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.     

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).     

Pulmonary function changes in Chinese hamsters exposed six months to diesel exhaust

Chinese hamsters were exposed for eight hours per day to automotive diesel exhaust emissions which were diluted with air (18 to 1) and had a particulate level of 6.4 mg/m³. Pulmonary function measurements were made after six months exposure. Body weight (BW), lung weight (LW), vital capacity (VC), residual volume by water displacement (RV_w) and by gas dilution (RV_D), alveolar volume (V_A), and carbon monoxide transfer factor (D_LCO) were measured. LW showed a significant increase in the diesel exposed animals (P < 0.01) while VC, RV_W, and D_LCO showed decreases (P < 0.01). Static deflation volume-pressure curves showed depressed deflation volumes for diesel exposed animals when volumes were corrected for body weight and even greater depressed volumes when volumes were corrected for lung weight. However, when volumes were expressed as percent vital capacity, the diesel exposed animals had higher lung volumes at 0 and 5 cm H₂O. Results of the pathological examination of the lung tissue will be necessary for final analysis of our findings. However, preliminary interpretation indicates possible emphysematous changes which are compatible with the observed decrease in D_LCO.     

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.     

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.     

Effect of bioethanol and thermal barrier coating on the performance of dual fuel engine operating on Honge oil methyl ester (HOME) and producer gas induction

Alternative fuels have numerous advantages compared to fossil fuels as they are renewable, biodegradable; provide energy security and foreign exchange saving besides addressing environmental concerns and socio-economic issues as well. Renewable fuels can be used predominantly as fuel for both transportation and power generation applications. Improved engine performance with reduced engine exhaust emissions is a major research objective in engine development. Today, the use of biomass derived producer gas is more relevant for addressing rural power generation and is a promising technique for controlling both nitric oxide (NO_x) and soot emission levels. In view of this, exhaustive experiments on the use of Honge oil methyl ester (HOME)–Producer gas in a dual fuel engine have been carried out with an intension of improving its fuel efficiency. This paper mainly presents results on a single cylinder four stroke direct injection diesel engine operated in dual fuel mode using HOME–Producer gas combination with and without bio-ethanol addition and thermal barrier coating (TBC). Further, the results were compared with diesel–producer gas mode of operation. Experimental investigation on dual fuel operation using HOME+5% bioethanol (BE5)–Producer gas operation with TBC showed 12.35% increased brake thermal efficiency with decreased hydrocarbon and carbon monoxide emissions and increased NO_x emission levels compared to HOME–Producer gas mode of operation.     

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.     

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.     

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.     

Characteristics of PM10, PM2.5, CO2 and CO monitored in interiors and platforms of subway train in Seoul, Korea

This study was performed to investigate the concentration of PM₁₀ and PM_2.5 inside trains and platforms on subway lines 1, 2, 4 and 5 in Seoul, KOREA. PM₁₀, PM_2.5, carbon dioxide (CO₂) and carbon monoxide (CO) were monitored using real-time monitoring instruments in the afternoons (between 13:00 and 16:00). The concentrations of PM₁₀ and PM_2.5 inside trains were significantly higher than those measured on platforms and in ambient air reported by the Korea Ministry of Environment (Korea MOE). This study found that PM₁₀ levels inside subway lines 1, 2 and 4 exceeded the Korea indoor air quality (Korea IAQ) standard of 150 μg/m³. The average percentage that exceeded the PM₁₀ standard was 83.3% on line 1, 37.9% on line 2 and 63.1% on line 4, respectively. PM_2.5 concentration ranged from 77.7 μg/m³ to 158.2 μg/m³, which were found to be much higher than the ambient air PM_2.5 standard promulgated by United States Environmental Protection Agency (US-EPA) (24 h arithmetic mean: 65 μg/m³). The reason for interior PM₁₀ and PM_2.5 being higher than those on platforms is due to subway trains in Korea not having mechanical ventilation systems to supply fresh air inside the train. This assumption was supported by the CO₂ concentration results monitored in tube of subway that ranged from 1153 ppm to 3377 ppm. The percentage of PM_2.5 in PM₁₀ was 86.2% on platforms, 81.7% inside trains, 80.2% underground and 90.2% at ground track. These results indicated that fine particles (PM_2.5) accounted for most of PM₁₀ and polluted subway air. GLM statistical analysis indicated that two factors related to monitoring locations (underground and ground or inside trains and on platforms) significantly influence PM₁₀ (p < 0.001, R² = 0.230) and PM_2.5 concentrations (p < 0.001, R² = 0.172). Correlation analysis indicated that PM₁₀, PM_2.5, CO₂ and CO were significantly correlated at p < 0.01 although correlation coefficients were different. The highest coefficient was 0.884 for the relationship between PM₁₀ and PM_2.5.     

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.     

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.     

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.     

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.     

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.     

Effects of EGR,swirl augmentation techniques on combustion of biodiesel/ethanol and their blends in a diesel engine

The diminishing resources and continuously increasing cost of petroleum in association with their alarming pollution levels from diesel engines have caused an interest in finding alternative fuels to diesel which are renewable and sustainable. Emission control and engine efficiency are two most important parameters in current engine design. The impending introduction of emission standards such as Euro IV and Euro V is forcing the research towards developing new technologies for combating engine emissions. The classification of Euro IV and V norms is applicable to heavy-duty engines in Europe, where as Euro 5 is applicable to light-duty engines. This paper presents the effects of exhaust gas recirculation (EGR), swirl augmentation techniques and ethanol addition on the combustion of Honge oil methyl ester (HOME) and its blends with ethanol in a diesel engine. From the experimental work conducted, it is found that the combustion of HOME plus up to 15% ethanol blend in a diesel engine operated with optimised parameters of injection timing 23° Before Top Dead Centre and compression ratio 17.5 results in acceptable combustion emissions and improved brake thermal efficiency (BTE). The addition of ethanol increased BTE with reduced hydrocarbons (HCs), CO and smoke emissions. However, NO _x emissions increased dramatically. Use of appropriate EGR reduces NO _x to acceptable levels. The implementation of swirl augmentation techniques further resulted in increased BTE and considerable reduction in tail pipe emissions such as smoke, HCs, CO and NO _x . The effect of swirl by providing grooves on the piston was taken into consideration to find the overall biodiesel engine performance, which gives scope for further studies.     

Honge oil methyl ester and producer gas-fuelled dual-fuel engine operated with varying compression ratios

Alternative and renewable fuels have numerous advantages compared with fossil fuels as they are renewable and biodegradable, besides providing food and energy security and foreign exchange savings and addressing environmental and socio-economic issues. Therefore, these renewable fuels can be used predominantly in compression ignition (CI) engines for transportation purposes and power generation applications. Today, the use of biomass-derived producer gas is more relevant for addressing rural power generation and is also a promising technique for controlling both NO_x and soot emission levels. Although a producer gas–biodiesel-operated dual-fuel diesel engine exhibits lower performance, they are independent from the use of fossil fuels. The lower performance of the engine could be due to the slow-burning and lower calorific value of producer gas. For this purpose, exhaustive experiments on the use of Honge oil methyl ester (HOME)–producer gas in a dual-fuel CI engine were carried out for the improvement of its fuel efficiency. This paper presents the effect of the compression ratio (CR) on the performance, combustion and exhaust emission characteristics of a single-cylinder, four-stroke, direct injection stationary diesel engine operated using HOME and producer gas in a dual-fuel mode. The results indicated that the HOME–producer gas combination exhibited lower brake thermal efficiency (BTE) with comparable emission levels with the diesel–producer gas combination at different CRs. Comparative measures of BTE, peak pressure, pressure–crank angle variation, heat release rate, smoke opacity, and hydrocarbon (HC), carbon monoxide (CO) and nitric oxide (NO_x) emission levels are presented and analysed.     

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.     

Evaluation of selected monitoring methods for formaldehyde in domestic environments

Varioys analytical methodologies for the monitoring of formaldehyde (CH₂O) concentrations in domestic environments have been developed and evaluated. A modified CEA Instruments, Inc., analyzer has near-real-time CH₃O-specific analysis capability with an 0.01 mg/m³ detection limit. A solid sorbent, 13X molecular sieve has been utilized in a pumped collection unit with a demonstrated 0.03–12.5 mg/m³ linear dynamic range using sampling periods of ≤ 15 min. The development of screening-type techniques has included (1) a semipermeable-membrane passive sampler for measurements of average CH₂O concentrations over 8–24-h periods, and (2) a visual colorimetric analysis method for semiquantitative CH₂O determinations using solid chemical reagents. A preliminary field evaluation has been completed. The results show excellent agreement between the new CH₂O monitoring methods and a reference sampling and analysis technique. A generation apparatus for the production of CH₂O vapor is also reported with a demonstrated linear dynamic range between 0.003 and 12.5 mg/m³.     

Physical characterization of diesel exhaust particles in exposure chambers

Since the deposition of particulate in the respiratory system is strongly influenced by particle size, a correct assessment of this parameter is important for any inhalation experiment studying the potential health effects of air pollutants. Measuring the distribution of particles according to their aerodynamic diameter and mechanical mobility diameter is crucial in analyzing the deposition of submicron particles in the lower respiratory system. Cascade impactor measurements of diluted diesel exhaust in 12.6 m³ animal exposure chambers in the GMR Biomedical Science Department showed that the mass median aerodynamic diameter of the aerosol was 0.2 μm with 88% of the mass in particles smaller than 1 μm. Diffusion battery measurements showed that the mass median mechanical mobility diameter was about 0.11 μm. Transmission electron micrographs of particles deposited on chamber surfaces revealed both agglomerates and nearly spherical particles. The particles in these chambers are similar in size and shape to diesel particles described elsewhere. The flux of diesel particles to food surfaces was measured. Calculations of the expected daily dose by inhalation and by feeding showed that the “worst case” dose by feeding was only about one-tenth the dose by breathing.