Nd

" For cancer potency equivalence factors and human cell mutagenicities, see Table 10.13. See text for more information on sampling times and dates collected.

h From Atkinson et al. (1988a). Highest and lowest 12-h concentrations measured in 1986-1987 at six sites in California with different emissions (see Fig. 10.21). The only measurable concentrations at the "clean air" site, Point Arguello, were benz[a]anthracene, 0.007; chrysene + triphenylene, 0.050; and benzo[e]pyrene, 0.007 ng m~3. Highest values are all from wintertime measurements in the Mammoth Lakes ski area; lowest values are generally at the residential site in Reseda. See also Atkinson and Arey (1997) and Table 10.15 for data collected in 1994 on concentrations of these carcinogenic PAHs in Riverside, California.

' From Fraser et al. (1998). Average and range of concentration (ng m~3) of 4-h samples collected every 6 h at four sites in southern California (Long Beach, central Los Angeles, Azusa, and Claremont) during hot weather and severe smog episode (peak 03 = 290 ppb, 1-h average), September 8-9, 1993. Concentrations of cyclopenta[ca!]pyrene (0.40) and benz[a]anthracene (0.25) are the sums of their vapor-phase (0.26 and 0.10) and particle-phase concentrations (0.14 and 0.15) ng m~3, respectively.

'' From Legzdins et al. (1994); mean and range of 24-h samples.

' From Halsall et al. (1994). Values reported are the 1992 annual averages of the sums of the vapor- and particle-phase concentrations of PAHs in samples collected weekly. The ranges given in parentheses are those for week-long samples taken biweekly.

' From Nielsen et al. (1996). Values are mean concentrations for seventy-six 24-h samples and 12 "short-term" samples in the heavily traveled street and fifty-one 24-h samples in the city park.

Only chrysene.

h Sum of benzol/c]fluoranthene and benzo[6]fluoranthene.

' Only dibenzo[a,c]anthracene.

'Sum of benzo[ A: ]fluoranthene and benzo[/]fluoranthene.

b. Human Cell Mutagenicity

The first studies of human cell mutagenicity of fine particles across an air basin were carried out by Hanni-gan et al. (1991, 1998) using the hfAlv2 human cell assay of Penman et al. (1994). Figure 10.23 shows the 1993 annual average hlAlv2 human cell mutagenic potencies (values in parentheses expressed as induced mutant fraction IMF (Xl06) per pg of equivalent organic carbon) and mutagen densities (values, no parentheses, expressed as induced mutant fraction (xlO6) per m3 of air). There were no systematic seasonal variations in the 1993 annual averages of the human cell mutagenic potencies of aerosols collected at any of the four urban sites, suggesting that no single, major seasonal emission source dominated the release of human cell mutagens into southern California air. Rather, they arise from a mixture of primary sources operating throughout the year, dominated by heavy motor vehicle emissions (e.g., see Schauer et al., 1996). In addition, as was the case for bacterial mutagens, if a significant portion of the human cell mutagens in Los Angeles air are formed in atmospheric reactions (e.g., 2-nitrofluoranthene), such reactions must take place basinwide, and in wintertime, as well as in the fall periods of maximum photochemical smog.

Although aerosols collected at the farthest downwind site (Rubidoux) show approximately the same mutagenic potency as the two upwind sites at central Los Angeles and Azusa (~0.14 induced mutant fraction (XlO6) per ¡jig of equivalent organic carbon), the downwind site has the highest annual loading of airborne fine particles and associated highest concentration of organic carbon. Hence the product of the mutagenic potency and the equivalent organic carbon loading results in the higher average mutagen density, 1.53 induced mutant fraction (XlO6) per m3 of air at the downwind site. Not surprisingly, given the sources in urban areas, the mutagen density for the samples collected at the regional background site (not an annual average) was a factor of ~ f 0 less than at any of the four onshore urban sites.

3. Bioassay-Directed Chemical Analysis for PAHs and PACs in Fine Ambient Aerosols Using a Human Cell Assay

In the studies of Hannigan et al. (1997) described in the previous subsection, the contributions of specific PAHs and PACs to the total human cell mutagenicity of fine ambient aerosols in southern California air were not identified and quantified. However, previous investigators had determined ambient levels of a number of carcinogenic PAHs (IARC classes 2A and 2B; see

Table 10.13) present in the polluted Los Angeles Air Basin (e.g., see Atkinson et al., 1988a; Atkinson and Arey, 1997; and Venkataraman and Friedlander, 1994b), many of which are mutagenic in the hfAfv2 cell line (Durant et al., 1996). Furthermore, such PAH human cell mutagens/animal carcinogens are ubiquitous, as illustrated in Table 10.24 for several urban sites in Canada, the United States, and western Europe. It could be expected that at least some of the biologically active PAHs in Table f0.24 would contribute significantly to the "total" human cell mutagenicities of the 1993 fine aerosols assayed by Hannigan et al. (1997).

As discussed earlier, with respect to bacterial mutagens, one of the applications of mutagen assays is their use in focusing analytical studies directed to determining the chemical composition. This approach was also used by Hannigan and co-workers (1998), who carried out a bioassay-directed chemical analysis using the hlAlv2 human cell line of a composite sample made up of a portion of every 24-h filter sample.

The approach is illustrated in Fig. 10.24. Briefly, HPLC was first used to separate the whole fraction extract into four portions: nonpolar 1 (PAHs and al-kanes); nonpolar 2 (high molecular weight PAHs and some smaller nitro-PACs, e.g., 9-nitroanthracene); semipolar (nitro-PAHs and other nitro-PACs and oxy-PACs, e.g., polycyclic ketones, quinones, aldehydes, amides, and aza-PACs); and polar (PAC anhydrides and acids, aliphatic aldehydes, and other organics). These four major fractions were then separated into a total of f9 subtractions. Those that were mutagenic have bold outlines; the exception is fraction 6a, which is double boxed and, along with fraction 7a, contains a significant amount of the moderately strong mutagen 6//-benzo[c<i]pyren-6-one.

The sum of the absolute potencies of these mutagenic subfractions, 243 + 35 induced mutant fraction (xi06)/mg of equivalent organic carbon, is significantly larger (~ 160%) than the value for the unfrac-tionated extract (150 + 31), a phenomenon that has been observed in other environmental human cell mutagenicity studies (e.g., Durant et al., 1994). Hannigan et al. (1998) proposed that this could be due to the presence of fewer interfering compounds in the less complex, fractionated material.

Hannigan et al. (1998) reported that most of the mutagenicity of the extract of the "whole," composite sample of respirable ambient aerosols was in the "whole" aromatics fraction, 83 vs 17% in the aliphatic portion. Table 10.25 summarizes the results of this bioassay-directed chemical analysis for the aromatics. As seen in Table 10.26, six PAHs, cyclopenta[c<i]pyrene,

142±30 47±7 12±5 16±9 14±4 9±4 5±2 10±3 5±2 13±3

Mutagenic potencies (IMF x 106 per mg EOC)

FIGURE 10.24 Flow chart for bioassay-directed chemical analysis of hlAlv2 human cell mutagens in composite sample of fine ambient aerosols collected in the year 1993 at four urban community monitoring stations in the Los Angeles Air Basin. Mutagenic potencies of the whole sample (150 + 31), the individual subfractions (la -» 16b), and the sum of the subfractions (243 + 35) are in units of induced mutant fraction, IMF, (X106) per mg of equivalent organic carbon, EOC. Standard deviations for subfractions are omitted for clarity. Fractions that were mutagenic have bold outlines or have a double box (adapted from Hannigan et al, 1998.)

142±30 47±7 12±5 16±9 14±4 9±4 5±2 10±3 5±2 13±3

Mutagenic potencies (IMF x 106 per mg EOC)

FIGURE 10.24 Flow chart for bioassay-directed chemical analysis of hlAlv2 human cell mutagens in composite sample of fine ambient aerosols collected in the year 1993 at four urban community monitoring stations in the Los Angeles Air Basin. Mutagenic potencies of the whole sample (150 + 31), the individual subfractions (la -» 16b), and the sum of the subfractions (243 + 35) are in units of induced mutant fraction, IMF, (X106) per mg of equivalent organic carbon, EOC. Standard deviations for subfractions are omitted for clarity. Fractions that were mutagenic have bold outlines or have a double box (adapted from Hannigan et al, 1998.)

BaP, benzo[g/z/]perylene, benzo[6]fluoranthene, in-deno[l,2,3-c<i]pyrene, and benzo[&]fluoranthene, contribute the major portion of the identifiable mutagenicity of the extract of the whole unfractionated sample, accounting for 8.6, 2.5, 1.7, 1.4, 1.2, and 0.8%, respectively, of the total mutagenicity of the whole sample. Two semipolar mutagenic PACs were also present at significant levels: 2-nitrofluoranthene, a product of atmospheric reactions, and 6//-benzo[c<f]pyren-6-one, a primary O-PAC pollutant present in exhaust emissions from diesel engines and non-catalyst-equipped cars (see Sections E and F). These account for an additional 0.8 and 1.6%, respectively, of the identified whole sample mutagenic potency (see Table 10.26).

The remaining PAHs and PACs in Table 10.25 are present at much smaller levels. Thus, overall only about 20% of the total whole composite sample mutagenicity is assignable to individual, "positively identified," quantified, and assayed PAHs and PACs. Furthermore, as seen from the mutagenicities of the extracts of the four samples produced in the first fractionation step [nonpolar 1 (66 ± f3), nonpolar 2 (39 + 12), semipolar (95 + 7), and polar (31 ± 12)], more than half of the total sample mutagenicity is found in the semipolar and polar fractions—in which only ~3% of the contribut ing PACs have been identified. The lack of closure may be due in part to the incomplete data base on the mutagenicity of individual compounds where pure samples have not been available for testing. For example, Hannigan et al. (1998) noted that all methyl isomers of PAHs tested to date in the hfAlv2 assay are more potent than the unsubstituted parent PAHs (e.g., 5-methylchrysene is 37 times more potent than chrysene itself, 0.63 vs 0.017, both relative to BaP = 1.00; Durant et al., 1996). This may be relevant because significant concentrations of four methylphenanthrene isomers (3-, 2-, 9-, and 1-) were present, but had not been tested.

However, there are several caveats that should be kept in mind before extrapolating these results to other parts of the world. First, as discussed in more detail in Chapter 16, since the mid-1960s reductions in emissions of volatile organic compounds and of respirable "soot" particles and the associated PAHs and PACs have occurred in California due to increasing controls on mobile, as well as stationary, sources. Standards for emissions from mobile sources are the most stringent in the world at the present time. Second, even over the time period from the mid-1980s to the mid-1990s when the bacterial and human cell mutagen studies discussed

TABLE 10.25 Mass Concentrations of Individual Genotoxic PAHs and PACs in the "Whole Sample" and in Subfractionated Extracts,' Their Absolute Mutagenic Potencies and Potencies Relative to Benzo[a]pyrene = 1.00, and Contributions of Each to the Overall Total Potency of a Composite Mixture of Ambient Aerosols Collected at Four Urban Sites in Southern California for One Year ( 1993) and Subjected to Bioassay-Directed Chemical Analysis Using the hlAlv2

Human Cell Mutagenicity Assay

TABLE 10.25 Mass Concentrations of Individual Genotoxic PAHs and PACs in the "Whole Sample" and in Subfractionated Extracts,' Their Absolute Mutagenic Potencies and Potencies Relative to Benzo[a]pyrene = 1.00, and Contributions of Each to the Overall Total Potency of a Composite Mixture of Ambient Aerosols Collected at Four Urban Sites in Southern California for One Year ( 1993) and Subjected to Bioassay-Directed Chemical Analysis Using the hlAlv2

Human Cell Mutagenicity Assay

Compound

Formula

Concentration (ng/mg of EOC)''

Mutagenic potency'

Potency relative to BaP = 1.00*

Contribution to potency of sample extracts IMF (xlO6) mg of EOC

Whole sample

Sub fraction

Whole sample

Sub fraction

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