The effect of diesel exhaust on sperm-shape abnormalities in mice

The sperm-shape abnormality bioassay in mice was used to determine whether chemical mutagens in diesel exhaust reach the testes. Strain A male mice (30 per group from 4 to 6 weeks of age) were exposed for 31 or 39 weeks to either diesel exhaust or clean air. After exposure, Eosin Y-stained, air-dried smears of cauda epididymal sperm were scored for changes in sperm-head abnormalities in three different laboratories. There was no difference in the proportion of abnormally shaped sperm in controls and mice exposed to diesel exhaust.     

In vivo detection of mutagenic effects of diesel exhaust by short-term mammalian bioassays

Male Chinese hamsters were exposed to diesel exhaust and clean air for six months at the Center Hill Facility of the U.S. Environmental Protection Agency in Cincinnati, Ohio. The animals were kept in specially constructed inhalation chambers and exposed to clean air or diesel exhaust for eight hours daily. The animals were sacrificed and slides prepared to study the mutagenic effects of diesel exhaust by four in vivo short term mammalian bioassays. Sperm morphology bioassay revealed a 2.67-fold increase in sperm abnormalities in the animals exposed to diesel exhaust as compared to those exposed to fresh air. Micronucleus bioassay revealed a 50% increase in the number of micronuclei in polychromatic erythrocytes obtained from animals exposed to diesel exhuast. However, no increase in sister chromatid exchange or chromosomal abnormalities was observed in bone marrow cells of animals treated with diesel exhaust. During these studies a decrease in mitotic index was observed in animals treated with diesel exhaust.     

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.     

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.     

Test of diesel exhaust emissions in the rat liver foci assay

In the initiation/promotion assay in rat liver, partial hepatectomy is used to enhance initiation, and a choline-devoid diet as promoter. The induction of carcinogenesis is determined by the focal appearance of gamma glutamyl transpeptidase (GGT) positive hepatocytes. We adopted this assay to diesel exhaust emission by performing a partial hepatectomy, and exposing the rats to either clean air or diesel exhaust emission. The rats were fed either a choline-devoid or a choline-supplemented diet for three or six months. The animals were sacrificed and liver sections stained for GGT were examined for the presence of foci of GGT(+) hepatocytes. The results indicate that diesel exhaust exposure does not result in a systemic dose of carcinogens sufficient to be detected in the liver foci assay.     

Carcinogenicity of diesel exhaust as tested in strain a mice

Groups of strain A mice were exposed to diesel exhaust by inhalation and diesel particulate by intraperitoneal injection. The animals were exposed from seven to eight weeks and then sacrificed 26–30 weeks postexposure. Other animals were exposed for up to seven months by the inhalation route. Some animals were promoted using urethane at a dose below which tumors would occur. There was no increase in incidence of pulmonary adenomas in the animals exposed to either diesel exhaust or diesel particulate over the control animals. In the animals which were promoted using urethane at a low dose, there was a significant increase in pulmonary adenomas. Diesel particulate was found in the lungs and bronchial lymph nodes of animals exposed to diesel exhaust 26–30 weeks after cessation of exposure.     

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).     

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.     

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.     

Pulmonary function testing of animals chronically exposed to diluted diesel exhaust for 267 days

Thw purpose of this work was to assess the potential effect that chronic inhalation of diesel exhaust may have on lung mechanics and lung volumes. Noninvasive pulmonary function tests have been conducted repeatedly on 25 rats exposed to diesel exhaust at a particulate concentration of 1500 μg/m³, for 20 h/day, days/week for 267 days. The same tests were conducted on 25 clean air control animals. When the data is normalized, there are no apparent functionally significant changes occurring in the lungs that may be attributed to the chronic inhalation of diesel exhaust.     

Behavioral alterations due to diesel exhaust exposure

Several experiments examining the effects of diesel exhaust on the behavior of rats are reported. Animals were exposed either as adults or neonates. The spontaneous locomotor activity (SLA), measured in standard running wheel cages, of adult rats exposed for 8 h/day, 7 days/week was significantly less than that of controls. Experiments involving diesel exhaust exposure to neonatal rats indicated that adult rats, exposed to diesel exhaust during their neonatal lives, were significantly less active as measured by SLA. Adult rats, exposed to 20 h diesel per day as neonates, were placed in skinner boxes after the SLA experiment described above had been completed. The exhaust exposed animals showed significantly decreased acquisition of a food reinforced bar pressing task. All animals that learned this task extinguished at the same rate. The results of the neonatal diesel exhaust experiments support the hypothesis that diesel exhaust exposure during development of an organism can lead to behavioral differences in adulthood.     

A review of in vitro testing systems applicable to diesel health effects research

In vitro mutagenicity and carcinogenicity testing techniques are currently being used to assess the potential risk to man of exposure to diesel exhaust emissions. This paper examines general considerations of such systems, the types of in vitro tests currently available, the advantages and disadvantages of each cell line and type of test, the limitations of in vitro techniques, the alternative human cell lines that could be utilized for diesel health effects studies, and recommendations for future research employing in vitro methods.     

Introduction to and experimental conditions during the first year of the GMR chronic inhalation studies of diesel exhaust

A chronic exposure study was initiated to determine the effects of diesel exhaust on the health of experimental animals. For this purpose, test atmospheres of clean air (control) or freshly diluted diesel exhaust at concentrations of 250, 750, and 1500 μg/m³ were supplied to four 12.6 m³ inhalation chambers which housed rats and guinea pigs. Diesel aerosol size and concentration, as well as chamber temperature and relative humidity, were continually monitored and controlled to maintain the exposure dose levels and an environment of 22±2°C and 50%±20% relative humidity. The concentrations of CO and NO_x were found to be 5.8±1.0 mg/m³ and 7.9±1.0 mg/m³ above ambient in the chamber containing 1500 μg/m³ of particulate. Animals were supplied from the chambers, on a random basis, for both intramural and extramural studies throughout the exposure period. The experiment ran uninterrupted for over twelve months with mean diesel particle mass concentrations within 2% of the target values.     

A study of diesel emissions on Drosophila

A sex-linked recessive lethal test was performed on male fruit flies of the species Drosophila melanogaster, (Oregon-R strain), exposed to an approximate five-fold dilution of exhaust from a diesel engine. The eight hour exposure was achieved by drawing diluted diesel exhaust from a three cubic meter stainless steel exposure chamber housing laboratory animals through a two liter reaction flask modified for use with Drosophila. A preconditioned sampling bag was used to collect the emissions after passing through the exposure chamber containing the flies. Results of analyses performed on the diesel exhaust mixture showed the following: carbon dioxide—0.17%, carbon monoxide—12.2 ppm, hydrocarbons—11.6 ppm, nitrogen oxide—3.8 ppm, nitrogen dioxide—2.9 ppm, sulphur dioxide—1.0 ppm, and particulates—2.18 mg/m³. Two broods of the F₂ generation were investigated for the occurrence of recessive lethal events. These broods approximated the developing gametogenic stages of mature sperm (P₁ matings on days 2 and 3 postexposure) and spermatocytes (P₁ matings on days 8 and 9). Additionally, the F₃ generation was evaluated for the occurrence of mosaic recessive lethal events which might escape detection in the F₂ generation. An equal number of F₂ and F₃ flies for both broods served as concurrent controls. Results indicate that, under the conditions tested, the diesel exhaust did not increase the mutation frequency of the exposed flies (F₂ rate = 0.30%, F₃ rate = 0%) when compared to the concurrent controls (F₂ rate = 0.37%, F₃ rate = 0.15%).     

Factors influencing NOx emission of a stationary diesel engine fuelled with crude rice bran oil methyl ester blend – Taguchi approach

The main objective of this work is to control the NO_x emission of a stationary diesel engine fuelled with crude rice bran oil methyl ester blend with less sacrifice on smoke density and brake thermal efficiency (BTE) and also to investigate the factors influencing the objective. Fuel injection timing, percentage of exhaust gas recirculation and fuel injection pressure are chosen as the promising factors for the objective and NO_x emission, smoke density and BTE are considered as response variables. Tests were conducted as per Taguchi’s L₉ orthogonal array and the most influencing factor for each response variable and also the significance of each factor on the same was found out through analysis of variance (ANOVA). Response graph was drawn for each response variable and from the results of response graph and ANOVA the optimum combination of the factor levels in achieving the objective was obtained and the same was confirmed experimentally.     

Pulmonary function evaluation of cats after one year of exposure to diesel exhaust

Adult male, inbred, disease-free cats of uniform age and size were exposed eight hours per day, seven days per week to a 1 : 18 dilution of diesel exhaust emissions. After one year of exposure, the animals were removed from the chambers for measurement of lung volumes, forced expiratory flow rates, dynamic compliance and resistance, diffusing capacity, and nitrogen washout. No important changes in pulmonary function were detected with the exception of a decrease in closing volume (P < 0.05). The inability to detect decrements in pulmonary function may have been due to insufficient cocentration of exhaust, insufficient exposure length, or to the use of a species resistant to diesel exhaust. To test these possibilities, the cats are being exposed for an additional year, and another species, hamsters, are being exposed for future testing at exhaust dilutions of 1 : 18 and 1 : 9.     

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.     

Effect of hydrogen addition to CNG in a biodiesel-operated dual-fuel engine

Alternative fuels for diesel engine applications are gaining more prominence as they have numerous advantages compared to fossil fuels. They are renewable, biodegradable; provide food and energy security and foreign exchange savings. They address environmental concerns and socio-economic issues as well. Gaseous fuels such as compressed natural gas and hydrogenated compressed natural gas (HCNG) appear more attractive fuels for diesel engine applications operated in dual-fuel mode. Such dual fuel engines can replace considerable amount of liquid-injected pilot fuels by gaseous fuels besides being friendly to the environment. A small quantity of liquid fuel injected towards the end of the compression stroke initiates combustion of the inducted gas in the dual-fuel engines. The main advantage of dual-fuel engines is their lower nitrogen oxides (NO_x) and particulate emissions. Hence renewable fuels such as biodiesels and gaseous fuels can be used predominantly for transportation and power generation applications. Gaseous fuels are clean burning and are more economical as well. A suitable carburettor was designed to supply a stoichiometric mixture of air and HCNG to the modified diesel engine operated in dual-fuel mode. The biodiesel used in this study is derived from Honge oil called the Honge oil methyl ester (HOME). This paper presents the performance, combustion and exhaust emission characteristics of a single cylinder, four stroke, direct injection, stationary diesel engine operated on HOME and HCNG in dual-fuel mode. From the results it is observed that HOME–HCNG combination gave lower brake thermal efficiency (BTE) and improved emission levels when compared with diesel/HOME in single fuel operation. Lower smoke and particulate matter were obtained with dual-fuel operation. Comparative measures of BTE, peak pressure, pressure–crank angle variation, smoke opacity, hydrocarbon, carbon monoxide and NO_x emissions have been made and analysed.     

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.     

Performance of a twin cylinder diesel engine in dual fuel mode using woody biomass producer gas

Due to energy crisis and shortage of fossil fuel, there is a growing interest in alternative fuel for internal combustion engine. Producer gas presents a very promising alternative fuel to diesel since it is a renewable and clean burning fuel having properties similar to that of diesel. In this study, a twin cylinder dual fuel diesel engine is experimentally optimized for maximum diesel saving and lower emissions, without any undue vibration of engine using woody biomass producer gas. The test is carried out to study the performance and emission parameters of the engine in diesel mode and dual fuel mode at different gas flow rates under different load conditions. The study reveals that maximum diesel savings is found to be 83% at optimum gas flow rate and 8 kW load. Carbon monoxide, hydrocarbon and carbon dioxide emissions in dual fuel mode were higher compared with diesel mode at all test ranges. However, the main pollutants, such as nitrogen oxide and smoke, decrease substantially in the dual fuel mode compared with the diesel mode. Lower brake thermal efficiency and higher brake-specific energy consumption as well as exhaust gas temperature are observed in dual fuel mode compared with diesel mode.