Toxicity of Chemical Components of Fine Particles Inhaled by Aged Rats: Effects of Concentration

Abstract

This study tested the hypothesis that exposure to mixtures containing fine particles and ozone (O₃) would cause pulmonary injury and decrements in functions of immunological cells in exposed rats (22–24 months old) in a dose-dependent manner. Rats were exposed to high and low concentrations of ammonium bisulfate and elemental carbon and to 0.2 ppm O₃. Control groups were exposed to purified air or O₃ alone. The biological end points measured included histopathological markers of lung injury, bronchoalveolar lung fluid proteins, and measures of the function of the lung’s innate immuno-logical defenses (macrophage antigen-directed phagocytosis and respiratory burst activity). Exposure to O₃ alone at 0.2 ppm did not result in significant changes in any of the measured end points. Exposures to the particle mixtures plus O₃ produced statistically significant changes consistent with adverse effects. The low-concentration mixture produced effects that were statistically significant compared to purified air but, with the exception of macrophage Fc receptor binding, exposure to the high-concentration mixture did not. The effects of the low- and high-concentration mixtures were not significantly different. The study supports previous work that indicated that particle + O₃ mixtures were more toxic than O₃ alone.     

Screening loblolly pine seedling responses to SO2 and O3: analysis of families differing in resistance to fusiform rust disease

Open pollinated families of loblolly pine differing in resistance to fusiform rust disease were screened in laboratory studies for responses to gaseous air pollutants. Twenty families were given acute exposures (2 fumigations for 4 h each) to SO(2) (0.4-1.0 ppm), O(3) (0.25 ppm), SO(2) (0.4-1.0 ppm) + O(3) (0.25 ppm) and control. Analyses of variance were performed to evaluate the treatment effects of these air pollutants on percent foliar injury, and to determine whether the families responded differentially to the air pollution treatments. Treatment effects were significant, with the combination treatment of SO(2) + O(3) producing a higher percentage of foliar injury than the controls; however, injury levels were very low and may not be of biological significance. Subsequently, twelve families were grown in two soil types for exposure to chronic levels of SO(2) (0.06 ppm), O(3) (0.07 ppm), SO(2) (0.06 ppm) + O(3) (0.07 ppm) and control. The families were then ranked for decreased primary shoot growth, shoot dry weight, root dry weight, total plant dry weight and root/shoot ratio after exposure to air pollution treatments. Air pollution treatments as a main effect were significant for only one of five growth parameters measured, that of primary shoot growth. The main effect of family, and the interaction of family and air pollution treatments, were significant for most growth parameters measured. In general, O(3) alone and in combination with SO(2) reduced growth more than SO(2) alone. Fumigation with O(3) reduced growth of two families in comparison with control groups, whereas SO(2) alone produced decreased growth in one family and stimulated growth in three families. Treatment with O(3) alone produced higher root/shoot ratios than fumigation with charcoal-filtered air in two families. Overall, families which were fast growers under control conditions maintained their ranking after exposure to air pollution. Families producing less growth in charcoal-filtered air also produced less growth under various air pollution regimes. Results indicated that these families exhibited a high degree of resistance to air pollution injury. Growth responses of seedlings may not reflect family differences in long-term productivity. No relationship was apparent between fusiform rust resistance and growth reductions due to air pollutants.     

In vitro evaluation of the mutagenic and cytostatic effect of Tamaron,Lannate and Manzate alone and in mixture

Pesticides are often used in agriculture, especially in floriculture. They are frequently applied in binary or ternary mixtures. Nevertheless, their impact on the genetic material has been scarcely explored. In this study, the mutagenic and cytostatic effect of three widely used pesticides, alone and combined, were analyzed. Briefly, lymphocytes cultures were obtained from peripheral blood samples of five healthy donors to determine the sister chromatid exchange and the replicative index (RI). Then, lymphocytes were exposed to Tamaron (100 ppm), Lannate (200 ppm) and Manzate (300 ppm) alone and combined. For the binary mixtures, the concentrations used were 50 ppm of Tamaron, 100 ppm of Lannate and 150 ppm of Manzate. For the ternary mixtures the following concentrations were used: Tamaron (33 ppm), Lannate (70 ppm) and Manzate (100 ppm). Finally, differential staining was performed. It was found that the frequency of SCE/cell showed a significant difference (P ≤ 0.05) between the control (2.66) and the individual treatments of Tamaron (4.87), Lannate: (5.12) and Manzate (4.23). Also, the values of the SCE in the binary mixture of Tamaron+Lannate (5.57), Tamaron+Manzate (6.06) and Lannate+Manzate (6.22) and the ternary mixture (6.63) were statistically different compared to the control. In the RI there was a significant difference between the control (1.98) and the Manzate (1.87). RI differences were also statistically significant (P ≤ 0.05) in mixtures of Tamaron+Lannate (1.64), Tamaron+Manzate (1.63), Lannate+Manzate (1.69) and total mixture (1.53). Therefore, it is suggested that these pesticides alone and in mixtures have both mutagenic and cytostatic synergistic effect in human lymphocytes in vitro.     

The influence of ozone exposure dynamics on the growth and yield of kidney bean

A field experiment was conducted in open-top chambers to assess the importance of peak exposure concentration and exposure frequency on the responses of kidney bean plants to O3. There were five treatments in the study: charcoal-filtered air, constant exposure to 0.05 ppm O3 (131 microg m(-3)) daily. fluctuating exposure to 0.08 ppm O3 on three alternate days, cluster exposure to 0.08 ppm O3 on three consecutive days, and peak exposure to 0.12 ppm O3 on two consecutive days. Exposures lasted 4 h and produced an average weekly exposure-period concentration of approximately 0.05 ppm in the O3-addition treatments and 0.025 ppm in the charcoal-filtered treatment. Exposures began on June 23 and terminated on September 8. Plants were harvested weekly and assessed for the number, area, and dry mass of leaves; dry mass of stems; dry mass of roots; the number of pods; and the incidence of foliar O3 injury. Yield was assessed at the end of the study. There were no consistent differences between the plants receiving charcoal-filtered air and those receiving O3 exposure. Significant differences were detected among the treatments for several of the growth variables assessed at the interim harvests, but in the final two harvests these differences had mostly disappeared. There were no significant effects of the O3-addition treatments on yield when compared to the plants receiving charcoal-filtered air. This indicates that there were no cumulative impacts on plants exposed to 0.12 ppm O3 for 4 h on two consecutive days followed by filtered air compared to plants receiving charcoal-filtered air. The seasonal 7-h average concentrations of O3 in the peak and filtered air treatments were approximately 0.040 and 0.025 ppm, respectively.     

Indirect effects of air pollutants: Changes in plant/parasite interactions

The impact of air pollutants on plant/parasite-interactions has been investigated. It could be demonstrated that fumigation of Vicia faba L. with 0.15 ppm SO2 (400 microg m(-3)) or 0.2 ppm NO2 (400 microg m(-3)) during 7 days caused changes in plant metabolism which resulted in higher growth rates of the aphid Aphis fabae Scop. feeding on these plants. Fumigation of V. faba with 0.085 ppm O3 during 2 or 3 days, however, caused decreased aphid growth on fumigated plants. That result could be reversed by higher O3 concentrations or through the presence of NOx during O3 fumigation. Ambient air comprising a mixture of pollutant gases had a strong enhancing effect on aphid performance. Thus, the growth of A. fabae on field bean plants was significantly higher in ambient summertime. London air than in charcoal-filtered air. Similarly, the growth of Macrosiphon rosae L. on rose bushes (Rosa sp., cv. Nina Weibull) was improved in ambient summertime Munich air; the increase in growth rate averaged about 20%.     

Reduction of net photosynthesis in oats after treatment with low concentrations of ozone

Oats (Avena sativa L. cv Titus) were exposed to low concentrations of O3 in an assimilation chamber system. Net photosynthesis (net CO2 uptake), measured before and after O3 fumigation, showed significantly different responses for leaves of different age. The oldest active leaf was the most sensitive to O3. Net photosynthesis was depressed after 2 h with 0.075 ppm (150 microg m(-3)) O3. For leaves exposed to 0.150 ppm (300 microg m(-3)) O3 for 2 h, net photosynthesis was reduced significantly for 4 h, after which recovery occurred, nearly reaching the preexposure level 19 h after the exposure. Dark respiration was initially more than doubled after exposure to 0.130 ppm (260 microg m(-3)) O3. There was no visible injury after any of the experiments. The results indicate that O3 may cause crop losses through effects on photosynthesis even in Scandinavia, where a typical O3 episode lasts 1 to 2 h, and the concentration seldom exceeds 0.150 ppm.     

Differences in ability of NO2 absorption in various broad-leaved tree species

Absorption of nitrogen dioxide (NO(2)) by various broad-leaved tree species was determined by the (15)N dilution method. The tree seedlings were continuously exposed to 0.3 ppm (microl litre(-1)) NO(2) or the mixture of 0.3 ppm NO(2) and 0.1 ppm O(3) for 30 days. The total amount of NO(2)-nitrogen absorbed by a seedling during the 30-day exposure period primarily depended on the size of the seedling. Among the tested tree species, three cultivars of Populus showed the highest rate of NO(2) absorption per unit leaf area, reaching as much as 0.3 mg N per dm(2) per day. The absorption rates for Populus cultivars were more than four times greater than those for Viburnum or Cinnamomum which had the lowest rate. A highly significant correlation was recognised between the rate of NO(2) absorption and the stomatal conductance among the species. Three cultivars of Populus which had the highest rates of NO(2) absorption were most susceptible to the mixture of NO(2) and O(3). On the contrary, Cinnamomum, Viburnum and Quercus, which showed the lowest rate of NO(2) absorption, were very tolerant to the mixed gas. These results indicate that the species difference in susceptibility to the mixture of NO(2) and O(3) was mainly determined by the difference in rate of absorption of these gases. Exposure to NO(2) alone had no detrimental effect on the tested tree species.     

The individual and combined effects of ozone and simulated acid rain on growth, gas exchange rate and water-use efficiency of Pinus armandi Franch

The seedlings of Pinus armandi Franch. were exposed to ozone (O(3)) at 300 ppb for 8 h a day, 6 days a week, and simulated acid rain of pH 3.0 or 2.3, 6 times a week, alone or in combination, for 14 weeks from 15 June to 20 September 1993. The control seedlings were exposed to charcoal-filtered air and simulated rain of pH 6.8 during the same period. Significant interactive effects of O(3) and simulated acid rain on whole plant net photosynthetic rate were observed, but not on other determined parameters. The exposure of the seedlings to O(3) caused the reductions in the dry weight growth, root dry weight relative to the whole plant dry weight, net photosynthetic rate, transpiration rate in light, water-use efficiency and root respiration activity, and increases in shoot/root ratio, and leaf dry weight relative to the whole plant dry weight without an appearance of acute visible foliar injury, but did not affect the dark respiration rate and transpiration rate in the darkness. The decreased net photosynthetic rate was considered to be the major cause for the growth reduction of the seedlings exposed to O(3). On the other hand, the exposure of the seedlings to simulated acid rain reduced the net photosynthetic rate per unit chlorophyll a + b content, but did not induce the significant change in other determined parameters.     

Variable Responses of Soybeans to Mixtures of Ozone and Sulfur Dioxide

Foliar injury and shoot fresh weight responses of soybeans (Glycine max L.) ‘Lee 68’ and ‘Dare’ exposed to mixtures of ozone (O₃) and sulfur dioxide (SO₂) were greater than additive (synergistic), less than additive (antagonistic), or additive. The result depended on the concentrations of O₃ and SO₂, the exposure duration, and the amount of injury caused by each gas singly. Synergism usually occurred when injury from O₃ or SO₂ singly was slight to moderate. Antagonism usually occurred when injury from either gas singly was severe. In many cases of antagonism, the injury and fresh weight effects of the mixture were less than those from SO₂ alone, suggesting that O₃ can sometimes protect soybeans from SO₂.     

Growth and nutritive quality of Poa pratensis as influenced by ozone and competition

Interspecific plant competition has been hypothesized to alter effects of early-season ozone (O3) stress. A phytometer-based approach was utilized to investigate O3 effects on growth and nutritive quality of Poa pratensis grown in monoculture and in mixed cultures with four competitor-plant species (Anthoxanthum odoratum, Achillea millefolium, Rumex acetosa and Veronica chamaedrys). Mesocosms were exposed during April/May 2000-2002 to charcoal-filtered air+25 ppb O3 (control) or non-filtered air+50 ppb O3 (elevated O3). Biomass production was not affected by O3, but foliar injury symptoms were observed in May 2002. Early-season O3 exposure decreased relative food value of P. pratensis by an average of 8%, which is sufficient to have nutritional implications for its utilization by herbivores. However, forage quality response to O3 was not changed by interspecific competition. Lack of injury and nutritive quality response in P. pratensis harvested in September may reflect recovery from early-season O3 exposure.     

Yield loss assessments for cultivars of broccoli, lettuce, and onion exposed to ozone

The effects of the photochemical oxidant air pollutant ozone (O(3)) on growth and yield of three garden crops, broccoli (Brassica oleracea L.), lettuce (Lactuca sativa L.), and onion (Allium cepa L.) were studied in an open-top chamber experiment conducted in the field in southern California. Four cultivars each of leaf lettuce, broccoli, and globe onion were exposed to charcoal-filtered air (CF), non-filtered (NF) air, or NF plus 1.5 times ambient O(3) concentration from 4 weeks after germination in January or February until harvest. Exposures lasted 31 days for lettuce, 55 to 78 days for broccoli, and 105 days for onion. Results showed that despite severe O(3) injury to outer leaves, lettuce yields were not affected by O(3). Broccoli also was resistant to O(3) and no growth reduction was observed at ambient O(3) concentrations. Onions were more susceptible to O(3), but only one cv. 'Rio Bravo' had significant yield losses (ca. 5%) at ambient O(3) levels. These results suggest that, in general, concentrations of O(3) in the winter and spring may be below the threshold for adverse effects on yields of broccoli, lettuce and onion.     

Effects of chronic ozone exposure on growth, root respiration and nutrient uptake of rice plants

To clarify the response of growth and root functions to low concentrations of ozone (O(3)), rice plants (Oryza sativa L.) were exposed to O(3) at 0.0 (control), 0.05 and 0.10 ppm for 8 weeks from vegetative to early heading stages. Exposure to 0.05 ppm O(3) tended to slightly stimulate the dry weight of whole plants up to 5 weeks and then slightly decrease the dry weight of whole plants. However, these effects were statistically significant only at 6 weeks. Exposure to 0.10 ppm O(3) reduced the dry weight of whole plants by 50% at 5 and 6 weeks, and thereafter the reduction of the dry weight of whole plants was gradually alleviated. Those changes in dry weight can be accounted for by a decrease or increase in the relative growth rate (RGR). The changes in the RGR caused by 0.05 and 0.10 ppm O(3) could be mainly attributed to the effect of O(3) on the net assimilation rate. Root/shoot ratio was lowered by both 0.05 and 0.10 ppm O(3) throughout the exposure period. The root/shoot ratio which had severely decreased at 0.10 ppm O(3) in the first half period of exposure (1-4 weeks) became close to the control in the latter part of exposure (5-8 weeks). Time-course changes in NH(4)-N root uptake rate were similar to those in the root/shoot ratio especially for 0.10 ppm O(3). On the other hand, root respiration increased from the middle to later periods. Since it is to be supposed that plants grown under stressed conditions change the ratio of plant organ weight to achieve balance between the proportion of shoots to roots in the plant and their activity for maintaining plant growth, these changes in root/shoot ratio and nitrogen uptake rate under long-term exposure can be considered to be an adaptive response to maintain rice growth under O(3) stress.     

Physiological and biochemical stress responses in grassland species are influenced by both early-season ozone exposure and interspecific competition

The effects of two-year early season ozone exposure on physiological and biochemical stress response were investigated in model plant communities. Achillea millefolium and Veronica chamaedrys target plants were grown in monocultures and in mixed cultures with Poa pratensis (phytometer) and exposed in open-top chambers over two years for five weeks to charcoal-filtered (CF) air plus 25 nl l(-1) O3 (control) and non-filtered (NF) air plus 50 nl l(-1) O3. Significant O3 effects were detected in different physiological and biochemical parameters, evidencing interspecific differences in metabolic stress responses and a strong influence of the competition factor. O3 induced strong oxidative effects in Achillea irrespective to the different growth modality. Veronica showed less O3-induced effects in monoculture than when grown in competition with the phytometer. Poa exhibited a different behaviour against O3 depending on the species in competition, showing an overall higher sensitivity to O3 when in mixture with Achillea.     

Effects of nitrogen dioxide on leaf chlorophyll and nitrogen content of soybean

One-month-old soybean (Glycine max [L.] Merrill), cultivar 'Williams', plants were exposed to nitrogen dioxide (0.1, 0.2, 0.3 and 0.5 ppm) and carbon filtered air (control), 7 h per day, for 5 days, under a controlled environment. Leaf chlorophyll content (Ch a, Ch b, and total Ch content) and foliar nitrogen content (%N) were determined before and after the exposure. The influence of NO(2) treatments up to 0.3 ppm on leaf chlorophyll content was negligible although a stimulatory effect was evident in Ch a and total Ch content with 0.2 ppm NO(2). Marked decline in Ch content was observed with 0.5 ppm treatment; the reductions in Ch a and total Ch were 45% and 47%, respectively. Foliar-N contents of plants treated with 0.2 and 0.3 ppm NO(2) were higher than the control; plants exposed to 0.5 ppm NO(2) showed a 41% reduction in foliar-N compared to pre-exposure values.     

Dose-response studies with the antiozonant ethylenediurea (EDU), applied as a soil drench to two growth substrates, on greenhouse-grown varieties of Phaseolus vulgaris L

To study plant growth and yield effects of the antiozonant ethylenediurea (EDU), which is frequently used for ozone crop loss assessments, dose-response studies were carried out with potted bean plants under greenhouse conditions in winter and spring. Two cultivars of Phaseolus vulgaris L., differing in sensitivity to ozone (O(3)), were grown in unfiltered air on a sandy loam rich in organic matter and on a vermiculite-clay mixture. Four treatments of EDU at concentrations from 300 to 800 mg liter(-1) were given as a soil drench during plant development. Foliar symptoms of EDU phytoxicity were observed at all doses, and plant biomass, particularly pod dry weight, was considerably reduced to increasing doses of EDU. Primary and first trifoliate leaf weight in EDU-treated plants increased as did the number of buds, indicating an extension of vegetative growth and a delay of reproductive processes. 'BBL 290' beans, which are O(3)-sensitive, were injured by EDU more than the O(3)-tolerant 'BBL 274'. The phytotoxic effects of EDU were more pronounced in the synthetic growth substrate than in field soil. In a second experiment, EDU was applied in concentrations from 100 to 400 mg liter(-1) to 'BBL 290' plants, exposed to filtered air or simulated levels of O(3) pollution. In field soil, plant growth and biomass partitioning in filtered air was only slightly altered by EDU, although leaf injury due to EDU occurred. In the vermiculite-clay mix, the biomass of most plant organs, particularly that of roots, was linearly reduced with increasing EDU doses. O(3) did not cause any alteration in plant biomass in field soil-grown and EDU-treated plants. Ozone leaf injury, which affected 67% of primary leaf area in non-treated plants, was completely suppressed by EDU doses as low as 100 mg liter(-1). This indicates that low concentrations of EDU, which do not affect plant growth in field soil, provide sufficient protection from O(3) injury. The need for careful EDU dose-response studies prior to field assessments is emphasized.     

Effect of ozone on growth of mosses on disturbed forest soil

Mixed forest floor organic matter and upper mineral soil from a 1580 m elevation conifer-hardwood stand in the Pisgah National Forest, NC, was placed in 4-cm diameter x 21-cm deep tubes and exposed to 0.00, 0.08, 0.16, 0.24 or 0.32 microl O3/liter air (ppm). Twelve tubes in each of three replications/treatment were fumigated in continuously-stirred tank reactors in a greenhouse for 6 h/day on 4 consecutive days/week. Soil was watered 3 days/week with deionized water amended with ions and adjusted to pH 4.3 with H2SO4 + HNO3 (70 meq SO4(2-): 30 meq NO3(-)). After 10 weeks the amount of soil surface covered by moss (predominantly Ditrichum pusillum, but also D. lineare, and Pohlia nutans) was estimated visually and assigned a rating on a scale of: 1 = 0-25%; 2 = 26-50%; 3 = 51-75%; 4 = 76-100%. Linear regression analysis revealed a significant (p < 0.001) negative relationship between coverage ratings and O3 concentration. Surface coverage in tubes exposed to 0.32 or 0.24 ppm was about half of that for 0.00 ppm (mean ratings of 1.1, 1.4 and 2.6, respectively). Coverage differences appeared to be due in part to O3 suppression of plant numbers. Linear regression analysis also revealed a significant (p < 0.001) negative relationship between heights of D. pusillum plants (measured after 12 weeks treatment) and O3 concentration.     

Tibouchina pulchra (Cham.) Cogn., a native Atlantic Forest species, as a bio-indicator of ozone: visible injury

Tibouchina pulchra saplings were exposed to carbon filtered air (CF), ambient non-filtered air (NF) and ambient non-filtered air+40 ppb ozone (NF+O3) 8 h per day during two months. The AOT40 values at the end of the experiment were 48, 910 and 12,895 ppb h(-1), respectively, for the three treatments. After 25 days of exposure (AOT40=3871 ppb h(-1)), interveinal red stippling appeared in plants in the NF+O3 chamber. In the NF chamber, symptoms were observed only after 60 days of exposure (AOT40=910 ppb h(-1)). After 60 days, injured leaves per plant corresponded to 19% in NF+O3 and 1% in the NF treatment; and the average leaf area injured was 7% within the NF+O3 and 0.2% within the NF treatment. The extent of leaf area injured (leaf injury index) was mostly explained by the accumulated exposure of ozone (r2=0.89; p<0.05).     

Interaction between ozone and winter stress

In some countries, ozone (O3) is primarily a summer pollutant, but in much of Europe, elevated concentrations occur outside the growing season so perennials and over-wintering annuals may be subjected to the combined stresses of pollution, plus chilling, freezing, and winter desiccation. It is recognised that some air pollutants modify the response of plants to environmental stress, but little is known of interactions involving O3. This paper is part of a programme concerned with the effects of O3 on resistance to chilling, freezing, and winter desiccation. Pea (Pisum sativum L.) was used as a convenient model to confirm that O3 affects freezing resistance. The experiment also served as a further evaluation of the use of induced chlorophyll fluorescence kinetics to detect latent O3 injury. Two cultivars, 'Feltham First' and 'Conquest', were fumigated for 7 days, 7 h day(-1). Diffusive resistance and induced fluorescence were recorded daily during the period, then the plants were hardened at 4 degrees C day/2 degrees C night before exposure to 0, -2, -4, -6 and -8 degrees C. Ozone (0.075 ppm; 150 microg O3 m(-3)) caused stomatal closure in both cultivars, but the response was more rapid in 'Conquest'. There were also rapid effects on fluorescence kinetics, and it was concluded that FR, the rate of rise of induced fluorescence, is a useful parameter for indicating latent injury and for distinguishing between cultivars of different sensitivity. Exposure to O3 increased freezing injury and led to greater electrolyte leakage. The freezing resistance of 'Feltham First' was more affected than that of 'Conquest', probably because of the slower stomatal response to the pollutant leading to greater flux of O3 to the internal tissues. It is concluded that interactions involving pollutants and winter stress have implications for crop loss assessment. Perennials and over-wintering annuals should be exposed to the full range of environmental stresses.     

Oxidant air pollution effects on plants of Joshua Tree National Monument

Joshua Tree National Monument (JOTR) is located about 100 km east of the Los Angeles Basin, site of the heaviest concentration of photochemical oxidant (O(3)) air pollution in the US. This investigation was conducted to measure O(3) concentrations in JOTR and to determine the effects of O(3) on vegetation in the park. Potentially phytotoxic concentrations of O(3) were recorded in JOTR in 1984 and 1985, but peak concentration occurred at night, when most plant species would be less sensitive to O(3). No O(3) effects were observed on permanent vegetation observation plots in JOTR in 1984 or 1985. Controlled exposures of native summer annual and woody perennial species to O(3) showed that most did not develop visible O(3) injury symptoms except at concentrations higher than those expected in the park. However, Rhus trilobata Nutt. was injured at 0.10 ppm O(3), 4 h per day for 4 days. This species would be a useful bioindicator to assess the effects of O(3) on native desert plants.