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Mutagenicity of bitumen and asphalt fumes
Toxicology in Vitro 17 (2003) 403–412 www.elsevier.com/locate/toxinvit
Mutagenicity of bitumen and asphalt fumes P.R. Heikkila¨a,*, V. Va¨a¨na¨nenb, M. Ha¨meila¨b, K. Linnainmaab a Department of Epidemiology and Biostatistics, Finnish Institute of Occupational Health, Topeliuksenkatu 41 aA, 00250 Helsinki, Finland Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 aA, 00250 Helsinki, Finland
b
Accepted 1 April 2003
Abstract The mutagenicity of asphalt fumes was tested with the Salmonella bioassays. The aim was to investigate if recycled additives modify the genotoxicity of emissions. Recycling of old asphalt is increasing, and we studied also the mutagenicity of emissions sampled during the re-use of asphalt. The composition of vapours and fumes were analysed by gas chromatography and by liquid chromatography. Bitumens containing coal fly ash (CFA) or waste plastics were heated to the paving temperatures in the laboratory. In the field, bitumen fumes were collected during paving of stone mastic asphalts (lime or CFA as a filler), remixing of stone mastic asphalt (lime or CFA as a filler), and of asphalt concrete. All the lab-generated vapour fractions were non-mutagenic. The particulate fractions were mutagenic with TA98 in the presence of the S9 activation. In addition, the lab-fumes from bitumen containing waste plastics were positive with both strains without S9. Only particulate fractions sampled in the field were tested. They were mutagenic with and without metabolic activation with both strains. The mutagenic potency of the field samples was higher than that of the lab-generated fumes without S9, and the remixing fumes were more mutagenic than the normal paving and lab-generated fumes with S9. The use of inorganic additive, CFA, did not change the mutagenicity of the fumes, whereas the organic additive, waste plastics, increased the mutagenicity of the laboratory emissions significantly. # 2003 Elsevier Ltd. All rights reserved. Keywords: Bitumen; Mutagenicity; Asphalt; Salmonella; Coal fly ash
1. Introduction Bitumen is a complex mixture of petroleum products, used mainly for road paving and for roofing. Asphalt is a mixture of bitumen and mineral matter such as stone, sand, or filler. Bitumen (referred to as asphalt in the USA) contains high proportions of high-molecular-weight paraffinic and naphthenic hydrocarbons, whereas coal tar materials are composed mainly of aromatic hydrocarbons (Concawe, 1992). The re-utilisation of road-making materials is increasing. Asphalt can be recycled by various methods e.g. the old asphalt layer can be removed by cold milling and then transferred to an asphalt plant or it can be scarified as hot and mixed in situ with virgin materials (Concawe, 1992). Recycling operations can lead to increased polycyclic aromatic hydrocarbon and bitumen fume exposures in paving crews (Burtsyn et al., 2000). Also several waste materials and industrial * Corresponding author. Tel.: +358-9-47472215; fax: +358-94773149. E-mail address: pirjo.heikkila@occuphealth.fi (P.R. Heikkila¨). 0887-2333/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00045-6
by-products such as glass, coal fly ash, and rubber tires have been used as asphalt modifiers (Butler et al., 2000). Epidemiologic studies of lung cancer among pavers exposed to bitumen fumes have yielded contradictory (Partanen and Boffetta, 1994) results. Many studies have limitations in the design or they failed to control adequately for smoking and possible confounding exposures such as coal tar, diesel exhaust and silica. In the recent IARC European epidemiological study of cancer mortality among asphalt workers, the SMR of lung cancer was slightly higher among the workers employed in jobs entailing exposure to bitumen (1.17, 95% CI 1.04–1.30) than among construction workers (1.01, 95% CI 0.89–1.15) (Boffetta et al., 2003a). After adjusting for coal tar pitch and with 15-year lag, lung cancer mortality slightly increased (SMR=1.23, 95% CI 1.02–1.48) among road pavers (Boffetta et al., 2003b). In animal studies, laboratory-generated bitumen roofing fume (Thayer et al., 1981; Niemeyer et al., 1988; Sivak et al., 1989; Sivak et al., 1997), raw roofing bitumen (Sivak et al., 1989; Sivak et al., 1997), and bitumen
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based paints (Robinson et al., 1984) have shown to be carcinogenic, when applied dermally to mice. In the study of Emmet et al. (Emmett et al., 1981), however, raw roofing bitumen was not carcinogenic after dermal treatments. No animal studies have examined the carcinogenic potential of fumes sampled during paving operations or that of laboratory-generated paving bitumen condensates. Mutagenic potential of laboratory generated bitumen fume and vapour condensates have been tested in several in vitro and in vivo genotoxicity assays. Positive results have been obtained in Ames Salmonella mutagenicity studies. Data have been reviewed in a document published by National Institute for Occupational Safety and Health in the USA (NIOSH, 2000). Generally, the findings have been positive only in the presence of a metabolic activation system (Machado et al., 1993; De Meo et al., 1996; Reinke et al., 2000). Only one study has investigated mutagenic properties of asphalt emissions sampled during asphalt production (Reinke et al., 2000). Asphalt fume condensates collected above the storage tank were not mutagenic (Reinke et al., 2000). Otherwise there has been no published data of the mutagenicity of asphalt emissions collected during the road paving. Further, no studies are available from the situations when recycled materials have been used in asphalts or when old asphalt layers have been recycled. Emissions from asphalt are complex mixture containing hundreds of different compounds. When assessing occupational exposure levels among bitumen workers the air concentration of bitumen fumes and polycyclic aromatic hydrocarbons are generally monitored (Burstyn, Kromhout, Boffetta, 2000; Burstyn, Kromhout et al., 2000). Possible health effects and biological response of fumes emitted from asphalts with different composition or paving temperature are difficult to judge only from the air concentration results. Therefore, testing of possible biological effects will give valuable data in assessing of biological significance of occupational exposures. The aim of this study was to compare the mutagenicity of emissions from the asphalts containing recycled materials, such as waste plastics or coal fly ash, to those from the normal asphalt mixes. The experiments were carried out both in the laboratory and field conditions. In addition, we investigated the mutagenicity of emissions during normal paving and recycling of asphalts.
temperatures used in paving of asphalt mixes (Table 1). The content of toxic metals in coal fly ash was lower than the limit values for contaminated soil (the manufacturer’s data). Waste plastics (WP) composed of polyethylene and polypropylene (79%), polystyrene (11%), polyvinyl chloride (7%) and polymethyl metacrylate (4%). WP was grounded into particles (d=2–3 mm), and mixed with bitumen at 170 C for 24 h. The sampling of emissions started when the temperature of the bitumen mixes was 140 C in all the experiments. The material samples were heated in a glass vessel (700 ml) equipped with a cover, where the sampling filters and adsorbent tubes were attached. The heating method is a modification of the laboratory rig presented by Brandt et al. (1985). About 200 g of deep frozen bitumens was crushed and poured into the vessel which was heated up in an electric mantle. The temperatures were well controlled with a digital thermometer (Elma 1300, NiCrNi, type K), and during sampling, the mixtures were stirred at a constant rate. Emissions were collected on the six parallel samplers per a batch. Sampling times ranged from 1 to 2 h. Both vaporous and particulate fractions of bitumens were sampled. The amount of total particles, vaporous and particulate bitumen emissions, and 15 PAHs were quantified in the collected samples. 2.2. Field generated samples Bitumen fume samples were collected on teflon filters during paving of the following asphalt mixtures: stone mastic asphalts (SMA) (5.7% B80+10% lime as a filler or 6% B80+10% coal fly ash as a filler, remixing of stone mastic asphalt (REMSMA) containing lime (L) or coal fly ash (CFA), and during remixing of asphalt concrete (REMAC) (Table 2). The bitumen fume samples were collected from the breathing zones of the workers. The sampling periods were from 6 to 8 h. In remixing, old pavement was softened by 2–3 heaters, scarified, and mixed in situ with virgin asphalt. Heaters were warmed up with liquefied gas at the remixing of SMAs, and with heavy heating oil at the remixing of asphalt concrete (AC). At the remixing site of AC, heaters were out of condition, and the temperature of road surface rose over 300 C when the recommended temperature range is below 250 C. 2.3. Collection and analysis of air samples
2. Materials and methods 2.1. Laboratory-generated emissions Bitumen (grade B120, Fortum, Kulloo, Finland) and bitumen mixtures containing of waste plastics, or pulverised coal fly ash (CFA) (Fortum, Naantali power plant, Finland) were heated in the laboratory at the
The fumes and vapours were collected on filter cassettes, loaded with SKC Teflon filters followed by SKC XAD-2 tubes (Table 3). The bitumen fume samples on the Teflon filters were eluted with 2 ml of tetrachloroethylene (Merck, p.a.) in an ultrasonic bath for 30 min, and analysed with Fourier-transform infrared (FTIR) spectrometer (Nicolet 20 SXC/60, Nicolet,
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Temperature of bitumen ( C)
Mutagenicity tests
Bitumen B120/ (B120vapours and B120particles)
170
Bitumen B80+coal fly ash (66%)/ (B80-CFAvapours and B80-CFAparticles) Bitumen B120+waste plastics (10%)/ (B120-WPvapours and B120-WPparticles)
170
Salmonella typhimurium TA98 and YG1024 with and without S9 Salmonella typhimurium TA98 and YG1024 with and without S9 Salmonella typhimurium TA98 and YG1024 with and without S9
170–180
Table 2 Mixtures, paving/heating temperatures of asphalt and genotoxicity tests used for the samples collected at the paving sites Asphalt + additive (w-%)/ (fractions studied)
Temp. of paved/ remixed asphalt ( C)
Mutagenicity tests
Stone mastic asphalt+lime (10%)/ (SMA-Lparticles) Stone mastic asphalt+coal fly ash (10%)/(SMA-CFAparticles) Remixing of SMA+lime (10%)/ (REM-SMA-Lparticles)* Remixing of SMA+coal fly ash (10%)/ (REM-SMA-CFAparticles)* Remixing of asphalt cement/(REM-ACparticles)+
175–200
Salmonella typhimurium TA98 with and without S9 mix, and YG1024 without S9 mix Salmonella typhimurium TA98 with and without S9 mix, and YG1024 without S9 mix Salmonella typhimurium TA98 and YG1024 with and without S9 mix Salmonella typhimurium TA98 with and without S9 mix, and YG1024 without S9 mix Salmonella typhimurium TA98 without S9 mix
170–210 180–207/150–260 190/160–250 160/310–350
*heaters of old asphalt operated with liquefied gas. + heaters of old asphalt operated with fuel oil.
Table 3 Fractions sampled and methods of sampling and analysis Fraction sampled
Sampling Media
Flow Rate (l/min)
Analytic Method
Bitumen fume (particulate)
Open-face 37 mm cassette with Teflon filter (SKC 225-1705, 1 mm) XAD-2 (SKC 226-30-06)
1.5
Extraction with tetrachloroethylene; FTIR spectrometry Extraction with dichloromethane; GC with FID Extraction: filters with cyclohexane and adsorbent with acetonitrile HPLC with fluorescence detector
Semi volatile organic compounds (SVOC) PAH particles PAH vapours
Open-face 37 mm cassette with Teflon filter (SKC 225-1707, 2 mm) XAD-2 (OrboTM-43 Supelco)
1.5 1
FTIR=Fourier-transform infrared. GC=gas chromatograph. FID=flame ionization detector. HPLC=high performance liquid chromatograph.
Madison, Wisc. USA) equipped with a mercury-cadmium-tellurium detector (Heikkila¨ et al., 2002). The sorbent layers of the XAD-2 were eluted with 2 ml of dichloromethane (Merck, p.a.) in an ultrasonic bath for 2 h, and analysed by a Hewlett Packard 5890 gas chromatograph (GC), equipped with a flame ionisation detector (FID), and automatic sampler 7672A (Hewlett Packard, Palo Alto, CA, USA) (Heikkila¨ et al., 2002). The PAH compounds were collected on teflon filters connected to XAD-2 adsorbent tubes (Table 3). The filters were extracted with 5 ml cyclohexane (Merck, p.a.) for 30 min in an ultrasonic bath, and the adsorbents were extracted with 2 ml acetonitrile for 40 min in
an ultrasonic bath. The extracts were filtered with Gelman GHP Acrodisc 13 (0.45 mm, P/N 4556) before analysing. The samples were analysed by reversed-phase high-performance liquid chromatography (HPLC). The HPLC system was HP 1100 (Hewlett Packard, Waldronn, Karlsruhe, Germany) and consisted of a vacuum degassed, a high-pressure gradient pump, an autosampler, column thermostat and a scanning fluorescence detector (Kuusima¨ki et al., 2002). Fifteen PAH compounds listed in the NIOSH Manual of Analytical Methods Method 5506 (National Institute for Occupational Safety and Health, 1998) (except acenaphthylene) were quantified, the quantitation limit for PAHs was from 0.002 to 0.008
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mg/m3 for the filters, and from 0.01 to 0.22 mg/m3 for the XAD-2 adsorbents. For qualitative analysis of the vapour and fume, material samples were heated in a laboratory rig, sampled on XAD-2 and on the Teflon filter and desorbed with 2 ml of dichloromethane. The components were separated and characterised by chemical class with the HRGC-MS system (Micromass AutoSpecQ). The ionisation techniques used was EI (electron ionisation, 70 eV). The analytical column was Ultra 2 (cross linked 5% Ph Me Silicone), 25 m0.32 mm I.D., 0,17 mm phase silica capillary column. The carrier gas (helium) pressure was set at 15 psi. The injector temperature was 260 C. The column temperature was programmed as follows: 40 C for 1 min, an increase of 5 C/min up to 270 C and held at 270 C for 13 min. The identification of components was based on the Wiley/NBS Registry of Mass Spectral Data, interpretation of the authentic spectra and the retention time and the elution information from the literature. The concentration of chlorinated aromatic compounds in the emissions from bitumen-waste plastics mixtures (B120-WP) was also determined (Ruokoja¨rvi, Ettala et al., 1995; Ruokojarvi, Ruuskanen et al., 1995). The samples were collected on XAD-2 (SKC 226-30-05) which were eluted with toluene in a Soxhlet for 20 h. After the Soxhlet extraction chlorophenols were eluted with 0.1 M potassium carbonate, acetylated and eluted with n-hexane. The rest of the Soxhlet extraction was evaporated almost to dryness, n-hexane was added, the samples were purified with sulphuric acid and aluminium oxide columns. PAHs and chlorophenols were analysed by a GC (Hewlett Packard 6890, Palo Alto, CA, USA), equipped with a MS detector (Hewlett Packard 5973), the selective ion monitoring mode was used. The analytical column was HP-5MS (30 m0.25 mm0.25 mm). Polychlorinated dioxins and furanes were separated and identified by the HRGC-MS system (VG 70–250 SE). The analytical column was DB-Dioxin (60 m0.25 mm0,15 mm) with the resolution 10 000. Polychlorinated biphenyls and chlorobenzenes were separated and identified by a GC (Hewlett Packard 5890, Palo Alto, CA, USA) equipped with an EC detector, the column was HP-1 (25 m0.32 mm0.52 mm). 2.4. Mutagenicity in the Ames Salmonella tests Mutagenicity of the bitumen and asphalt fumes and vapours were investigated by the Ames Salmonella bacteria assay. The laboratory generated vapours and fumes were first extracted with 4 ml dichloromethane in an ultrasonic bath for 20 min from the adsorbent tubes and filters. Then, 1 ml dimethyl sulphoxide (DMSO) was added, after which dichloromethane was evaporated. From field samples, only particulate fractions were investigated. The Teflon filters were extracted with 2 ml of tetrachloroethylene (C2Cl4, Merck, p.a.) in an
ultrasonic bath for 30 min. The extractions from the same paving site were combined. Tetrachloroethylene was evaporated gently under nitrogen flow, and the residue was dissolved in 1 ml DMSO. Five concentrations of each extract (0.031–0.5 mg/plate) were tested using the frame-shift mutation strains TA98 and YG1024. TA98 was selected because it is known that bitumen fumes contain PAHs (Heikkila¨ et al., 2002) that cause frame-shift mutations at the presence of oxidative metabolic activation. Salmonella typhimurium YG1024 is a derivative of S. typhimurium TA98 with a high level of N-hydroxyarylamine O-acetyltransferase (OAT) activity. This strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes (Einisto et al., 1991). YG1024 was selected because paving workers are exposed not only to bitumen emissions but also e.g., to diesel exhausts from paving machines which are known to contain nitroarenes (Hayakawa et al., 1997). The tests were performed by standard protocols according to OECD guideline (OECD, 1997). As positive control compounds, 4-nitroquinoline (4-NQO, direct mutagen) and 2-aminoanthracene (2-AA, mutagenic with metabolic activation) were applied. Of the laboratory samples, both vaporous and particulate fractions were tested in the presence and in the absence of metabolic activation (S9). Rat liver S9 was prepared from male Wistar rats, pre-treated with Aroclor 1254. S9 mix contained 1 ml S9 fraction and 5.4 ml phosphate buffer (167 mmol), 0.18 ml KCl (1.65 mol), 0.9 ml MgSO4 (80 mmol), 0.9 ml glucose-6-phosphate (50 nmol), and 1.2 ml NADP (40 mmol). S9 mix was filtered before use. Because of the limited amount of the field samples, only a part of the samples could be tested with and without S 9 and with both strains (Table 2). Duplicate cultures were run for each fraction. The mutagenic potency evaluations were based on the slope of the linear regression (B, when y=A+Bx) of two parallel test results. The test result was interpreted to be positive if the slope was > 0 and P-value < 0.05. The slopes and P-values were calculated with SAS/STAT1 package and with the SALM software program that is available in ftp ftp.bios.unc.edu (Kim and Margolin, 1999). The SALM-program gave identical results for the slopes and P-values with SAS/ STAT1.
3. Results 3.1. Composition and concentration of bitumen emissions Vapour and particulate fractions of the laboratory generated emissions were analysed qualitatively. The main components in the vapour fractions were C8H18C19H40, and in the particulate fractions, C10H22-C29H90 straight-chain, aliphatic hydrocarbons. The vapour
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fractions contained also alkyl benzenes, hydrated naphthalenes, indane and alkyl indanes, biphenyl and alkyl biphenyls, several 2–3 ring PAHs and their alkylated derivatives (the main vaporous PAHs being phenanthrene and alkyl phenanthrenes, naphthalene, and alkyl naphthalenes,), alkyl benzothiophenes, and dibenzothiophenes. In addition, the vapours from B120-WP contained alkyl phenols, styrene, and alkyl styrenes. The main PAHs in all the particulate fractions were fluorene, phenanthrene, pyrene and their alkyl derivatives. The particulate fractions contained also biphenyl and alkyl biphenyls, several alkyl benzothiophenes, dibenzothiophene and alkyl dibenzothiophenes. In the laboratory experiments, the concentrations of bitumen vapours ranged from 10 mg/m3 (B80-CFA) to 280 mg/m3 (B120), bitumen fume from 2 mg/m3 (B120WP) to 5 mg/m3 (B80-CFA), and 15 PAHs from 20 mg/ m3 (B80-CFA) to 100 mg/m3 (B120), respectively. At the paving sites, the air concentrations of bitumen vapours ranged from 0.7 mg/m3 (REM-AC) to 2.2 mg/m3 (REM-SMA-CFA), bitumen fume from 0.1 mg/m3 (REM-AC) to 0.6 mg/m3 (SMA-L and SMA-CFA), and 15 PAHs from 5.4 mg/m3 (REM-SMA-CFA) to 13 mg/m3 (REM-AC). The proportions of quantified vaporous and particulate 15 PAHs in the fractions tested are presented in Table 4. The proportion of PAHs in the vapour fraction of bitumen emissions was lower in the laboratory samples (0.01–0.15%) than in the paving samples (0.23– 1.7%), while the proportions of particulate PAHs in the laboratory and field fumes were at the same magnitude (0.069–0.81%) except in the remixing of asphalt concrete (1.5%), when the temperature of old asphalt rose over the recommended value ( > 300 C). Due to the small amount of PVC in the recycled plastics, the content of chlorinated aromatic compounds in the emissions from B120-WP was analysed. The
emissions contained traces of the following compounds: 2- and 4-chlorophenols, dichlorophenols, trichlorophenols, tetrachlorophenols and pentachlorophenols, penta- and hexachlorobenzenes, chlorinated biphenyls. The concentrations of dioxins and furans were below the detection limit of the method. 3.2. Mutagenicity in the Ames Salmonella tests 3.2.1. Laboratory samples The mutagenicity of the laboratory generated samples is presented in Table 5. All the vapour fractions were negative with both strains in the presence and absence of the S9 activation. All the particulate samples (B120particle, B80-CFAparticle and B120-WPparticle) showed mutagenic activity with TA98 + S9-mix. In addition, the fume fraction B80-CFAparticle was weakly (slope =21) positive also without S9-mix. The fume fraction of bitumen-waste plastic mixture (B120-WPparticle) was the only lab-sample that was positive with both strains, TA98 and YG1024, in the absence of the S9 activation (Fig. 1a). The mutagenic index (slope) of the B120-WPparticle fraction was at the same magnitude with and without S9 with TA98, whereas the YG1024 strain gave negative response with the oxidative S9 activation. Bitumen fumes (B120particle) showed a positive result with YG1024+S9, but the variation of the parallel test results was so large that no judgement on the mutagenicity can be made. 3.2.2. Field samples The results of the field samples are presented in Table 6. All the fume samples tested were positive with the both strains with and without the S9 activation. Due to the small amounts of the collected samples, however, all the tests could not be performed with both strains with and without the oxidative metabolic activation.
Table 4 The proportions of vaporous polycyclic aromatic hydrocarbons (PAHs) to semivolatile organic compounds (SVOC), and particulate PAHs and 4–6 aromatic ring PAHs to bitumen fume (BF) in the emissions sampled in the laboratory and during road paving Generation/mass
PAHvapours/SVOC (%)
PAHparticle/BF (%)
PAH4-6 (%)
Laboratory/B120 Laboratory/B80-CFA Laboratory/B120-WP Paving/ SMA-L Paving/ SMA-CFA Remixing/ SMA-L Remixing/ SMA-CFA Remixing/ AC
0.03 0.15 0.01 0.29 0.28 0.71 0.23 1.7
0.81 0.14 0.14 0.16 0.16 0.14 0.069 1.5
0.029 0.051 0.030 0.037 0.025 0.073 0.036 1.2
Bxx=bitumen type. SMA=stone mastic asphalt. L=lime. CFA=coal fly ash. WP=waste plastic. AC=asphalt concrete.
rings/BF
PAH4-6 rings/PAHparticles (%) 4 36 21 23 16 52 52 80
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Table 5 Mutagenicity (revertants/plate) of the bitumen emissions generated in the laboratory Bitumen type and fraction tested
Strain TA98 B120vapour B120particle B80-CFAvapour B80-CFAparticle B120-WPvapour B120-WPparticle Strain YG1024 B120vapour B120particle B80-CFAvapour B80-CFAparticle B120-WPvapour B120-WPparticle
Dose (mg/plate)
Slopest error
Probability
S9 mix
Background control*
0.031
0.063
0.13
0.25
0.50
– + – + – + – + – + – +
20 55 22 31 20 53 22 31 20 53 19 31
17 36 21 45 20 43 23 47 20 43 22 43
19 34 23 41 20 61 25 58 24 51 29 52
19 42 23 48 23 50 24 50 19 52 32 50
21 44 26 48 20 49 26 54 18 51 36 62
17 38 26 56 24 50 33 65 19 57 55 66
3.55 1014 9.65 3712 6.87 4.417 213 4615 4.47 1610 678 5713
0.54 0.48 0.10 0.01 0.33 0.81 <0.01 0.01 0.56 0.12 <0.01 <0.01
– + – + – + – + – + – +
21 126 44 64 35 126 44 64 40 112 41 64
22 87 40 70 31 110 36 59 39 104 39 63
14 91 42 65 34 95 36 58 31 102 50 68
15 100 39.5 76 30 89 41 58 30 93 50 73
18 112 40 73 31 89 46 63 35 81 58 69
20 107 50 85 33 88 44 59 37 93 78 71
1.05 1025 147 3842+ 1.25 5531 1110 1.419 1.48 3622 749 1310
0.85 0.68 0.08 0.18 0.81 0.15 0.32 0.94 0.86 0.13 <0.01 0.22
CFA=coal fly ash (65.7w-%). WP=waste plastic (10w-%). *The sampling media were used as the background controls: XAD-2 for vapours and Teflon filters for particles (Table 3). + The standard error of the slopes in the parallel tests was so large that the mutagenicity cannot be judged reliably although the mean result was positive. The mutagenicity of positive controls, 4-NQO without S9 and 2-AA with S9 were 439-625 and 1224-2462 revertants/plate, respectively.
Table 6 Mutagenicity (revertants/plate) of particulate fractions of emissions sampled during laying of different asphalts Strains and asphalt types Strain TA98 SMA-Lparticles SMA-CFAparticles REM-SMA-Lparticles REM-SMA-CFAparticles REM-ACparticles Strain YG1024 SMA-Lparticles SMA-CFAparticles REM-SMA-Lparticles REM-SMA-CFAparticles
Dose (mg/plate)
Slopest error
P-value
S9 mix – + – + – + – + +
Background control* 14 31 14 31 14 31 14 31 31
0.031 15 39 15 40 19 25 18 32 49
0.063 18 31 13 45 29 38 22 37 49
0.13 22 40 21 30 26 49 26 37 82
0.25 30 53 22 50 27 47 26 63 138
0.50 28 53 27 61 41 71 31 75 271
308 4611 287 5313 449 8111 286 9510 47916
P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01
– – – + –
34 34 34 87 34
31 28 38 95 41
38 29 43 108 43
40 35 41 97 48
43 43 63 127 53
35+ 45 64 197 61
4021 338 6310 21027 4814
P=0.04 P<0.01 P<0.01 P<0.01 P<0.01
*The sampling media were used as the background controls: XAD-2 for vapours and Teflon filters for particles (Table 3). + The highest dose omitted in the testing of mutagenicity (toxic). The mutagenicity of positive controls, 4-NQO without S9 and 2-AA with S9, were 428-513 and 642-846 revertants/plate, respectively. SMA-L=stone mastic asphalt containing lime as a filler, SMA-CFA=stone mastic asphalt containing coal fly ash as a filler, REM=remixing.
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The mutagenic index of REM-ACparticle with TA98+S9 was highest, but unfortunately, the sample was too small to carry the test without S9. The surface temperature of old asphalt was above 300 C, and thus exceeded the standard values due to a failure in a heater. In general, the dose ranges were non-toxic. The highest dose of SMALparticle appeared toxic with YG1024, and this result was therefore omitted. The remixing fume of SMA-L was mutagenic with YG1024 + S9-mix in contrast to the corresponding laboratory fumes, but unfortunately, we could not test the other field samples with YG1024 in the presence of the S9 system due to small amounts of the samples. The mutagenic indices of the laboratory generated fumes were lower than those of the normal paving fumes when tested with both strains without S9 (except B120WP) (Fig. 1a). The mutagenic indices of the remixing fumes were higher than those of normal paving and labgenerated fumes when tested with TA98 and YG1024 at the presence of S9-mix (Fig. 1b). However, in the absence of S9, the indices of the fumes from the normal paving and remixing sites did not differ significantly (Fig. 1a). Only field samples and B120-WPparticle contained compounds that were mutagenic with YG1024 without S9 (Fig. 1a). None of the negative slopes did differ significantly from zero (Tables 5 and 6), thus the negative slopes were due to random variations, not indicators of toxic effects. 3.2.3. Correlation with PAHs PAHs possess mutagenic activity only in the presence of oxidative metabolic activation. Consequently, their possible effect on the mutagenic activity was calculated only for TA98+S9 (Table 7). The concentration of PAHs or 4-6 ring PAHs in the lab fumes did not explain the higher mutagenicity of the B120-WP fumes. In contrast to the laboratory results, a significant or almost significant correlation was seen between the mutagenic indices and the proportion of PAHs, and 4–6 ring PAHs in the field fumes. The correlation was not seen when the field samples were tested without the results of REM-AC. The main particulate PAHs in the laboratory fumes were 2–3 ring PAHs such as fluorene, and the main 4–6 ring PAHs pyrene, benz(a)anthracene and chrysene, respectively. The proportions of 4–6 ring PAHs in particulate PAHs were higher in the remixing fumes (52–80%) than measured in the laboratory or paving fumes (4–36%) (Table 4).
4. Discussion The laboratory-generated bitumen condensates tested in the earlier studies have been mixtures of vapour and fume fractions (Machado et al., 1993; De Meo et al., 1996; Reinke et al., 2000). We studied the effects of vapour and particulate fractions separately. All the laboratory generated vapour fractions were negative;
Fig. 1. (a) and (b). Mutagenic indices (slopes) of particulate emissions from bitumens and asphalts. Emissions were sampled in the laboratory (bitumen and its mixtures containing coal fly ash (CFA) or waste plastics (WP)), during paving of stone mastic asphalt (SMAs), during remixing of SMA (REMSMA) and during remixing of asphalt concrete (REMAC). SMAs contained lime (L) or CFA as an additive. Because of the limited amount of field samples, only fumes from one paving site, REMSMA-L, could be tested with YG1024+S9.
mutagenic compounds in bitumen emissions were bound to particles. Therefore, vapour fractions were not included in the field studies. The mutagenicity results of the particulate fractions generated in the laboratory are in accordance with the earlier studies, which show that lab-generated bitumen emissions have been mutagenic in bacterial tests in the presence of the S9 activation (Machado et al., 1993; De Meo et al., 1996; Reinke et al., 2000). No previous studies on the mutagenicity of fumes from paving sites are available. The fumes from the paving and remixing sites showed as or even more mutagenic than the corresponding laboratory samples with TA98 + S9 activation (Fig. 1b). Our results are in
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Table 7 The correlation coefficients and P-values between the proportion of polycyclic aromatic hydrocarbons (PAH) in bitumen fume and the mutagenicity indices with the strain TA98 in the presence of oxidative metabolic activation, S9 Laboratory fumes Particulate PAHs in bitumen fume 4–6 ring PAHs in bitumen fume
0.835 0.015
Field fumes
Field fumes without REMAC
Laboratory + field fumes
0.986 (P<0.01) 0.995 (P=0.059)
0.820 0.416 (P=0.226)
0.833 (P=0.019) 0.992 (P=0.059)
REMAC=Remixing of asphalt concrete.
conflict with Reinke et al. (2000) who concluded that the asphalt fumes sampled above a hot mix asphalt storage tank (140–157 C) were less mutagenic with TA98 at the presence of S9 than the laboratory-generated fumes. The temperature of asphalt mixes at the paving sites were higher in our than in Reinke’s study (Table 2). The higher temperature likely results different composition of bitumen fumes. The composition of the remixing fumes differed from the laboratory and paving samples regarding the concentration of 4–6 ring PAHs. The content of PAHs, and 4–6 ring PAHs explained, at least partly, the mutagenic potency of the remixing fumes with TA98+S9 (Table 7). However, the complexity of bitumen fumes makes the interpretation of the results difficult. Fumes contained e.g., PAHs, alkylated PAHs, polycyclic aromatic sulphur compounds (PASH) with 1–2 aromatic rings and their alkyl derivatives. In contrast to our findings, Reinke with his co-workers have found mutagenic PASHs with 3–4 aromatic rings in the fumes (Reinke et al., 2000). PASHs with 1–2 aromatic rings such as dibenzothiophenes have not shown mutagenic activity (McFall et al., 1984). Many of the identified compounds such as 4–6 ring PAHs, alkylated PAHs, some methylated PASHs (McFall et al., 1984) and some 3–4 ring PASHs (Pelroy et al., 1983) can exhibit mutagenicity upon oxidative metabolism of the aromatic ring. These compounds likely explain the positive results with TA98+S9. However, these compounds do not explain the mutagenicity found in the absence of S9. Our results suggest that, as a contrast to the laboratory fumes, the fumes from paving and remixing sites contain direct acting mutagens (Fig. 1a). The compounds identified in the fumes do not, however, explain these findings. One field sample was far more mutagenic than the lab samples with YG1024 in the presence of S9. Unfortunately, only one field sample (REMSMA-L) could be tested with YG1024+S9 due to a limited amount of fumes. The concentration of bitumen fume was relatively low at the paving and remixing sites, and although several parallel samples were collected, the amount of fume sampled remained insufficient for all the tests. Therefore, we cannot judge whether remixing fumes with OAT active strain YG1024 in the presence of the S9 activation are more mutagenic than normal paving fumes.
We studied the effects of two recycled products, coal fly ash and waste plastics, on the mutagenicity of bitumen emissions. The mutagenic potency of the fractions from mixtures including CFA did not differ significantly from regular bitumens or asphalts. Coal fly ash itself has shown variable results in numerous bacterial mutagenicity studies (Griest et al., 1982; Mumford and Lewtas, 1982; Morris et al., 1989). However, the waste plastic mixture fumes (B120-WP) were significantly more mutagenic than the other lab-generated fractions in the absence of S9 mix. The mutagenicity of B120-WP with the S9 activation, however, did not differ significantly from the lab-fumes of bitumen or those of bitumen-coal fly ash mixture (Fig. 1b). At the test temperature (170 C), waste plastics partly decomposed. The identified decomposition products could not explain the positive results, however. Many of aromatic chlorinated compounds are not mutagenic (Schoeny et al., 1979; Schoeny, 1982; Safe, 1989), and styrene is not mutagenic without the S9 activation (Loprieno et al., 1978). Thus the mutagens in the emission of B120-WP remained unknown. In summary, our data indicate that the laboratory generated bitumen fumes pose lower mutagenic potency than the fumes emitted at normal paving. Further, the fumes from remixing operations may have higher ability to induce mutagenic activity than those from normal paving operations. The use of waste plastics in bitumen increased the mutagenic potency of fumes significantly. There are efforts to find re-uses for waste plastics, road construction and paving being possible alternatives considered. If waste plastics or other organic recycled material will be used in asphalts, more research is needed to assess their effects on workers’ exposure and health. Also, sorting of waste plastics before its possible use in asphalt is important in order to take away plastics that may decompose to hazardous compounds in heating. Our results demonstrated that the use of coal fly ash in asphalts did not increase the mutagenicity of bitumen emissions.
Acknowledgements The authors gratefully acknowledge the assistance of Satu Suhonen and Marjaleena Aatamila in the laboratory analyses, the assistance of Erkki Nykyri in statistical calculations, and the co-operation of Asko Saarela,
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Petri Peltonen and the participating companies. This research was conducted in association to the project of Finnish Research Programme on Environmental Health (SYTTY), and was supported by the Academy of Finland and the Finnish Work Environment Fund.
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Mutagenicity of bitumen and asphalt fumes P.R. Heikkila¨a,*, V. Va¨a¨na¨nenb, M. Ha¨meila¨b, K. Linnainmaab a Department of Epidemiology and Biostatistics, Finnish Institute of Occupational Health, Topeliuksenkatu 41 aA, 00250 Helsinki, Finland Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 aA, 00250 Helsinki, Finland
b
Accepted 1 April 2003
Abstract The mutagenicity of asphalt fumes was tested with the Salmonella bioassays. The aim was to investigate if recycled additives modify the genotoxicity of emissions. Recycling of old asphalt is increasing, and we studied also the mutagenicity of emissions sampled during the re-use of asphalt. The composition of vapours and fumes were analysed by gas chromatography and by liquid chromatography. Bitumens containing coal fly ash (CFA) or waste plastics were heated to the paving temperatures in the laboratory. In the field, bitumen fumes were collected during paving of stone mastic asphalts (lime or CFA as a filler), remixing of stone mastic asphalt (lime or CFA as a filler), and of asphalt concrete. All the lab-generated vapour fractions were non-mutagenic. The particulate fractions were mutagenic with TA98 in the presence of the S9 activation. In addition, the lab-fumes from bitumen containing waste plastics were positive with both strains without S9. Only particulate fractions sampled in the field were tested. They were mutagenic with and without metabolic activation with both strains. The mutagenic potency of the field samples was higher than that of the lab-generated fumes without S9, and the remixing fumes were more mutagenic than the normal paving and lab-generated fumes with S9. The use of inorganic additive, CFA, did not change the mutagenicity of the fumes, whereas the organic additive, waste plastics, increased the mutagenicity of the laboratory emissions significantly. # 2003 Elsevier Ltd. All rights reserved. Keywords: Bitumen; Mutagenicity; Asphalt; Salmonella; Coal fly ash
1. Introduction Bitumen is a complex mixture of petroleum products, used mainly for road paving and for roofing. Asphalt is a mixture of bitumen and mineral matter such as stone, sand, or filler. Bitumen (referred to as asphalt in the USA) contains high proportions of high-molecular-weight paraffinic and naphthenic hydrocarbons, whereas coal tar materials are composed mainly of aromatic hydrocarbons (Concawe, 1992). The re-utilisation of road-making materials is increasing. Asphalt can be recycled by various methods e.g. the old asphalt layer can be removed by cold milling and then transferred to an asphalt plant or it can be scarified as hot and mixed in situ with virgin materials (Concawe, 1992). Recycling operations can lead to increased polycyclic aromatic hydrocarbon and bitumen fume exposures in paving crews (Burtsyn et al., 2000). Also several waste materials and industrial * Corresponding author. Tel.: +358-9-47472215; fax: +358-94773149. E-mail address: pirjo.heikkila@occuphealth.fi (P.R. Heikkila¨). 0887-2333/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0887-2333(03)00045-6
by-products such as glass, coal fly ash, and rubber tires have been used as asphalt modifiers (Butler et al., 2000). Epidemiologic studies of lung cancer among pavers exposed to bitumen fumes have yielded contradictory (Partanen and Boffetta, 1994) results. Many studies have limitations in the design or they failed to control adequately for smoking and possible confounding exposures such as coal tar, diesel exhaust and silica. In the recent IARC European epidemiological study of cancer mortality among asphalt workers, the SMR of lung cancer was slightly higher among the workers employed in jobs entailing exposure to bitumen (1.17, 95% CI 1.04–1.30) than among construction workers (1.01, 95% CI 0.89–1.15) (Boffetta et al., 2003a). After adjusting for coal tar pitch and with 15-year lag, lung cancer mortality slightly increased (SMR=1.23, 95% CI 1.02–1.48) among road pavers (Boffetta et al., 2003b). In animal studies, laboratory-generated bitumen roofing fume (Thayer et al., 1981; Niemeyer et al., 1988; Sivak et al., 1989; Sivak et al., 1997), raw roofing bitumen (Sivak et al., 1989; Sivak et al., 1997), and bitumen
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based paints (Robinson et al., 1984) have shown to be carcinogenic, when applied dermally to mice. In the study of Emmet et al. (Emmett et al., 1981), however, raw roofing bitumen was not carcinogenic after dermal treatments. No animal studies have examined the carcinogenic potential of fumes sampled during paving operations or that of laboratory-generated paving bitumen condensates. Mutagenic potential of laboratory generated bitumen fume and vapour condensates have been tested in several in vitro and in vivo genotoxicity assays. Positive results have been obtained in Ames Salmonella mutagenicity studies. Data have been reviewed in a document published by National Institute for Occupational Safety and Health in the USA (NIOSH, 2000). Generally, the findings have been positive only in the presence of a metabolic activation system (Machado et al., 1993; De Meo et al., 1996; Reinke et al., 2000). Only one study has investigated mutagenic properties of asphalt emissions sampled during asphalt production (Reinke et al., 2000). Asphalt fume condensates collected above the storage tank were not mutagenic (Reinke et al., 2000). Otherwise there has been no published data of the mutagenicity of asphalt emissions collected during the road paving. Further, no studies are available from the situations when recycled materials have been used in asphalts or when old asphalt layers have been recycled. Emissions from asphalt are complex mixture containing hundreds of different compounds. When assessing occupational exposure levels among bitumen workers the air concentration of bitumen fumes and polycyclic aromatic hydrocarbons are generally monitored (Burstyn, Kromhout, Boffetta, 2000; Burstyn, Kromhout et al., 2000). Possible health effects and biological response of fumes emitted from asphalts with different composition or paving temperature are difficult to judge only from the air concentration results. Therefore, testing of possible biological effects will give valuable data in assessing of biological significance of occupational exposures. The aim of this study was to compare the mutagenicity of emissions from the asphalts containing recycled materials, such as waste plastics or coal fly ash, to those from the normal asphalt mixes. The experiments were carried out both in the laboratory and field conditions. In addition, we investigated the mutagenicity of emissions during normal paving and recycling of asphalts.
temperatures used in paving of asphalt mixes (Table 1). The content of toxic metals in coal fly ash was lower than the limit values for contaminated soil (the manufacturer’s data). Waste plastics (WP) composed of polyethylene and polypropylene (79%), polystyrene (11%), polyvinyl chloride (7%) and polymethyl metacrylate (4%). WP was grounded into particles (d=2–3 mm), and mixed with bitumen at 170 C for 24 h. The sampling of emissions started when the temperature of the bitumen mixes was 140 C in all the experiments. The material samples were heated in a glass vessel (700 ml) equipped with a cover, where the sampling filters and adsorbent tubes were attached. The heating method is a modification of the laboratory rig presented by Brandt et al. (1985). About 200 g of deep frozen bitumens was crushed and poured into the vessel which was heated up in an electric mantle. The temperatures were well controlled with a digital thermometer (Elma 1300, NiCrNi, type K), and during sampling, the mixtures were stirred at a constant rate. Emissions were collected on the six parallel samplers per a batch. Sampling times ranged from 1 to 2 h. Both vaporous and particulate fractions of bitumens were sampled. The amount of total particles, vaporous and particulate bitumen emissions, and 15 PAHs were quantified in the collected samples. 2.2. Field generated samples Bitumen fume samples were collected on teflon filters during paving of the following asphalt mixtures: stone mastic asphalts (SMA) (5.7% B80+10% lime as a filler or 6% B80+10% coal fly ash as a filler, remixing of stone mastic asphalt (REMSMA) containing lime (L) or coal fly ash (CFA), and during remixing of asphalt concrete (REMAC) (Table 2). The bitumen fume samples were collected from the breathing zones of the workers. The sampling periods were from 6 to 8 h. In remixing, old pavement was softened by 2–3 heaters, scarified, and mixed in situ with virgin asphalt. Heaters were warmed up with liquefied gas at the remixing of SMAs, and with heavy heating oil at the remixing of asphalt concrete (AC). At the remixing site of AC, heaters were out of condition, and the temperature of road surface rose over 300 C when the recommended temperature range is below 250 C. 2.3. Collection and analysis of air samples
2. Materials and methods 2.1. Laboratory-generated emissions Bitumen (grade B120, Fortum, Kulloo, Finland) and bitumen mixtures containing of waste plastics, or pulverised coal fly ash (CFA) (Fortum, Naantali power plant, Finland) were heated in the laboratory at the
The fumes and vapours were collected on filter cassettes, loaded with SKC Teflon filters followed by SKC XAD-2 tubes (Table 3). The bitumen fume samples on the Teflon filters were eluted with 2 ml of tetrachloroethylene (Merck, p.a.) in an ultrasonic bath for 30 min, and analysed with Fourier-transform infrared (FTIR) spectrometer (Nicolet 20 SXC/60, Nicolet,
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Temperature of bitumen ( C)
Mutagenicity tests
Bitumen B120/ (B120vapours and B120particles)
170
Bitumen B80+coal fly ash (66%)/ (B80-CFAvapours and B80-CFAparticles) Bitumen B120+waste plastics (10%)/ (B120-WPvapours and B120-WPparticles)
170
Salmonella typhimurium TA98 and YG1024 with and without S9 Salmonella typhimurium TA98 and YG1024 with and without S9 Salmonella typhimurium TA98 and YG1024 with and without S9
170–180
Table 2 Mixtures, paving/heating temperatures of asphalt and genotoxicity tests used for the samples collected at the paving sites Asphalt + additive (w-%)/ (fractions studied)
Temp. of paved/ remixed asphalt ( C)
Mutagenicity tests
Stone mastic asphalt+lime (10%)/ (SMA-Lparticles) Stone mastic asphalt+coal fly ash (10%)/(SMA-CFAparticles) Remixing of SMA+lime (10%)/ (REM-SMA-Lparticles)* Remixing of SMA+coal fly ash (10%)/ (REM-SMA-CFAparticles)* Remixing of asphalt cement/(REM-ACparticles)+
175–200
Salmonella typhimurium TA98 with and without S9 mix, and YG1024 without S9 mix Salmonella typhimurium TA98 with and without S9 mix, and YG1024 without S9 mix Salmonella typhimurium TA98 and YG1024 with and without S9 mix Salmonella typhimurium TA98 with and without S9 mix, and YG1024 without S9 mix Salmonella typhimurium TA98 without S9 mix
170–210 180–207/150–260 190/160–250 160/310–350
*heaters of old asphalt operated with liquefied gas. + heaters of old asphalt operated with fuel oil.
Table 3 Fractions sampled and methods of sampling and analysis Fraction sampled
Sampling Media
Flow Rate (l/min)
Analytic Method
Bitumen fume (particulate)
Open-face 37 mm cassette with Teflon filter (SKC 225-1705, 1 mm) XAD-2 (SKC 226-30-06)
1.5
Extraction with tetrachloroethylene; FTIR spectrometry Extraction with dichloromethane; GC with FID Extraction: filters with cyclohexane and adsorbent with acetonitrile HPLC with fluorescence detector
Semi volatile organic compounds (SVOC) PAH particles PAH vapours
Open-face 37 mm cassette with Teflon filter (SKC 225-1707, 2 mm) XAD-2 (OrboTM-43 Supelco)
1.5 1
FTIR=Fourier-transform infrared. GC=gas chromatograph. FID=flame ionization detector. HPLC=high performance liquid chromatograph.
Madison, Wisc. USA) equipped with a mercury-cadmium-tellurium detector (Heikkila¨ et al., 2002). The sorbent layers of the XAD-2 were eluted with 2 ml of dichloromethane (Merck, p.a.) in an ultrasonic bath for 2 h, and analysed by a Hewlett Packard 5890 gas chromatograph (GC), equipped with a flame ionisation detector (FID), and automatic sampler 7672A (Hewlett Packard, Palo Alto, CA, USA) (Heikkila¨ et al., 2002). The PAH compounds were collected on teflon filters connected to XAD-2 adsorbent tubes (Table 3). The filters were extracted with 5 ml cyclohexane (Merck, p.a.) for 30 min in an ultrasonic bath, and the adsorbents were extracted with 2 ml acetonitrile for 40 min in
an ultrasonic bath. The extracts were filtered with Gelman GHP Acrodisc 13 (0.45 mm, P/N 4556) before analysing. The samples were analysed by reversed-phase high-performance liquid chromatography (HPLC). The HPLC system was HP 1100 (Hewlett Packard, Waldronn, Karlsruhe, Germany) and consisted of a vacuum degassed, a high-pressure gradient pump, an autosampler, column thermostat and a scanning fluorescence detector (Kuusima¨ki et al., 2002). Fifteen PAH compounds listed in the NIOSH Manual of Analytical Methods Method 5506 (National Institute for Occupational Safety and Health, 1998) (except acenaphthylene) were quantified, the quantitation limit for PAHs was from 0.002 to 0.008
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mg/m3 for the filters, and from 0.01 to 0.22 mg/m3 for the XAD-2 adsorbents. For qualitative analysis of the vapour and fume, material samples were heated in a laboratory rig, sampled on XAD-2 and on the Teflon filter and desorbed with 2 ml of dichloromethane. The components were separated and characterised by chemical class with the HRGC-MS system (Micromass AutoSpecQ). The ionisation techniques used was EI (electron ionisation, 70 eV). The analytical column was Ultra 2 (cross linked 5% Ph Me Silicone), 25 m0.32 mm I.D., 0,17 mm phase silica capillary column. The carrier gas (helium) pressure was set at 15 psi. The injector temperature was 260 C. The column temperature was programmed as follows: 40 C for 1 min, an increase of 5 C/min up to 270 C and held at 270 C for 13 min. The identification of components was based on the Wiley/NBS Registry of Mass Spectral Data, interpretation of the authentic spectra and the retention time and the elution information from the literature. The concentration of chlorinated aromatic compounds in the emissions from bitumen-waste plastics mixtures (B120-WP) was also determined (Ruokoja¨rvi, Ettala et al., 1995; Ruokojarvi, Ruuskanen et al., 1995). The samples were collected on XAD-2 (SKC 226-30-05) which were eluted with toluene in a Soxhlet for 20 h. After the Soxhlet extraction chlorophenols were eluted with 0.1 M potassium carbonate, acetylated and eluted with n-hexane. The rest of the Soxhlet extraction was evaporated almost to dryness, n-hexane was added, the samples were purified with sulphuric acid and aluminium oxide columns. PAHs and chlorophenols were analysed by a GC (Hewlett Packard 6890, Palo Alto, CA, USA), equipped with a MS detector (Hewlett Packard 5973), the selective ion monitoring mode was used. The analytical column was HP-5MS (30 m0.25 mm0.25 mm). Polychlorinated dioxins and furanes were separated and identified by the HRGC-MS system (VG 70–250 SE). The analytical column was DB-Dioxin (60 m0.25 mm0,15 mm) with the resolution 10 000. Polychlorinated biphenyls and chlorobenzenes were separated and identified by a GC (Hewlett Packard 5890, Palo Alto, CA, USA) equipped with an EC detector, the column was HP-1 (25 m0.32 mm0.52 mm). 2.4. Mutagenicity in the Ames Salmonella tests Mutagenicity of the bitumen and asphalt fumes and vapours were investigated by the Ames Salmonella bacteria assay. The laboratory generated vapours and fumes were first extracted with 4 ml dichloromethane in an ultrasonic bath for 20 min from the adsorbent tubes and filters. Then, 1 ml dimethyl sulphoxide (DMSO) was added, after which dichloromethane was evaporated. From field samples, only particulate fractions were investigated. The Teflon filters were extracted with 2 ml of tetrachloroethylene (C2Cl4, Merck, p.a.) in an
ultrasonic bath for 30 min. The extractions from the same paving site were combined. Tetrachloroethylene was evaporated gently under nitrogen flow, and the residue was dissolved in 1 ml DMSO. Five concentrations of each extract (0.031–0.5 mg/plate) were tested using the frame-shift mutation strains TA98 and YG1024. TA98 was selected because it is known that bitumen fumes contain PAHs (Heikkila¨ et al., 2002) that cause frame-shift mutations at the presence of oxidative metabolic activation. Salmonella typhimurium YG1024 is a derivative of S. typhimurium TA98 with a high level of N-hydroxyarylamine O-acetyltransferase (OAT) activity. This strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes (Einisto et al., 1991). YG1024 was selected because paving workers are exposed not only to bitumen emissions but also e.g., to diesel exhausts from paving machines which are known to contain nitroarenes (Hayakawa et al., 1997). The tests were performed by standard protocols according to OECD guideline (OECD, 1997). As positive control compounds, 4-nitroquinoline (4-NQO, direct mutagen) and 2-aminoanthracene (2-AA, mutagenic with metabolic activation) were applied. Of the laboratory samples, both vaporous and particulate fractions were tested in the presence and in the absence of metabolic activation (S9). Rat liver S9 was prepared from male Wistar rats, pre-treated with Aroclor 1254. S9 mix contained 1 ml S9 fraction and 5.4 ml phosphate buffer (167 mmol), 0.18 ml KCl (1.65 mol), 0.9 ml MgSO4 (80 mmol), 0.9 ml glucose-6-phosphate (50 nmol), and 1.2 ml NADP (40 mmol). S9 mix was filtered before use. Because of the limited amount of the field samples, only a part of the samples could be tested with and without S 9 and with both strains (Table 2). Duplicate cultures were run for each fraction. The mutagenic potency evaluations were based on the slope of the linear regression (B, when y=A+Bx) of two parallel test results. The test result was interpreted to be positive if the slope was > 0 and P-value < 0.05. The slopes and P-values were calculated with SAS/STAT1 package and with the SALM software program that is available in ftp ftp.bios.unc.edu (Kim and Margolin, 1999). The SALM-program gave identical results for the slopes and P-values with SAS/ STAT1.
3. Results 3.1. Composition and concentration of bitumen emissions Vapour and particulate fractions of the laboratory generated emissions were analysed qualitatively. The main components in the vapour fractions were C8H18C19H40, and in the particulate fractions, C10H22-C29H90 straight-chain, aliphatic hydrocarbons. The vapour
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fractions contained also alkyl benzenes, hydrated naphthalenes, indane and alkyl indanes, biphenyl and alkyl biphenyls, several 2–3 ring PAHs and their alkylated derivatives (the main vaporous PAHs being phenanthrene and alkyl phenanthrenes, naphthalene, and alkyl naphthalenes,), alkyl benzothiophenes, and dibenzothiophenes. In addition, the vapours from B120-WP contained alkyl phenols, styrene, and alkyl styrenes. The main PAHs in all the particulate fractions were fluorene, phenanthrene, pyrene and their alkyl derivatives. The particulate fractions contained also biphenyl and alkyl biphenyls, several alkyl benzothiophenes, dibenzothiophene and alkyl dibenzothiophenes. In the laboratory experiments, the concentrations of bitumen vapours ranged from 10 mg/m3 (B80-CFA) to 280 mg/m3 (B120), bitumen fume from 2 mg/m3 (B120WP) to 5 mg/m3 (B80-CFA), and 15 PAHs from 20 mg/ m3 (B80-CFA) to 100 mg/m3 (B120), respectively. At the paving sites, the air concentrations of bitumen vapours ranged from 0.7 mg/m3 (REM-AC) to 2.2 mg/m3 (REM-SMA-CFA), bitumen fume from 0.1 mg/m3 (REM-AC) to 0.6 mg/m3 (SMA-L and SMA-CFA), and 15 PAHs from 5.4 mg/m3 (REM-SMA-CFA) to 13 mg/m3 (REM-AC). The proportions of quantified vaporous and particulate 15 PAHs in the fractions tested are presented in Table 4. The proportion of PAHs in the vapour fraction of bitumen emissions was lower in the laboratory samples (0.01–0.15%) than in the paving samples (0.23– 1.7%), while the proportions of particulate PAHs in the laboratory and field fumes were at the same magnitude (0.069–0.81%) except in the remixing of asphalt concrete (1.5%), when the temperature of old asphalt rose over the recommended value ( > 300 C). Due to the small amount of PVC in the recycled plastics, the content of chlorinated aromatic compounds in the emissions from B120-WP was analysed. The
emissions contained traces of the following compounds: 2- and 4-chlorophenols, dichlorophenols, trichlorophenols, tetrachlorophenols and pentachlorophenols, penta- and hexachlorobenzenes, chlorinated biphenyls. The concentrations of dioxins and furans were below the detection limit of the method. 3.2. Mutagenicity in the Ames Salmonella tests 3.2.1. Laboratory samples The mutagenicity of the laboratory generated samples is presented in Table 5. All the vapour fractions were negative with both strains in the presence and absence of the S9 activation. All the particulate samples (B120particle, B80-CFAparticle and B120-WPparticle) showed mutagenic activity with TA98 + S9-mix. In addition, the fume fraction B80-CFAparticle was weakly (slope =21) positive also without S9-mix. The fume fraction of bitumen-waste plastic mixture (B120-WPparticle) was the only lab-sample that was positive with both strains, TA98 and YG1024, in the absence of the S9 activation (Fig. 1a). The mutagenic index (slope) of the B120-WPparticle fraction was at the same magnitude with and without S9 with TA98, whereas the YG1024 strain gave negative response with the oxidative S9 activation. Bitumen fumes (B120particle) showed a positive result with YG1024+S9, but the variation of the parallel test results was so large that no judgement on the mutagenicity can be made. 3.2.2. Field samples The results of the field samples are presented in Table 6. All the fume samples tested were positive with the both strains with and without the S9 activation. Due to the small amounts of the collected samples, however, all the tests could not be performed with both strains with and without the oxidative metabolic activation.
Table 4 The proportions of vaporous polycyclic aromatic hydrocarbons (PAHs) to semivolatile organic compounds (SVOC), and particulate PAHs and 4–6 aromatic ring PAHs to bitumen fume (BF) in the emissions sampled in the laboratory and during road paving Generation/mass
PAHvapours/SVOC (%)
PAHparticle/BF (%)
PAH4-6 (%)
Laboratory/B120 Laboratory/B80-CFA Laboratory/B120-WP Paving/ SMA-L Paving/ SMA-CFA Remixing/ SMA-L Remixing/ SMA-CFA Remixing/ AC
0.03 0.15 0.01 0.29 0.28 0.71 0.23 1.7
0.81 0.14 0.14 0.16 0.16 0.14 0.069 1.5
0.029 0.051 0.030 0.037 0.025 0.073 0.036 1.2
Bxx=bitumen type. SMA=stone mastic asphalt. L=lime. CFA=coal fly ash. WP=waste plastic. AC=asphalt concrete.
rings/BF
PAH4-6 rings/PAHparticles (%) 4 36 21 23 16 52 52 80
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Table 5 Mutagenicity (revertants/plate) of the bitumen emissions generated in the laboratory Bitumen type and fraction tested
Strain TA98 B120vapour B120particle B80-CFAvapour B80-CFAparticle B120-WPvapour B120-WPparticle Strain YG1024 B120vapour B120particle B80-CFAvapour B80-CFAparticle B120-WPvapour B120-WPparticle
Dose (mg/plate)
Slopest error
Probability
S9 mix
Background control*
0.031
0.063
0.13
0.25
0.50
– + – + – + – + – + – +
20 55 22 31 20 53 22 31 20 53 19 31
17 36 21 45 20 43 23 47 20 43 22 43
19 34 23 41 20 61 25 58 24 51 29 52
19 42 23 48 23 50 24 50 19 52 32 50
21 44 26 48 20 49 26 54 18 51 36 62
17 38 26 56 24 50 33 65 19 57 55 66
3.55 1014 9.65 3712 6.87 4.417 213 4615 4.47 1610 678 5713
0.54 0.48 0.10 0.01 0.33 0.81 <0.01 0.01 0.56 0.12 <0.01 <0.01
– + – + – + – + – + – +
21 126 44 64 35 126 44 64 40 112 41 64
22 87 40 70 31 110 36 59 39 104 39 63
14 91 42 65 34 95 36 58 31 102 50 68
15 100 39.5 76 30 89 41 58 30 93 50 73
18 112 40 73 31 89 46 63 35 81 58 69
20 107 50 85 33 88 44 59 37 93 78 71
1.05 1025 147 3842+ 1.25 5531 1110 1.419 1.48 3622 749 1310
0.85 0.68 0.08 0.18 0.81 0.15 0.32 0.94 0.86 0.13 <0.01 0.22
CFA=coal fly ash (65.7w-%). WP=waste plastic (10w-%). *The sampling media were used as the background controls: XAD-2 for vapours and Teflon filters for particles (Table 3). + The standard error of the slopes in the parallel tests was so large that the mutagenicity cannot be judged reliably although the mean result was positive. The mutagenicity of positive controls, 4-NQO without S9 and 2-AA with S9 were 439-625 and 1224-2462 revertants/plate, respectively.
Table 6 Mutagenicity (revertants/plate) of particulate fractions of emissions sampled during laying of different asphalts Strains and asphalt types Strain TA98 SMA-Lparticles SMA-CFAparticles REM-SMA-Lparticles REM-SMA-CFAparticles REM-ACparticles Strain YG1024 SMA-Lparticles SMA-CFAparticles REM-SMA-Lparticles REM-SMA-CFAparticles
Dose (mg/plate)
Slopest error
P-value
S9 mix – + – + – + – + +
Background control* 14 31 14 31 14 31 14 31 31
0.031 15 39 15 40 19 25 18 32 49
0.063 18 31 13 45 29 38 22 37 49
0.13 22 40 21 30 26 49 26 37 82
0.25 30 53 22 50 27 47 26 63 138
0.50 28 53 27 61 41 71 31 75 271
308 4611 287 5313 449 8111 286 9510 47916
P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01 P<0.01
– – – + –
34 34 34 87 34
31 28 38 95 41
38 29 43 108 43
40 35 41 97 48
43 43 63 127 53
35+ 45 64 197 61
4021 338 6310 21027 4814
P=0.04 P<0.01 P<0.01 P<0.01 P<0.01
*The sampling media were used as the background controls: XAD-2 for vapours and Teflon filters for particles (Table 3). + The highest dose omitted in the testing of mutagenicity (toxic). The mutagenicity of positive controls, 4-NQO without S9 and 2-AA with S9, were 428-513 and 642-846 revertants/plate, respectively. SMA-L=stone mastic asphalt containing lime as a filler, SMA-CFA=stone mastic asphalt containing coal fly ash as a filler, REM=remixing.
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The mutagenic index of REM-ACparticle with TA98+S9 was highest, but unfortunately, the sample was too small to carry the test without S9. The surface temperature of old asphalt was above 300 C, and thus exceeded the standard values due to a failure in a heater. In general, the dose ranges were non-toxic. The highest dose of SMALparticle appeared toxic with YG1024, and this result was therefore omitted. The remixing fume of SMA-L was mutagenic with YG1024 + S9-mix in contrast to the corresponding laboratory fumes, but unfortunately, we could not test the other field samples with YG1024 in the presence of the S9 system due to small amounts of the samples. The mutagenic indices of the laboratory generated fumes were lower than those of the normal paving fumes when tested with both strains without S9 (except B120WP) (Fig. 1a). The mutagenic indices of the remixing fumes were higher than those of normal paving and labgenerated fumes when tested with TA98 and YG1024 at the presence of S9-mix (Fig. 1b). However, in the absence of S9, the indices of the fumes from the normal paving and remixing sites did not differ significantly (Fig. 1a). Only field samples and B120-WPparticle contained compounds that were mutagenic with YG1024 without S9 (Fig. 1a). None of the negative slopes did differ significantly from zero (Tables 5 and 6), thus the negative slopes were due to random variations, not indicators of toxic effects. 3.2.3. Correlation with PAHs PAHs possess mutagenic activity only in the presence of oxidative metabolic activation. Consequently, their possible effect on the mutagenic activity was calculated only for TA98+S9 (Table 7). The concentration of PAHs or 4-6 ring PAHs in the lab fumes did not explain the higher mutagenicity of the B120-WP fumes. In contrast to the laboratory results, a significant or almost significant correlation was seen between the mutagenic indices and the proportion of PAHs, and 4–6 ring PAHs in the field fumes. The correlation was not seen when the field samples were tested without the results of REM-AC. The main particulate PAHs in the laboratory fumes were 2–3 ring PAHs such as fluorene, and the main 4–6 ring PAHs pyrene, benz(a)anthracene and chrysene, respectively. The proportions of 4–6 ring PAHs in particulate PAHs were higher in the remixing fumes (52–80%) than measured in the laboratory or paving fumes (4–36%) (Table 4).
4. Discussion The laboratory-generated bitumen condensates tested in the earlier studies have been mixtures of vapour and fume fractions (Machado et al., 1993; De Meo et al., 1996; Reinke et al., 2000). We studied the effects of vapour and particulate fractions separately. All the laboratory generated vapour fractions were negative;
Fig. 1. (a) and (b). Mutagenic indices (slopes) of particulate emissions from bitumens and asphalts. Emissions were sampled in the laboratory (bitumen and its mixtures containing coal fly ash (CFA) or waste plastics (WP)), during paving of stone mastic asphalt (SMAs), during remixing of SMA (REMSMA) and during remixing of asphalt concrete (REMAC). SMAs contained lime (L) or CFA as an additive. Because of the limited amount of field samples, only fumes from one paving site, REMSMA-L, could be tested with YG1024+S9.
mutagenic compounds in bitumen emissions were bound to particles. Therefore, vapour fractions were not included in the field studies. The mutagenicity results of the particulate fractions generated in the laboratory are in accordance with the earlier studies, which show that lab-generated bitumen emissions have been mutagenic in bacterial tests in the presence of the S9 activation (Machado et al., 1993; De Meo et al., 1996; Reinke et al., 2000). No previous studies on the mutagenicity of fumes from paving sites are available. The fumes from the paving and remixing sites showed as or even more mutagenic than the corresponding laboratory samples with TA98 + S9 activation (Fig. 1b). Our results are in
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Table 7 The correlation coefficients and P-values between the proportion of polycyclic aromatic hydrocarbons (PAH) in bitumen fume and the mutagenicity indices with the strain TA98 in the presence of oxidative metabolic activation, S9 Laboratory fumes Particulate PAHs in bitumen fume 4–6 ring PAHs in bitumen fume
0.835 0.015
Field fumes
Field fumes without REMAC
Laboratory + field fumes
0.986 (P<0.01) 0.995 (P=0.059)
0.820 0.416 (P=0.226)
0.833 (P=0.019) 0.992 (P=0.059)
REMAC=Remixing of asphalt concrete.
conflict with Reinke et al. (2000) who concluded that the asphalt fumes sampled above a hot mix asphalt storage tank (140–157 C) were less mutagenic with TA98 at the presence of S9 than the laboratory-generated fumes. The temperature of asphalt mixes at the paving sites were higher in our than in Reinke’s study (Table 2). The higher temperature likely results different composition of bitumen fumes. The composition of the remixing fumes differed from the laboratory and paving samples regarding the concentration of 4–6 ring PAHs. The content of PAHs, and 4–6 ring PAHs explained, at least partly, the mutagenic potency of the remixing fumes with TA98+S9 (Table 7). However, the complexity of bitumen fumes makes the interpretation of the results difficult. Fumes contained e.g., PAHs, alkylated PAHs, polycyclic aromatic sulphur compounds (PASH) with 1–2 aromatic rings and their alkyl derivatives. In contrast to our findings, Reinke with his co-workers have found mutagenic PASHs with 3–4 aromatic rings in the fumes (Reinke et al., 2000). PASHs with 1–2 aromatic rings such as dibenzothiophenes have not shown mutagenic activity (McFall et al., 1984). Many of the identified compounds such as 4–6 ring PAHs, alkylated PAHs, some methylated PASHs (McFall et al., 1984) and some 3–4 ring PASHs (Pelroy et al., 1983) can exhibit mutagenicity upon oxidative metabolism of the aromatic ring. These compounds likely explain the positive results with TA98+S9. However, these compounds do not explain the mutagenicity found in the absence of S9. Our results suggest that, as a contrast to the laboratory fumes, the fumes from paving and remixing sites contain direct acting mutagens (Fig. 1a). The compounds identified in the fumes do not, however, explain these findings. One field sample was far more mutagenic than the lab samples with YG1024 in the presence of S9. Unfortunately, only one field sample (REMSMA-L) could be tested with YG1024+S9 due to a limited amount of fumes. The concentration of bitumen fume was relatively low at the paving and remixing sites, and although several parallel samples were collected, the amount of fume sampled remained insufficient for all the tests. Therefore, we cannot judge whether remixing fumes with OAT active strain YG1024 in the presence of the S9 activation are more mutagenic than normal paving fumes.
We studied the effects of two recycled products, coal fly ash and waste plastics, on the mutagenicity of bitumen emissions. The mutagenic potency of the fractions from mixtures including CFA did not differ significantly from regular bitumens or asphalts. Coal fly ash itself has shown variable results in numerous bacterial mutagenicity studies (Griest et al., 1982; Mumford and Lewtas, 1982; Morris et al., 1989). However, the waste plastic mixture fumes (B120-WP) were significantly more mutagenic than the other lab-generated fractions in the absence of S9 mix. The mutagenicity of B120-WP with the S9 activation, however, did not differ significantly from the lab-fumes of bitumen or those of bitumen-coal fly ash mixture (Fig. 1b). At the test temperature (170 C), waste plastics partly decomposed. The identified decomposition products could not explain the positive results, however. Many of aromatic chlorinated compounds are not mutagenic (Schoeny et al., 1979; Schoeny, 1982; Safe, 1989), and styrene is not mutagenic without the S9 activation (Loprieno et al., 1978). Thus the mutagens in the emission of B120-WP remained unknown. In summary, our data indicate that the laboratory generated bitumen fumes pose lower mutagenic potency than the fumes emitted at normal paving. Further, the fumes from remixing operations may have higher ability to induce mutagenic activity than those from normal paving operations. The use of waste plastics in bitumen increased the mutagenic potency of fumes significantly. There are efforts to find re-uses for waste plastics, road construction and paving being possible alternatives considered. If waste plastics or other organic recycled material will be used in asphalts, more research is needed to assess their effects on workers’ exposure and health. Also, sorting of waste plastics before its possible use in asphalt is important in order to take away plastics that may decompose to hazardous compounds in heating. Our results demonstrated that the use of coal fly ash in asphalts did not increase the mutagenicity of bitumen emissions.
Acknowledgements The authors gratefully acknowledge the assistance of Satu Suhonen and Marjaleena Aatamila in the laboratory analyses, the assistance of Erkki Nykyri in statistical calculations, and the co-operation of Asko Saarela,
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Petri Peltonen and the participating companies. This research was conducted in association to the project of Finnish Research Programme on Environmental Health (SYTTY), and was supported by the Academy of Finland and the Finnish Work Environment Fund.
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