Application of the Salmonella mutagenicity assay and determination of polycyclic aromatic hydrocarbons in workplaces exposed to petroleum pitch and petroleum coke

Summary Workplaces of an Italian carbon electrode factory, exposed to petroleum pitch and petroleum coke, were studied using a coupled chemical and biological approach to evaluate occupational mutagenic/carcinogenic hazards. Analytical procedures for the determination of polycyclic aromatic hydrocarbons (PAH) and Salmonella/microsome mutagenicity tests (with TA98 and TA 100 strains) were performed on both industrial ingredients (pitch and coke) and airborne particulate matter of the working environment, after fractionating by sequential Soxhlet extractions with four organic solvents of increasing polarity (benzene, chloroform, methanol and acetone). The results showed: (a) the presence of extraordinarily high PAH (carcinogenic and noncarcinogenic) contents in the benzene extracts of petroleum pitch (3.6 wt% of total PAH) and of airborne particulate samples (up to 0.35 wt% of total PAH), in correlation with very high indirect (after metabolic activation) mutagenic responses of benzene extracts with strain TA98; (b) very high indirect mutagenic responses in the other extracts of the airborne particulate samples (especially with strain TA98); (c) the production during the processing at high temperatures of directly acting mutagens (without metabolic activation) which were absent in the starting materials and their release in the air of workplaces. The comparison of chemical analytical and mutagenicity data has proved to be an interesting approach for better defining the relative health hazards due to occupational exposure to potentially mutagenic/carcinogenic petroleum products.     

Atmospheric Deposition of Polycyclic Aromatic Hydrocarbons in an Urban and a Suburban Area of Korea from 2002 to 2004

Atmospheric bulk samples (wet and dry) were collected monthly during 2002 to 2004 from an urban and a suburban area in Korea for assessment of depositional flux and seasonal variations in the concentrations of polycyclic aromatic hydrocarbons (PAHs). PAH depositional flux ranged from 64.1 to 610 μg/m²/y for the urban area and from 65 to 460 μg/m²/y for the suburban area. The fluxes of PAHs measured in this study were comparable with those reported for urban and suburban areas in other countries. The fluxes of particulates and PAHs were higher in winter than in summer, consistent with the greater per capita consumption of fossil fuel in winter than in summer. Ambient temperature played a major role in the seasonal variability in PAH fluxes. Photochemical degradation of PAHs appears to occur during the summer months. The relationship of PAH depositional fluxes with major air pollutants, such as ozone, sulfur dioxide, carbon monoxide, nitrogen dioxide, and presence of particulate matter up to 10 μm in size (PM₁₀), was also investigated. Dominant PAH compounds in both the urban and the suburban locations were benzo[g,h,i]perylene, pyrene, and indeno[1,2,3-c,d]pyrene. Based on the PAH diagnostic ratios and a factor analysis, the major sources of PAHs in the urban and the suburban regions were found to be similar. Diesel exhaust, coal combustion, and gasoline emissions contributed predominantly to atmospheric PAH contamination.     

国产汽车尾气提取物的致突变性研究

采用ＴＡ９８和ＴＡ１００两种菌株检测汽车尾气中颗粒及气体冷凝提取物的致突变性，所有样品均有明显的致突变效应，以诱导ＴＡ９８菌株回变为主。综合比较，由于柴油车颗粒排放量高，每Ｌ排放物诱导的菌落回变数最高，除柴油车和吉普车外，其余汽油车尾气的间接诱变作用均以气体部分为主。     

Correlations between mutagenicity of airborne particles and concentrations of air pollutants]

With highly efficient aspirometry method there were sampled airborne particles in different parts of Wroc?aw in winter and summer. Organic compounds adsorbed on the particles were extracted for 8 hours in Soxhlet apparatus. Concentration of PAHs and benzo(a)pyrene was determined with GC-MS. Mutagenicity of particles was examined with Ames test. Concentrations of airborne particles ranged from 17-144 mg/m3, and organic compounds adsorbed on the particles--1.1-28.6 mg/m3. Concentrations of PAHs from EPA list ranged from 8.3-1211.6 ng/m3, benzo(a)pyrene's ones--from 4.5-709 ng/m3. Airborne particles sampled in many different locations of Wroc?aw in winter and summer displayed mutagenic activity. Air volumes polluted with the particles resulting in mutagenic effect in Ames test in TA 98 strain without activation with fraction S9 ranged from 0.25-42.5 m3. They displayed correlation with concentration of airborne particles (correlation index -0.35), organic compounds adsorbed on the particles (correlation index -0.58), PAHs from EPA list (correlation index -0.52) and benzo(a)pyrene (correlation index -0.52). Physiochemical indexes of air pollution only approximately indicate health hazards caused by mutagenes and cancergenes adsorbed on airborne particles. Therefore monitoring of air pollution should be supplemented with testing their mutagenicity with Ames test.     

Mutagenicity and amounts of polycyclic aromatic hydrocarbons in marijuana smoking tar extracted from pipes]

In spite of strict laws prohibiting it, criminal cases involving marijuana abuse are increasing, resulting in a serious social problem in Japan. The most common method of ingestion is smoking, since abusers feel that this method produces quick psychological effects controversial. Marijuana is known to induce psychological addiction, but it's genotoxic effects are still argued. Marijuana smoking tar has been extracted from seized pipes. The amounts of polycyclic aromatic hydrocarbons (PAH) in the tar were weighed and the mutagenic activity was tested. The results are as follows: 1) Five of the seven PAHs were detected in marijuana tar, although benzo(e)pyrene and perylene were not. 2) There was more pyrene than any other substance detected in the tar, with fluoranthene having the next highest concentration. 3) Mutagenic activity was observed in all samples, with relative mutagenic potency ranging from 0.03 to 1.59. 4) The type of smoking pipe and burning temperature may affect the mutagenic activity and the amount of PAH. 5) Since marijuana smoking tar contains carcinogens, promoters, and mutagens, there is a risk of developing cancer just as in the case of tobacco.     

Particulate Polycyclic Aromatic Hydrocarbons (PAH) in the Atmosphere of Bizerte City,Tunisia

The particle-phase concentrations of polycyclic aromatic hydrocarbons (PAH) were determined in 13 air samples collected in an urban area of Bizerte (Tunisia) during 2009–2010. Atmospheric particulate samples were extracted by ultrasonic bath and analyzed by high-performance liquid chromatography with fluorescence detection. PAH were found in all the analyzed air samples and the most abundant compounds were pyrene, fluoranthene, benzo[g,h,i]perylene, benzo[b]fluoranthene, chrysene and benzo[a]pyrene. ∑14-PAH concentrations ranging from 9.38 to 44.81 ng m^?3 with mean value of 25.39 ng m^?3. PAH diagnostic ratio source analysis revealed gasoline and diesel vehicular emissions as major sources. The mean total benzo[a]pyrene toxicity equivalent calculated for samples was 3.66 ng m^?3 and the mean contribution of the carcinogenic potency of benzo[a]pyrene was determined to be 55.8 %. Concentrations of particulate PAH in Bizerte city atmosphere were approximately eight times greater than sampled at a nearby rural site.     

Polycyclic aromatic hydrocarbon (PAH) residues on skin in relation to air levels among roofers

To assess the utility of skin wipes as an index of exposure to polycyclic aromatic hydrocarbons (PAHs), simultaneous skin wipe and breathing zone air samples were obtained for 10 roofers. Samples were obtained during removal of an old coal-tar pitch roof and application of a new asphalt roof. Skin wipes were obtained immediately before and after the workshift. Anthracene was present in air but not skin samples. In air samples, the relative concentrations of seven PAHs were fluoranthene greater than pyrene greater than benzanthracene greater than benzo[a]pyrene greater than benzo[b]fluoranthene greater than benzo[ghi]perylene greater than benzo[k]fluoranthene. A similar pattern, or rank concentration, was observed in matching skin wipe samples. The amount (ng) of PAH (either total or individual compounds) found in skin wipes taken after the workday was significantly correlated with that in air samples (micrograms/m3, time-weighted average) for 8 of the 9 cases with air and skin samples taken the same day (r = 0.99 for total PAH). Skin wipe PAH residues were not significantly correlated with air samples taken 4 d earlier. These findings suggest that skin wipes can provide a useful measure of exposure to PAH.     

The mutagenic hazards of environmental PM2.5 in Turin

Owing to the large number of natural and anthropogenic sources, particulate matter (PM) may present several physical and chemical patterns in different areas. The finer PM2.5 fraction, which is now widely but not routinely measured in Europe, is considered to be the alveolar fraction of the ambient particles. Annual and winter mean concentrations of PM2.5 substantially vary in Europe, with higher concentrations in the South. The aims of this work were to (a) measure the PM2.5 levels in Turin over a long period, (b) evaluate mutagenic activities of organic extracts containing this collected complex mixture using the Ames test and (c) determine the level of polycyclic aromatic hydrocarbons (PAHs) in order to identify important mutagens in ambient air. Sampling was carried out from November 2001 to December 2004. The monthly mean of PM2.5 was 48.76+/-24.12 microg/m3. From the beginning to the end of the sample period there was a decrease in gravimetric levels, with annual means of 54.10+/-29.77 microg/m3 in 2002; 42.48+/-15.73 microg/m3 in 2003 and 45.89+/-24.92 microg/m3 in 2004. Samples were tested for mutagenicity using Salmonella typhimurium strains TA98 and TA100, with and without S9 mix metabolic activation. A positive genotoxic response was observed for TA98, with and without metabolic activation. The measured PAHs monthly mean level was 8.24+/-6.30 ng/m3, with values ranging from 0.20 to 21.38 ng/m3 Seasonal variation of gravimetric, mutagenic and PAH values was significant. The Salmonella assay results statistically correlated to PM2.5 and PAHs levels, but sometimes the mutagenic potencies were rather different despite an equal concentration of pollutant. The results confirm the usefulness of this biological approach to detect genotoxic properties of sampled PM2.5 and they show the variability of the mutagenic properties of the airborne mixture over time.     

Mutagenic activity of heated potato/oil systems

Mutagens detected with Salmonella typhimurium strain TA 98 in the presence of liver S9 mix were extracted from potato slices, but not pure potato starch, after frying in oil. No mutagenic activity was detected using strain TA 100, in the presence or absence of S9 mix with either fried potato slices or potato starch. Mutagenic activity was detected at frying temperatures of 140 degrees C and above. The mutagenic activity was limited to the outer portion of the fried potato slices and increased with frying time and temperature. Mutagenic activity ratios for extraction with both (NH4)2SO4/NH4OH and Na2SO4/NaOH were similar.     

Mutagenic activity of heated potato/oil systems

Mutagens detected with Salmonella typhimurium strain TA 98 in the presence of liver S9 mix were extracted from potato slices, but not pure potato starch, after frying in oil. No mutagenic activity was detected using strain TA 100, in the presence or absence of S9 mix with either fried potato slices or potato starch. Mutagenic activity was detected at frying temperatures of 140°C and above. The mutagenic activity was limited to the outer portion of the fried potato slices and increased with frying time and temperature. Mutagenic activity ratios for extraction with both (NH4)2SO4/ NH4 OH and Na2 SO4 /NaOH were similar.     

Testing for mutagens in filter samples from the work atmosphere of an aluminum plant

Filter extracts of airborne particles from a S?derberg potroom and an anode paste plant were tested for mutagenicity by the Salmonella reversion assay. The extracts were mutagenic to strains TA100 and TA98, mainly after metabolic activation, but positive results were obtained also without S9 mix in strain TA98. These findings indicate that the particulate phase of air from the potroom and the anode paste plant of aluminum plants contain mostly indirect mutagens of both the base-pair substitution and frameshift type, and--to a less degree--frameshift mutagens. The relationship between concentration and mutagenicity was more positive for the potroom extract than for the anode paste plant extract.     

Analysis of particle and vapour phase PAHs from the personal air samples of bus garage workers exposed to diesel exhaust

The levels of particle and vapour phase polycyclic aromatic hydrocarbons (PAHs) derived from the diesel exhaust compounds in bus garage work were measured in winter and in summer. Five personal air samples were collected from the breathing zones of 22 garage workers every other day of consecutive weeks. Control samples (n = 22) were collected from office workers in Helsinki. Fifteen PAHs in the air samples were analysed by HPLC using a fluorescence detector. Statistically significant differences were observed between total PAH levels of the exposed workers (2241 and 1245 ng/m(3)) and the control group (254 and 275 ng/m(3)) in both winter (P < 0.001) and summer (P < 0.001). Phenanthrene, pyrene, benzo[ghi]perylene and fluoranthene were the major compounds in the particle phase, and naphthalene, phenanthrene and fluorene in the vapour phase. About 98% of PAHs measured were related to the vapour phase compounds, whereas the high molecular weight PAH compounds were detected only in the particle phase. The PAH levels in the garages were twice as high (P < 0.001) in winter as in summer. Even though the exposure levels were low in the bus garages, the low level does not allow conclusions to be drawn about the possible adverse health effects due to exposure to diesel exhaust.     

Assessment of exposure to polycyclic aromatic hydrocarbons in police in Florence, Italy, through personal air sampling and biological monitoring of the urinary metabolite 1-hydroxypyrene

In this study, the authors evaluated exposure to airborne polycyclic aromatic hydrocarbons (PAHs) in workers exposed to exhaust gas from cars, and they assessed the efficiency of urinary 1-hydroxypyrene as an indicator of exposure to pyrene and PAHs. The authors selected 2 groups of police who worked in 2 areas in the city of Florence: 1 group was highly exposed to high-density traffic emissions during the winter and summer of 1997, and the 2nd group experienced low exposure to traffic emissions during the same period. Ambient monitoring was achieved with personal sampling of airborne PAHs during each workshift. Eight hydrocarbons were used as indicators of pollution caused by PAHs (e.g., pyrene, benzo[a]pyrene, benzo[a]anthracene, dibenzo[a,h]anthracene). Biological monitoring was performed through dosing of 1-hydroxypyrene (pyrene metabolite) in urine samples taken at the end of each workshift. The ambient monitoring revealed that PAH concentrations were influenced by both season of sampling and varying intensities of traffic in the different areas. The median concentration of benzo[a]pyrene in winter was twice as high in the high-density traffic area as in the low-density traffic area (i.e., 4.1 ng/m3 versus 1.8 ng/m3). In summer, the high-density traffic area experienced benzo[a]pyrene concentrations that were 6 times higher than in the low-density traffic area (i.e., 1.2 ng/m3 versus 0.2 ng/m3). Benzo[a]pyrene was also correlated highly (r(s) = .92, p < .0001) with the mixture of total PAHs analyzed, thus confirming its function as a good indicator of exposure to PAHs in an urban environment. Levels of urinary 1-hydroxypyrene appeared to be generally influenced by the intensity of traffic, especially during the winter (i.e., median value in winter was 199.2 ng/gm creatinine in the high-density traffic area and 120.5 ng/gm creatinine in the low-density traffic area). An analysis of the general data revealed that 1-hydroxypyrene was, to some degree, related to pyrene, benzo[a]pyrene, and airborne total PAHs, whereas analysis of separate data for the area and the season revealed an emergence of a closer correlation during the winter in the high-traffic area. Therefore, 1 -hydroxypyrene can be considered a good biological indicator of exposure to airborne PAHs in the urban environment, especially in winter and in high-density traffic areas.     

Increases in polycyclic aromatic hydrocarbon content and mutagenicity in a cutting fluid as a consequence of its use

Summary The aim of this study was to evaluate the relationships between the length of time a cutting fluid was used, its content in polycyclic aromatic hydrocarbons (PAHs) and its mutagenic potential. The PAH concentrations were determined by means of a high-resolution gas chromatograph-mass spectrometer in samples of new cutting fluid and in samples used for 3, 6 and 9 months. The following PAHs were measured: phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene + triphenylene, benzo [e] pyrene, benzo [a]-pyrene and perylene. Mutagenicitiy assays were carried out on the aforementioned samples using the Ames test. Salmonella typhimurium TA98 was used as an indicator to show up mutagens capable of inducing frame-shift genetic changes, and Escherichia coli WP2 uvrA was used as an indicator to detect mutagens capable of inducing base pair genetic changes. The mutagenic tests were carried out with and without microsomial activation, using 1:1, 1:10, 1:20 and 1: 50 dilutions of cutting fluid samples. An increase in the concentrations of total PAHs over time was observed in the samples of cutting fluid used for 3, 6 and 9 months. The highest percentage increase in PAH concentrations was observed in the 6-month-old sample (10 times the initial concentration, from 45 to 411.8 g of oil). None of the samples were mutagenic to S. typhimurium without metabolic activation or to E. coli with and without metabolic activation. All samples except for the 1:1 diluted sample showed moderate but significant mutagenic activity in the S. typhimurium test with metabolic activation. Mutagenicity rose to its highest levels (similar to those observed with the mutagenic compound 2-acetylaminofluorene, which was used as a positive control) in the 9-month-old sample and in the 6-month-old 1:20 and 1:50 diluted sample. This study shows an increase in the PAH concentrations and mutagenicity of cutting fluids following prolonged use. In order to minimize the risk caused by such an increase in PAH content, it is recommended that the cutting fluid be changed at least every 6 months.     

Urinary screening for potentially genotoxic exposures in a chemical industry.

Mutagenic activity, measured by the bacterial fluctuation assay and thioether concentration in urine from workers at a chemical plant producing pharmaceuticals and explosives, was determined before and after exposure. Of 12 groups only those exposed to trinitrotoluene (n = 14) showed a significant increase in mutagenic activity using Salmonella typhimurium TA 98 without any exogenous metabolic system. The same strain responded only weakly when the S-9 mix was used; with Escherichia coli WP2 uvrA no effect of exposure was observed. Urinary thioether concentration was higher among smokers than among non-smokers, but occupational exposure had no effect. Urinary mutagenicity testing may be a useful tool for screening potentially genotoxic exposures in complex chemical environments.     

Urinary screening for potentially genotoxic exposures in a chemical industry

Mutagenic activity, measured by the bacterial fluctuation assay and thioether concentration in urine from workers at a chemical plant producing pharmaceuticals and explosives, was determined before and after exposure. Of 12 groups only those exposed to trinitrotoluene (n = 14) showed a significant increase in mutagenic activity using Salmonella typhimurium TA 98 without any exogenous metabolic system. The same strain responded only weakly when the S-9 mix was used; with Escherichia coli WP2 uvrA no effect of exposure was observed. Urinary thioether concentration was higher among smokers than among non-smokers, but occupational exposure had no effect. Urinary mutagenicity testing may be a useful tool for screening potentially genotoxic exposures in complex chemical environments.     

Significance of dermal and respiratory uptake in creosote workers: exposure to polycyclic aromatic hydrocarbons and urinary excretion of 1-hydroxypyrene.

OBJECTIVES--To evaluate workers' exposure in a creosote impregnation plant by means of ambient and biological monitoring. METHODS--Naphthalene (vapour phase) and 10 large molecular polycyclic aromatic hydrocarbons (PAHs) (particulate phase) were measured in the breathing zone air during an entire working week. 1-Hydroxypyrene (1-HP) was measured in 24 hour urine as a metabolite of the pyrene found in neat (dermal exposure) and airborne creosote. RESULTS--Naphthalene (0.4-4.2 mg/m3) showed 1000 times higher concentrations in air than did the particulate PAHs. In total, the geometric mean (range) of three to six ring PAHs was 4.8 (1.2-13.7) micrograms/m3; pyrene 0.86 (0.23-2.1) micrograms/m3, and benzo(a)pyrene 0.012 (0.01-0.05) micrograms/m3. There was no correlation between pyrene and gaseous naphthalene. The correlations between pyrene and the other nine particulate PAHs were strong, and gave a PAH profile that was similar in all air samples: r = 0.83 (three to six ring PAHs); r = 0.81 (three ring PAHs); r = 0.78 (four to six ring PAHs). Dermal exposure was probably very high in all workers, because the daily output of urinary 1-HP exceeded the daily uptake of inhaled pyrene by < or = 50-fold. Urinary 1-HP concentrations were very high, even on Monday mornings, when they were at their lowest (4-22 mumol/mol creatinine). 1-HP seldom showed any net increase over a workshift (except on Monday) due to its high concentrations (16 to 120 mumol/mol creatinine) in the morning samples. 1-HP was always lower at the end of the shift (19 to 85 mumol/mol creatinine) than in the evening (27 to 122), and the mean (SD) change over the working week (47 (18)) was greater than the change over Monday (35 (32)). The timing of 1-HP sampling is therefore very important. CONCLUSIONS--Urinary 1-HP proved to be a good biomarker of exposure to three to six ring PAHs but not to airborne naphthalene. Hence, biomonitoring based on 1-HP has to be completed with exposure assessment for naphthalene as a marker for creosote volatiles that mainly enter the body through the lungs.     

Occupational exposure to polycyclic aromatic hydrocarbons in airborne particulate matter: validation and application of a gas chromatography-mass spectrometry analytical method

This study concerns the validation of an analytical method for the measurement of occupational exposure to trace levels of polycyclic aromatic hydrocarbons (PAHs) in airborne particulate matter (APM). Personal exposure to selected PAHs of five workers occupationally exposed to urban pollution in Rome, Italy, was evaluated. The samples were collected over 10 days evenly distributed during winter and summer of 2008. Polycyclic aromatic hydrocarbons were collected by a sampling pump and trapped in polytetrafluoroethylene filters; ultrasonic extraction was applied to extract PAH species from the matrix with toluene, and the concentrated extract was quantitatively analyzed by GC/MS. The analytical method was optimized and validated using a standard reference material of urban dust (SRM 1649a). Detection limits ranged from 0.8 ng per sample for indeno [1,2,3-cd] pyrene to 20.4 ng for sample for anthracene. Experimental results of the 50 personal samples collected showed that phenanthrene was the predominant polycyclic aromatic hydrocarbon [95% CI (32.42-41.13 ng m(-3))]; the highest benzo[a]pyrene concentration was 2.58 ng m(-3), approximately 2-fold higher than European annual target values (1 ng m(-3)). Seasonal variations of personal exposure to selected PAHs suggested higher emissions and reduced atmospheric reactivity of PAH compounds in winter. The analytical method was a suitable procedure for the determination of 13 of the 16 priority PAHs in APM personal samples and can be considered a useful tool to evaluate occupational exposure to low PAH levels.     

Effect of prenatal PAH exposure on birth outcomes and neurocognitive development in a cohort of newborns in Poland. Study design and preliminary ambient data

Preliminary data suggest an association between infant mortality rates and several measures of ambient air pollution, including dustfall and polycyclic aromatic hydrocarbons (PAH). The effects of airborne PAH components on fetal growth and early childhood development are of primary interest since we have previously demonstrated that these pollutants were associated with significant decrements in birthweight, length and head circumference in Polish newborn babies. The undertaken research combines a state-of-the-art environmental monitoring and molecular approaches with comprehensive neurodevelopment assessments. A further innovation is the incorporation of biomarkers (lead and antioxidants in cord blood) to control for potential confounding of exposure-effect. The mean overall concentrations of specific PAH compounds in Kraków were highest for benzo(b)fluoranthene (23 ng/m3), benzo(a)anthracene (16 ng/m3), indeno(l,2,3-cd)pyrene (14 ng/m3), chyresene (13 ng/m3) and benzo(a)pyrene (12 ng/m3). In general, concentrations of the PAH compounds measured by personal monitoring were considerably higher among the residents of the higher polluted area of the city. The comparison between the Kraków and New York City monitoring ambient data demonstrates that the concentrations and proportions of specific compounds in the total PAH mixture differ widely across these cities. Thus, one may expect that the PAH-related health risks would differ between the two areas not only because exposures are different, but also because the PAH profile differs. Presumably, this different profile is a consequence, at least in part, of variations in the sources of fossil and solid fuels used in production of energy. In Poland, solid fuels such as coal is used to a much greater extent than in New York City, where fossil fuels are the universal source of energy production. Auto emissions for example, are low in benzo(a)pyrene, whereas such emissions from refuse burning are high.     

Embryotoxicity of organic extracts from airborne participates in ambient air in the chicken embryo

A fraction containing polycyclic aromatic hydrocarbons (PAHs), prepared from an organic extract of airborne particulate matter, was separated into nine subfractions by high pressure liquid chromatography (HPLC). The embryotoxicity of each of these fractions was investigated and analysis for PAHs by capillary gas chromatography-mass spectrometry (GC-MS) was performed. The ninth subfraction, with coronene as its main component, had the strongest toxic effects on chicken embryos per m³ of air. Of the remaining eight subfractions, three had the greatest toxicity: the second fraction with benzofluoranthenes and benzo[e]pyrene as the main components, the fourth fraction having PAH-estimated compounds in small amounts, and the fifth fraction with indeno[l,2,3-cd]pyrene and benzo[ghi]perylene as the main ingredients had the greatest toxicity. These findings indicate PAHs to be responsible for embryotoxicity but their total amounts were not necessarily proportional to it. For further characterization of toxicity, the effects of each PAH and/or quantification of other embryotoxic compounds possibly present in small amounts should be investigated.