claxton, and S. Nesnow (eds.). Short- Term Bioassays in the Analysis of Complex Environmental Mixtures. II. Plenum, New York. Kopfler, F. C., W. E. Coleman, R. G. Melton, R. G. Tardiff, S. C. Lynch, and J. K. Smith. 1977. Extraction and identification of organic micropollutants: Reverse osmosis method. Ann. N.Y. Acad. Sci. 298:20-30. Kringstad, K. P., P. O. Ljungquist, F. de Souse, end e. M. Stromberg. 1981. Identificationandmuta-

SAMPLING AND CHEMICAL CHARACTERIZATION 95 genie properties of some chlorinated aliphatic compounds in the spent liquor from kraft pulp chlorin- ation. Environ. Sci. Technol. 15:562-566. Krost, K. J., E. D. Pellizzari, S. G. Walburn, and S. A. Hubbard. 1982. Collection and analysis of hazardous organic emissions. Anal. Chem. 54:810-817. Larson, R. A., L. L. Hunt, and D. W. Blankenship. 1977. Formation of toxic products from a #2 fuel oil by photooxidation. Environ. Sci. Technol. 11 :492-496. Later, D. W., and M. L. Lee. 1983. Chromatographic methods for the chemical and biological charac- terization of polycyclic aromatic compounds in synfuel materials, pp. 44-73. In C. W. Wright, W. C. Weimer, and W. D. Felix (eds.). Advanced Techniques in Synthetic Fuels Analysis. PNL-SA- 11552. CONF-811160. U.S. Department of Energy, Technical Information Center, Oak Ridge, Tenn. (Available from NTIS as DE83015528.) Later, D. W., and B. W. Wright. 1984. Capillary column gas chromatographic separation of amino polycyclic aromatic hydrocarbon isomers. J. Chromatography 289:183-193. Later, D. W., M. L. Lee, K. D. Bartle, R. C. Kong, and D. L. Vassilaros. 1981. Chemical class separation and characterization of organic compounds in synthetic fuels. Anal. Chem. 53:1612- 1620. Later, D. W., T. G. Andros, and M. L. Lee. 1983a. Isolation and identification of amino polycyclic aromatic hydrocarbons from coal-derived products. Anal. Chem. 55:2126-2132. Later, D. W., C. W. Wright, and B. W. Wilson. 1983b. The analytical chemistry of products from process strategies designed to reduce the biological activity of direct coal liquefaction materials. Am. Chem. Soc., Div. Fuel Chem. Preprints 28(5):273-284. Lee, P. N., K. Rothwell, and J. K. Whitehead. 1977. Fractionation of mouse skin carcinogens in cigarette smoke condensate. Br. J. Cancer 35:730-742. Lentzen, D. W., D. E. Wagoner, E. D. Estes, and W. F. Gutknecht. 1978. IERL-RTP Procedures Manual: Level 1 Environmental Assessment. 2nd ed. EPA/600/7-78/201. Industrial Environmental Research Lab, Research Triangle Park, N.C. (Available from NTIS as PB-293 795/1.) (279 pp.) Lippmann, M. 1985. Development of particle size-selective threshold limit values. Ann. Am. Conf. Ind. Hyg. 12:27-34. Mahlum, D. D. 1983a. Initiation/promotion studies with coal-derived liquids. J. Appl. Toxicol. 3:31-34. Mahlum, D. D. 1983b. Skin-tumor initiation activity of coal liquids with different boiling-point ranges. J. Appl. Toxicol. 3:254-258. Mahlum, D. D., and D. L. Springer. 1986. Teratogenic response of the rat and mouse to a coal liquid after dermal administration. Abstract No. 371. Toxicologist 6(1):94. Maruoka, S., and S. Yamanaka. 1980. Production of mutagenic substances by chlorination of waters. Mutat. Res. 79:381-386. McGill, W. B., and M. J. Rowell. 1980. Determination of oil content of oil contaminated soil. Sci. Total Environ. 14:245-253. McGuire, M. J., and Suffet, I. H. 1983. Treatment of Water by Granular Activated Carbon. Advances in Chemistry Series 202. American Chemical Society, Washington, D.C. Middleton, F. M., W. Grant, and A. A. Rosen. 1956. Drinking water taste and odor: Correlation with organic chemical content. Ind. Eng. Chem. 48:268-274. Miller, F. J., D. E. Gardner, J. A. Graham, R. E. Lee, Jr., J. Bachmann, and W. E. Wilson. 1979. Particle Size Considerations for Establishing a Standard for Inhaled Particles. U.S. Environmental Protection Agency, Research Triangle Park, N.C. Modell, M., R. P. deFilippi, and V. Krukonis. 1978. Regeneration of activated carbon with supercriti- cal carbon dioxide. Presented before the American Chemical Society, Division of Environmental Chemistry, Miami, Fla. Moller, M., and I. Alfheim. 1983. Mutagenicity of air samples from various combustion sources. Mutat. Res. 116:35-46.

96 COMPLEX MIXTURES Nguyen, T. V., J. C. Theiss, and T. S. Matney. 1982. Exposure of pharmacy personnel to mutagenic antineoplastic drugs. Cancer Res. 42:4792-4796. Novotny, M. 1985. Analytical chromatography: The current situation and future directions, pp. 318- 365. In B. L. Shapiro (ed.). New Directions in Chemical Analysis. Texas A&M University Press, College Station, Tex. (502 pp.) NRC (National Research Council), Panel on Quality Criteria for Water Reuse. 1982. Quality Criteria for Water Reuse. National Academy Press, Washington, D.C. Oelert, H. H. 1969. Atypical gel chromatography in the system Sephadex-isopropanol. Z. Anal. Chem. 244:91-101. (In German; English abstract.) Ogner, G., and M. Schnitzer. 1970. The occurrence of alkanes in fulvic acid, a soil humic fraction. Geochim. Cosmochim. Acta 34:921-928. Pederson, T. C. 1983. Biologically active nitro-PAH compounds in extracts of diesel exhaust particu- late, pp. 227-245. In D. Rondia, M. Cooke, and R. K. Haroz (eds.). Mobile Source Emissions Including Polycyclic Organic Species. D. Reidel, Dordrecht, Holland. Pederson, T. C., and J.-S. Siak. 1981. The role of nitroaromatic compounds in the direct-acting mutagenicity of diesel particle extracts. J. Appl. Toxicol. 1 :54-60. Pellizzari, E. D. 1982. Analysis for organic vapor emissions near industrial and chemical waste dis- posal sites. Environ. Sci. Technol. 16:781-785. Pellizzari, E. D., and K. J. Krost. 1984. Chemical transformations during ambient air sampling for organic vapors. Anal. Chem. 56: 1813-1819. Pellizzari, E. D., J. E. Bunch, R. E. Berkley, and J. McRae. 1976. Collection and analysis of trace organic vapor pollutants in ambient atmospheres: The performance of a Tenax GC cartridge sampler for hazardous vapors. Anal. Lett. 9:45-63. Pellizzari, E. D., L. W. Little, C. Sparacino, T. J. Hughes, L. Claxton, and M. D. Waters. 1978. Integrating microbiological and chemical testing into the screening of air samples for potential muta- genicity, pp. 331-351. In M. D. Waters, S. Nesnow, J. L. Huisingh, S. S. Sandhu, and L. Claxton (eds.). Application of Short-Term Bioassays in the Fractionation and Analysis of Complex Environ- mental Mixtures. Plenum, New York. Pellizzari, E., B. Demian, and K. Krost. 1984. Sampling of organic compounds in the presence of reactive inorganic gases with Tenax GC. Anal. Chem. 56:793-798. Penzer, G. 1980. Molecular emission spectroscopy (fluorescence and phosphorescence), pp. 70-114. In S. B. Brown (ed.). An Introduction to Spectroscopy for Biochemists. Academic Press, New York. Preidecker, B. L. B. 1980. Bacterial mutagenicity of particulates from Houston air. Environ. Mutagen. 2:75-83. Randall, L. G. 1984. Carbon dioxide based supercritical fluid chromatography: Column eff~ciencies and mobile phase solvent power, pp. 135-169. In S. Ahuja (ed.). Ultrahigh Resolution Chromatog- raphy. ACS Symposium Series 250. American Chemical Society, Washington, D.C. Rappaport, S. M., Y. Y. Wang, E. T. Wei, R. Sawyer, B. E. Watkins, and H. Rapoport. 1980. Isolation and identification of a direct-acting mutagen in diesel-exhaust particulates. Environ. Sci. Technol. 14:1505-1509. Sato, T., T. Momma, Y. Ose, T. Ishikawa, and K. Kato. 1983. Mutagenicity of Nagara River sedi- ment. Mutat. Res. 118:257-267. Schoeny, R. 1985. Exploratory Research on Mutagenic Activity of Coal-Related Materials Using Statistical Evaluation. DOE/PC/62999-6. Kettering Lab, Cincinnati, Ohio. (Available from NTIS as DE85013667/XAB.) (19 pp.) ~b Schoeny, R., D. Warshawsky, and G. Moore. 1986. Non-additive mutagenic responses by compo- nents of coal-derived materials. Am. Chem. Soc., Div. Fuels Chem. Preprints 31 (2): 147- 155. Schoeny, R., D. Warshawsky, L. Hollingsworth, M. Hund, and G. Moore. 1980. Mutagenicity of coal gasification and liquefaction products, pp. 461-475. In M. D. Waters, S. S. Sandhu, J. L. Huisingh, L. Claxton, and S. Nesnow (eds.). Short-Term Bioassays in the Analysis of Complex Environmental Mixtures II. Plenum, New York.

SAMPLING AND CHEMICAL CHARACTERIZATION 97 Selby, C., J. Calkins, and H. Enoch. 1983. Detection of photomutagens in natural and synthetic fuels. Mutat. Res. 124:53-60. Shapiro, J. 1961. Freezing-out, a safe technique for concentration of dilute solutions. Science 133:2063-2064. Silverstein, R. M., and G. C. Bassler. 1963. Spectrometric Identification of Organic Compounds. John Wiley & Sons, New York (377 pp.) Smith, R. D. 1983. New approaches combining chromatography and mass spectrometry for synfuel analysis, pp. 332-352. In C. W. Wright, W. C. Weimer, and W. D. Felix (eds.). Advanced Tech- niques in Synthetic Fuels Analysis. PNL-SA-11552. CONF-811160. U.S. Department of Energy, Technical Information Center, Oak Ridge, Tenn. (Available from NTIS as DE83015528.) Smith, R. D., and H. R. Udseth. 1983. Mass spectrometry with direct fluid supercritical injection. Anal. Chem. 55:2266-2272. Smith, R. D., H. R. Udseth, and H. T. Kalinoski. 1984. Capillary supercritical fluid chromatography/ mass spectrometry with electron impact ionization. Anal. Chem. 56:2971-2973. Springer, D. L., M. L. Clark, D. H. Willard, and D. D. Mahlum. 1982. Generation and delivery of coal liquid aerosols for inhalation studies. Am. Ind. Hyg. Assoc. J. 43:486-491. Springer, D. L., R. A. Miller, W. C. Weimer, H. A. Ragan, and R. L. Buschbom. 1984. Effects of Inhalation Exposure to SRC-II Heavy and Middle Distillates. PNL-5273. Battelle Pacific Northwest Labs, Richland, Wash. (Available from NTIS as DE85004327/XAB.) (68 pp.) Stamoudis, V. C. 1982. A gas chromatographic scheme, based on relative retention indices for rapid quantitative and qualitative analysis. Abstract No. 72 in Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy, March 8-13, 1982, Atlantic City, N.J. Abstracts (unpublished). Stamoudis, V. C., and J. C. Demirgian. 1985. Computer-Assisted Analysis of Energy-Related Com- plex Mixtures by Retention-Index Gas Chromatography. ANL/SER-5. Argonne National Lab, Argonne, Ill. (Available from NTIS as DE85013702/XAB.) (55 pp.) Stamoudis, V. C., D. A. Haugen, M. J. Peak, and K. E. Wilzbach. 1983. Biodirected chemical characterization of synfuel materials, pp.202-214. In C. W. Wright, W. C. Weimer, and W. D. Felix (eds.). Advanced Techniques in Synthetic Fuels Analysis. PNL-SA-11552. CONF-811160. U.S. Department of Energy, Technical Information Center, Oak Ridge, Tenn. (Available from NTIS as DE83015528.) Strommen, D. P., and K. Nakamoto. 1984. Laboratory Raman Spectroscopy. John Wiley & Sons, New York (138 pp.) Swain, A. P., J. E. Cooper, and R. L. Stedman. 1969. Large-scale fractionation of cigarette smoke condensate for chemical and biologic investigations. Cancer Res. 29:579-583. Talcott, R., and W. Harger. 1980. Airborne mutagens extracted from particles of respirable size. Mutat. Res. 79:177-180. Teranishi, K., K. Hamada, and H. Watanabe. 1978. Mutagenicity in Salmonella typhimurium mutants of the benzene-soluble organic matter derived from air-borne particulate matter and its five fractions. Mutat. Res. 56:273-280. Tokiwa, H., K. Morita, H. Takeyoshi, K. Takahashi, and Y. Ohnishi. 1977. Detection of mutagenic activity in particulate air pollutants. Mutat. Res. 48:237-248. Toste, A. P., D. S. Sklarew, and R. A. Pelroy. 1982. Partition chromatography—high-performance liquid chromatography facilitates the organic analysis of biotesting of synfuels. J. Chromatogr. 249:267-282. U.S. EPA (Environmental Protection Agency). 1971. National Primary and Secondary Ambient Air Quality Standards. Appendix B: Reference method for the determination of suspended particulates in the atmosphere (high volume method). Federal Register 36:8191-8193. U.S. EPA. 1982. Test Methods for Evaluating Solid Waste: Physical/Chemical Methods. 2nd ed. SW846. U.S. Environmental Protection Agency, Off~ce of Solid Waste, Washington, D.C. (Avail- able from NTIS as PB 87-120291.)

98 COMPLEX MIXTURES U.S. EPA. 1984. Calculation of Precision, Bias, and Method Detection Limit for Chemical and Physi- cal Measurements. EPA 600t4-85-058. U.S. Environmental Protection Agency, Office of Research and Development, Office of Monitoring Systems and Quality Assurance, Quality Assurance Man- agement and Special Studies Staff, Washington, D.C. U.S. EPA. 1985. Guidelines for Preparing Environmental and Waste Samples for Mutagenicity (Ames) Testing. Environmental Monitoring System Laboratory, Las Vegas, Nev. (Available from NTIS as PB 86-120144.) (255 pp.) van Hoof, F., and J. Verheyden. 1981. Mutagenic activity in the River Meuse in Belgium. Sci. Total Environ. 20:15-22. Vithayathil, A. J., B. Commoner, S. Nair, and P. Madyastha. 1978. Isolation of mutagens from bacte- rial nutrients containing beef extract. J. Toxicol. Environ. Health 4: 189-202. Watson, J. T., and Throck. 1985. Introduction to Mass Spectrometry. Raven Press, New York. (351 pp.) Weimer, W. C., and B. W. Wilson. 1983. Integration of chemical analysis and biological testing in the study of coal liquid toxicology. (Abstract.) Toxicol. Lett. 18(Suppl. 1):78. Wilk, M., J. Rochlitz, and H. Bende. 1966. Saulenchromatographie van polycyclischen aromatischen Kohlenwasserstoffen an lipophilem Sephadex LH-20. J. Chromatogr. 24:414-416. Wilson, B. W., R. Pelroy, and J. T. Cresto. 1980. Identification of primary aromatic amines in muta- genically active subfractions from coal liquefaction materials. Mutat. Res. 79: 193-202. Wilson, B. W., A. P. Toste, R. A. Pelroy, B. Vieux, and D. Wood. 1981. Accurate Mass/Metastable Ion Analysis of Higher-Molecular-Weight Nitrogen Compounds in Coal Liquids. CONF-801039-7. Battelle Pacific Northwest Labs, Richland, Wash. (Available from NTIS as PNL-SA-8852.) (21 pp.) Wood, E. J. 1980. Atomic absorption spectroscopy, pp. 320-335. In S. B. Brown (ed.). An Introduc- tion to Spectroscopy for Biochemists. Academic Press, New York. Wright, B. W., R. D. Smith, and H. R. Udseth. 1984. Approaches and applications of supercritical fluid chromatography and supercritical fluid chromatography-mass spectrometry techniques. Ab- stract No. 594 in The Pittsburgh Conference and Exposition, March 5-9, 1984, Atlantic City, N.J. Abstracts (unpublished). Wright, B. W., E. K. Chess, H. T. Kalinoski, R. D. Smith, and C. W. Wright. 1985. Comparative analysis of nitro-PAH by capillary gas and supercritical fluid chromatography methods. Abstract No. ANYL 8 in American Chemical Society, Abstracts of Papers, 189th ACS National Meeting, Miami Beach, Fla., April 28-May 3, 1985. American Chemical Society, Washington, D.C. Wright, C. W. 1984. Comparative analysis of four quantitative methods for coal liquids analysis using capillary column chromatography. Abstract No. 831 K in The Pittsburgh Conference and Exposition, March 5-9, 1984, Atlantic City, N.J. Abstracts (unpublished). Wright, C.W., and D. D. Dauble. 1986. Effects of Coal Rank on the Chemical Composition and Toxicological Activity of Coal Liquefaction Materials. PNL-5 805. U.S. Department of Energy, Washington, D.C. (Available from NTIS as DE86011015.) (67 pp.) Wynder, E. L., and D. Hoffmann. 1965. Some laboratory and epidemiological aspects of air pollution carcinogenesis. J. Air Pollut. Control Assoc. 15: 155-159. Wynder, E. L., and G. Wright. 1957. A study of tobacco carcinogenesis. I. The primary fractions. Cancer 10:255-271.

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In the laboratory, testing the toxic effects for a single compound is a straightforward process. However, many common harmful substances occur naturally as mixtures and can interact to exhibit greater toxic effects as a mixture than the individual components exhibit separately. Complex Mixtures addresses the problem of identifying and classifying complex mixtures, investigating the effect of exposure, and the research problems inherent in testing their toxicity to human beings. A complete series of case studies is presented, including one that examines the cofactors of alcohol consumption and cigarette smoke.

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