Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: Study design, sample generation, collection, and preparation

A major diesel emissions research program has been initiated by the U.S. Environmental Protection Agency to assess the human health risk associated with increased use of diesel automobiles. This program is intended to establish the mutagenic and carcinogenic potency of complex organics associated with diesel particles as well as comparative particle-bound organics from other environmental emissions for which human epidemiological data are available. The mobile source samples selected for this study were collected from a heavy-duty diesel engine, a series of light-duty diesel passenger cars, and a gasoline catalyst automobile. The comparative source samples incorporated into the study were cigarette smoke condensate, coke oven emissions, roofing tar emissions, and benzo(a)pyrene. The samples were tested using three mutagenic assays and four carcinogenic assays as prescribed by a test matrix. This report describes the study design, particle generation, and sample collection and preparation. A brief summary of the bioassays is also included.     

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: Summary and discussion of the results

The proposed conversion from gasoline powered automobiles to diesel powered vehicels has prompted the Environmental Protection Agency to evaluate the potential health effects associated with exposure to diesel emissions. At present, there is no direct epidemiological link between this exposure and human health. Therefore, a research program was constructed to compare the health effects associated with diesel emissions with those from other emission sources for which epidemiological information was available. The emission sources chosen were cigarette smoke, roofing tar, and coke oven. An additional comparative emission source which was a gasoline catalyst engine. Respirable particles from a variety of combustion sources have the potential of being carcinogenic and mutagenic. The objective of these studies was to determine the relative biological activity of the organic material adsorbed on these particles in both in vitro mutagenesis and in vitro and in vivo bioassays. The organic extracts from the following series of emission sources were quantitatively bioassayed in a matrix of tests for their carcinogenic and mutagenic activity: (1) a light-duty Oldsmobile diesel 350 engine; (2) a heavy-duty Caterpillar diesel engine; (3) a light-duty Nissan engine; (4) a Volkswagen Rabbit diesel engine; (5) cigarette smoke; (6) roofing tar; (7) coke oven; and (8) a gasoline catalyst Mustang. The test matrix consisted of the following bioassay: reverse mutation in Salmonella typhimurium; mitotic recombination in Saccharomyces cerevisiae; DNA damage in Syrian hamster embryo cells (SHE); sister chromatid exchange in CHO cells; gene mutation in L5178Y mouse lymphoma cells, Balb/c 3T3 mouse embryo fibroblasts and CHO cells; viral enhancement of SHE cells; oncogenic transformation in Balb/c 3T3 cells; and skin tumor initiation in SENCAR and C57 black mice. The results of this test matrix are discussed.     

Mutagenic and carcinogenic potency of extracts from diesel related environmental emissions: Simultaneous morphological transformation and mutagenesis in BALB/c 3t3 cells

Particulate extracts from six different environmental emission sources were assayed for genotoxic activity in mouse BALB/c 3T3 clone A31-1 cells. All compounds were tested simultaneously for both transforming and mutagenic (induction of ouabain-resistance) potential with and without exogenous metabolic activation in the form of a 9000 × g postmitochondrial hepatic supernatant fraction from Aroclor-1254 induced Fischer 344 rats. Dichloromethane particulate extracts from the exhaust of two light duty diesel engines (Oldsmobile and Nissan), one heavy duty diesel engine (Caterpillar) and one late model gasoline engine (Mustang II) were assayed in an identical manner to particulate extracts from the emissions of a roofing tar pot and a coke oven. No clear dose-dependent responses were observed, but several of the samples showed significant transforming and mutagenic activity. A qualitative ranking system showed the activity of these particulate extracts for either mutagenesis or transformation was: coke oven = Mustang II gasoline engine > Nissan diesel engine > roofing tar. Particulate extracts from the Oldsmobile diesel engine and the Caterpillar diesel engine showed essentially no activity.     

Mutagenic and carcinogenic potency of extracts of diesel and related environmental emissions: In vitro mutagenesis and oncogenic transformation

Extracts from emissions of four diesel engines, a gasoline engine and three related environmental samples were tested in four in vitro assay systems designed to detect carcinogenic or mutagenic activity of chemicals. Samples from three of four diesel extracts, the gasoline engine, and all three related samples were positive in an enhancement of viral transformation assay. Two diesel samples, the gasoline engine extract and extract from coke oven emissions were positive for mutation induction in Chinese hamster ovary cells. Only the gasoline engine extract and the coke oven sample were positive in a DNA fragmentation assay using alkaline sucrose gradients. Experiments using chemical transformation of Syrian hamster embryo cells as an assay method have not been completed.     

Mutagenic and carcinogenic potency of diesel and related environmental emissions: Salmonella bioassay

Due to the expected increase in the percentage of diesel vehicles in the United States, the Environmental Protection Agency must evaluate the health effects associated with exposure to diesel emissions. Respirable particles from a variety of combustion sources have the potential of being carcinogenic and mutagenic. The objective of these studies was to determine the relative biological activity of the organic material adsorbed on these particles in in vitro mutagenesis bioassays. The organic extracts from the following series of emission sources were bioassayed in the Salmonella assay for mutagenic activity: (1) a light-duty Oldsmobile diesel 350 engine; (2) a heavy-duty Caterpillar diesel engine; (3) a light-duty Nissan engine; (4) a Volkswagen Rabbit diesel engine; (5) cigarette smoke; (6) roofing tar; (7) coke oven; and (8) a gasoline catalyst Mustang. This paper provides a comparison of these sources within the Salmonella bioassay and also demonstrates how bacterial systems can be used as a quality assurance measure in in vivo testing.     

DNA-binding studies with diesel exhaust particle extract

This study examines whether chemical components from diesel exhaust particulates react with DNA to form covalently bound adducts. Experiments in this report describe the in vitro reaction of purified DNA with a dichloromethane extract of diesel exhaust particulates in the absence or presence of enzyme activation by rat liver microsomes. The reactivity of the particle extract was compared to that of benzo[a]pyrene metabolites using low temperature fluorescence techniques which detect small quantities of polycyclic aromatic compounds bound to DNA. Incubation of DNA with the particle extract in the presence of microsomal enzymes produced no detectable fluorescent adducts in contrast to model experiments using benzo[a]pyrene. However, addition of the particle extract to incubation mixtures containing benzo[a]pyrene markedly decreased formation of benzo[a]pyrene-DNA adducts because the particle extract inhibits microsomal enzymes which activate benzo[a]pyrene and other polycyclic aromatic hydrocarbons. In the absence of microsomal enzymes, fluorescent material was detected in DNA exposed to high concentrations of the particle extract, but probably not as a result of covalent binding because the mutagenic activity of the particle extract remained unchanged during prolonged incubation with DNA. This stability is in contrast to the rapid decrease in mutagenic activity of benzo[a]pyrene-4,5-oxide during incubation with DNA. Thus, direct mutation of bacteria by the particle extract may require activation by bacterial enzymes as is known to occur with nitroaromatic compounds.     

Salmonella/microsome mutagenicity assays of exhaust from diesel and gasoline powered motor vehicles

Motor vehicle exhaust from prechamber injection diesel and gasoline powered passenger cars, sampled during US FTP 1973 test cycles and comprising both particulate matter and compounds condensable at ambient temperature, has been assayed for mutagenicity in the Salmonella/microsome test. Mutagenic components were to a large extent active in the absence of the mammalian microsomal preparation. The mutagenicity of both particulate matter and condensate from diesel exhaust and condensate from gasoline exhaust was decreased in the presence of the microsomal preparation whereas the mutagenicity of particulate matter from gasoline exhaust was enhanced by microsomal activation. A comparison between the investigated diesel and gasoline exhaust samples shows that the mutagenic effect in the Salmonella test of the diesel exhaust is more than ten times higher than that of the gasoline exhaust. Fractionation with respect to polarity indicates that the mutagenic components mainly are distributed in neutral aliphatic, aromatic, and oxygenated fractions. Tests for mutagenic monofunctional nitroarenes by an anaerobic assay indicate that such compounds at most are marginally present in the exhaust samples as compared with their presence in airborne particulate matter collected in an urban environment.     

Detection and average content levels of carcinogenic and mutagenic compounds from the particulates on diesel and gasoline engine mufflers

Carcinogenic and mutagenic compounds, which were extracted from the particulates that adhered to inner surfaces of diesel and gasoline engine mufflers, were quantified by the series method of Soxhlet extraction, liquid-liquid partition, thin-layer chromatography, and spectrofluorometry. Mutagenic activity of their neutral and acidic fractions was tested in the improved Ames assay by the preincubation method with Salmonella typhimurium TA98 in the presence and absence of metabolic activation system (S-9 mix). The average content levels (μg/g tar) of polycyclic aromatic hydrocarbons from gasoline engine cars were greater than those from diesel engine vehicles. However, the levels of nitro derivatives of PAHs and polycyclic quinones from the diesel engines were greater than from the gasoline engines. Mutagenic activity of the diesel acidic fraction was the highest among the diesel and gasoline fractions, and was significantly higher in the absence of the S-9 mix. Furthermore, the relative value (R_{c = 0}) of infrared absorption of carbonyl stretching vibration to that of methylene asymmetric stretching vibration of the diesel acidic fraction was the highest among the diesel and gasoline fractions. These results strongly suggest that highly direct-acting mutagens in the acidic fraction are at higher levels in diesel emission particulates than those from gasoline, and that these mutagens are carboxylic acid, aldehyde, and alcohol derivatives of PAHs and NPAHs.     

Metaphase analysis, micronuclei assay, and urinary mutagenicity assay of mice exposed to diesel emissions

Female Swiss mice were exposed 8 h/day to diesel exhaust for 1, 3, and 7 weeks. Urine was collected overnight for 4 days prior to sacrifice while the mice continued to be exposed for eight hours during the day. The presence of mutagens was determined by the Ames Salmonella test. One hour prior to sacrifice each mouse received 1 mg/kg colcemide. After sacrifice, the marrow from each femur was obtained. The marrow from one femur was used to prepare slides for metaphase analysis and the other for micronuclei assay. Other mice received IP 50 mg/kg cyclophosphamide 24 h prior to sacrifice or 1 μmole/kg benzo(a)pyrene in each of four daily doses prior to sacrifice and served as positive controls. The Ames Salmonella assay of the unconcentrated urine after 1, 3, and 7 weeks and concentrated urine after 7 weeks exposure to diesel exhaust did not significantly vary from clean air controls. In the micronucleus test, and metaphase analysis, cyclophosphamide produced a strong positive response and the 7 week diesel exposure was not different from clean air controls.     

Genotoxicity of syrian hamster lung cells treated in vivo with diesel exhaust particulates

Polycyclic aromatic hydrocarbons extracted and concentrated from diesel exhaust particulates have been shown to be mutagenic and carcinogenic, but attempts to induce pulmonary tumors through chronic inhalation of diesel exhaust by experimental animals have failed. We have attempted to resolve this incongruity by measuring chromosomal damage in lung tissue of chronically exposed hamsters, using the highly sensitive test for genotoxic chemical agents, sister chromatid exchange (SCE) analysis. To determine the degree of responsiveness of the test system to both diesel exhaust particulates and benzo(a)pyrene (BaP), these agents were instilled intratracheally into anesthetized hamsters as suspensions in 0.25 ml volumes of Hank's balanced salt solution (HBSS). Lung tissues from these animals were subsequently cultured in vitro and chromosomes from the resulting cell divisions were scored for exchanges of chromatin between sister chromatids. Control animals, treated weekly with 0.25 ml of BSS for 10 weeks, showed an average value of 12 SCE's per cell, while animals treated weekly with 200 ng BaP over a 10-week period showed an average of 17 SCE's per cell. HBSS, given as a single treatment, also produced an average of 12 SCE's per cell in control animals, but animals treated with a single instillation of 12.5 μg BaP showed an average SCE value of 19. These data confirmed that the procarcinogen BaP can be metabolically activated by lung cells in vivo and also demonstrated the efficacy of using this technical approach to study the effect of chemical mutagens that enter the lungs. Diesel exhaust particulates, administered in a range from 0 to 20 mg per hamster over a 24 h exposure period, produced a linear SCE dose-response ranging from 12 to 26 SCE's per metaphase. This curve suggested that a concentration of 3 mg of diesel particulates per hamster would not produce a statistically significant increase in SCE's above control values. One group of 8 hamsters, chronically exposed to diesel exhaust particulates for 3 months showed an average of 12 SCE's per cell. This was equivalent to a set of 5 control animals which also showed an average of 12 SCE's per cell. Although the scope of this study was limited, the data demonstrated that diesel exhaust particulates can induce genotoxic damage but a 3-month exposure to 6 mg/m³ of diesel exhaust particulates was insufficient to produce measurable mutagenic changes in lung cells. This negative response is consistent with the results from other studies in which similar exposures failed to produce pulmonary tumors.     

The mutagenic activity of thirty polycyclic aromatic hydrocarbons (PAH) and oxides in urban airborne particulates

Data are presented on the mutagenicity of an organic extract of a composite sample of urban air particulates and of thirty PAH compounds in such samples, including four quinone derivatives, isolated quantitatively by thin-layer chromatography and identified by fluorescence or other spectral techniques. Mutagenic activity was determined by the Ames assay, using histidine auxotrophs of Salmonella typhimurium strains TA98, TA100, TA1537 and TA1538. All compounds were dissolved in dimethyl sulfoxide (DMSO) which was the least toxic of eight organic solvents tested.The mutagenic activity of a benzene extract of suspended particulates from the air of Hamilton, Ontario was significantly greater with strain TA98 than with strain TA100, suggesting the presence of more frame-shift acting mutagens. The mutagenic response of this extract was similar with and without S-9 activation.Mutagenic tests on the 30 PAH compounds indicated that only the benzo(a)pyrene quinones were direct acting mutagens. All of the chemical compounds were tested with and without S-9 activation. The following PAH showed unequivocal mutagenic activity, with S-9 activation: benz(a)anthracene, benzo-(a)pyrene, benzo(ghi)perylene, benzo(rst)pentaphene, benzophenanthrene, chrysene, 1, 2, 3, 4-dibenzanthracene, 2, 3, 6, 7-dibenzanthracene, and 3-methyl cholanthrene. The quinones of 1, 6-; 3, 6-; and 6, 12-benzo(a)pyrenes showed weak mutagenic activity but 3, 6-benzo(a)pyrene elicited also a photodynamic response.     

红枫湖地表水中多环芳烃的分布及来源

将固相微萃取与气相色谱联用，对贵阳红枫湖水样中16种美国环境保护署优控的多环芳烃进行分析。结果表明：红枫湖水中16种多环芳烃总量为0167 1~0336 4 μg/L，与国内其它水系相比，湖中存在多环芳烃轻度污染。7种（萘、荧蒽、苯并(b)荧蒽、苯并(k)荧蒽、苯并(a)芘、茚并(1,2,3-cd)芘和苯并(ghi)苝）多环芳烃的总量未超出中国城市供水行业对多环芳烃规定的限值，但作为饮用水源，红枫湖水中的苯并(a)芘含量已超出我国标准GB3838-2002中生活饮用水地表水源地的苯并(a)芘限值，并且苯并(a)蒽、〖JX-*9〗〖SX（B-25x〗〖HT7，5”〗艹〖〗〖HT6”，5”〗屈〖HT5”〗〖SX）〗〖JX*9〗、苯并(b)荧蒽、苯并(k)荧蒽、苯并(a)芘、茚并(1,2,3-cd)芘的含量也超过了美国环境保护署地表水水质标准限值。通过多环芳烃特征参数的比值，分析了红枫湖水中多环芳烃的污染来源。污染源分析表明，湖中多环芳烃的主要来源为燃烧源，包括木材、煤以及化石燃料的燃烧，同时也有一部分多环芳烃是来源石油类物质的输入.     

红枫湖地表水中多环芳烃的分布及来源

将固相微萃取与气相色谱联用,对贵阳红枫湖水样中16种美国环境保护署优控的多环芳烃进行分析。结果表明：红枫湖水中16种多环芳烃总量为0167 1~0336 4 μg/L,与国内其它水系相比,湖中存在多环芳烃轻度污染。7种（萘、荧蒽、苯并(b)荧蒽、苯并(k)荧蒽、苯并(a)芘、茚并(1,2,3-cd)芘和苯并(ghi)苝）多环芳烃的总量未超出中国城市供水行业对多环芳烃规定的限值,但作为饮用水源,红枫湖水中的苯并(a)芘含量已超出我国标准GB3838-2002中生活饮用水地表水源地的苯并(a)芘限值,并且苯并(a)蒽、〖JX-*9〗〖SX（B-25x〗〖HT7,5”〗艹〖〗〖HT6”,5”〗屈〖HT5”〗〖SX）〗〖JX*9〗、苯并(b)荧蒽、苯并(k)荧蒽、苯并(a)芘、茚并(1,2,3-cd)芘的含量也超过了美国环境保护署地表水水质标准限值。通过多环芳烃特征参数的比值,分析了红枫湖水中多环芳烃的污染来源。污染源分析表明,湖中多环芳烃的主要来源为燃烧源,包括木材、煤以及化石燃料的燃烧,同时也有一部分多环芳烃是来源石油类物质的输入.     

Enhanced susceptibility to infection in mice after exposure to dilute exhaust from light duty diesel engines

A series of experiments was conducted in which groups of mice were first exposed for various durations to diluted exhaust from light duty diesel engines and then briefly to an infectious aerosol generated by nebulizing cultures of a bacterial pathogen (Streptococcus). Typically, postinfection mortality was significantly greater in groups exposed to exhaust than in their corresponding control groups exposed to purified air only. Data of recent diesel and of past diesel- and catalyst-treated gasoline engine exhaust experiments suggest a somewhat greater excess mortality from (enhanced susceptibility to) bacterial infection in mice exposed to diesel exhaust than in those exposed to catalytic gasoline exhaust. Limited data on acute tests of NO₂ and acrolein vapor alone suggest that the infectivity-enhancing effect of diesel exhaust could be accounted for in large part by these components. Exposures to diesel exhaust, NO₂, or acrolein did not enhance the mortality response to a viral pathogen (A/PR8-34).     

南京某地农业土壤中有机污染分布状况研究

对某大型矿业企业周边农业土壤中两类POPs物质——15种多环芳烃和有机氯农药（DDTs和HCHs）的残留量进行了调查。结果表明,PAHs检出率为100%,总残留量范围为312.2~27 580.9 μg·kg-1,且以四环以上多环芳烃组分为主。不同样区土壤PAHs残留量受常年风向影响明显。农业土壤中有机氯农药六六六和滴滴涕均有检出,但残留水平不高,分别为3.60 μg·kg-1(0.81~9.43 μg·kg-1)和11.13 μg·kg-1(3.31~43.81 μg·kg-1)。土壤有机氯农药的残留以p,p′ DDE和p,p′ DDT为主。土壤中HCHs的各异构体组分含量特征为α>β>γ>δ。部分采样点土壤仍可能有新的有机氯污染物来源。     

Benzo[a]pyrene metabolism in mice exposed to diesel exhaust: II. Metabolism and excretion

In this study we examined the effect of diesel exhaust (DE) exposure on in vivo metabolism of benzo[a]pyrene (BaP). DE-exposed and unexposed A/Jax mice of group B were instilled intratracheally with ³H-BaP. At each time point of 2, 24, and 168 h after instillation five mice were killed and the liver, lungs, and testes were removed and frozen. Aliquots of the organs were homogenized in 2 ml water and each received 3 volumes of cold ethanol. Radioactivity in supernatant and precipitate was measured. The supernatant extracts were subjected to HPLC analysis on ALOX-T and on Zorbax ODS. The ALOX-T method was a modification of Autrup's procedure for conjugate assay (Biochem. Pharmacol.28, 1727, 1979). Fractions were (a) free BaP; (b) nonconjugated primary metabolites; (c) sulfate conjugates; (d) glucuronides, glutathiones, and other conjugates. By 2 h after instillation primary metabolites were found in liver and lung, but very little was conjugated. The unconjugated BaP was mainly in the form of free BaP and phenolic metabolite(s). The lungs of DE-exposed mice had less capacity to dispose of “bound” BaP 1 week after instillation.     

Benzo[a]pyrene metabolism in mice exposed to diesel exhaust: I. Uptake and distribution

In this study we examined the effect of diesel exhaust (DE) exposure on the disposition of a typical polycyclic aromatic hydrocarbon. DE-exposed and nonexposed A/Jax mice were divided into three groups and each mouse instilled intratracheally with benzo[a]pyrene (BaP). One group (A) received ¹⁴C-BaP, and at intervals of 2, 24, and 168 h, three mice from the group were killed and quick frozen for whole body autoradiography. Sagittal sections were cut at 0.5 mm intervals and autoradiograms prepared. Adjacent sections were studied so that radioactive areas were matched to specific organs. The second group (B) received ³H-BaP and at 2, 24, and 168 h these mice were killed. Livers, lungs, and testes were weighed and frozen. From these tissues metabolites were analyzed; these data are reported in the next paper. Histofluorescent examination of tissues from mice instilled with nonradioactive BaP (group C) confirmed that BaP was present in the lung. The autoradiography data are the basis for elucidating the BaP distribution in the mouse. Within 2 h after instillation radioactivity was detected in the entire animal, with most in lungs, liver, and GI tract. By 24 h after instillation considerable radioactivity had redistributed to the GI tract. At 168 h after instillation only a trace of label was found in the GI mucosa.     

Deposition and biological availability of diesel particles and their associated mutagenic chemicals

To estimate the human health risk of inhaled diesel particles, it is necessary to know their deposition and retention in the respiratory tract and the rate of dissociation of mutagenic compounds associated with the particles. The deposition of a chain aggregate aerosol of ⁶⁷Ga₂O₃ with size and shape characteristics similar to diesel exhaust particles has been evaluated using Beagle dogs. Approximately one-third of the inhaled activity is deposited in the respiratory tract with most of the particles deposited in the lung. The mutagenic activity present in dichloromethane, dog serum, dog lung lavage fluid, saline, dipalmitoyl lecithin (DPL) and albumin following incubation of these fluids with diesel exhaust particles was determined in the Ames Salmonella system. As observed by other investigators, large quantities of mutagenic activity were removed by dichloromethane. A very small amount of mutagenic activity was removed by the serum and lavage fluid over a 3-day incubation period. No activity was detected following elution with the other solvents. The finding that minimal mutagenic activity could be demonstrated in the biological media following incubation with diesel exhaust particles may be due to a lack of removal of mutagens from the particles or an inactivation of removed mutagens by protein binding or other processes.     

Residues and source identification of persistent organic pollutants in farmland soils irrigated by effluents from biological treatment plants

Sewage and industrial effluents from biological treatment plant have been widely used for agricultural irrigation in north part of China. However, effluents after biological treatment still contain heavy metals and persistent organic contaminants. The persistent organic contaminants accumulated in soil may transfer through the food chains and cause adverse health effects on human or biological effects on soil fauna and flora after long-term application. In present study, field surveys were carried out in the farmlands irrigated by effluents from biological treatment plants that receive sewage wastewater and industrial discharges. Residues of polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs) in the soils irrigated using both ground water and effluents were compared. The origins of PAHs in the soils were discussed. The results showed that wastewater irrigation could cause accumulation of PAHs in soils close to the pollution discharge. Significantly higher concentrations of PAHs were observed in the sampling sites close to the entrance of main channel in contrast to those along branches and the reference sites. There was no significant relationship between the accumulation of persistent organic pollutants and organic matter content in soil (TOC). Soil contamination of these persistent organic pollutants as affected by effluent irrigation was characterized by the dominant accumulation of high-molecular-weight PAHs (HMW-PAHs). In the case study, concentration of benzo[a]pyrane (BaP, 45.6 ng/g), indeno[1,2,3-cd]pyrene (IcP, 86.3 ng/g), benzo[g,h,i]perlene (BgP, 66.9 ng/g) could exceed the limits of the soil quality standard for biodegraded soils. In identification of the sources, the IcP/BgP values of PAHs in soils were more close to that in air particulates from coal/coke source (1.09+/-0.03 ng/g) [Dickhut RM, Canuel EA, Gustafson KE, Liu K, Arzayus KM, Walkers E, et al. Automotive sources of carcinogenic polycyclic aromatic hydrocarbons associated with particulate matter in the chesapeake bay region. Environ Sci Technol 2000;34:4635-40]. Therefore, both of the PAHs residues in effluents and emission from a nearby coal/coke plant were responsible. Also in this case study, low levels of the OCPs were observed and were not of significant concern in this wastewater irrigation area. Among the different OCPs analyzed, DDTs (mean 8.41 ng/g) and HCHs (mean 2.91 ng/g) were the major components. From the ratios of DDT/DDTs and beta-HCH/HCHs, it indicated that OCPs residues should be from historical usage.     

The influence of benzo(a)pyrene on the growth of wheat and corn

Corn seeds responded to soaking in aqueous solutions of benzo(a)pyrene with increased root growth. Growth stimulation decreased from 14% to 0 with increasing concentrations (0.0005–0.02 ppm), and with increasing soaking times (6 and 12 hr). Shoot growth and dry weights of shoots were not affected. Wheat did not respond as distinctly as corn to similar treatments. A decrease in the growth of shoots and roots with increasing BaP concentrations was not statistically significant.