The effect of ozone and season on the pool sizes of cyclitols in Scots pine (Pinus sylvestris)

Summary The effect of 100 and 200 g · m^-3 (50 and 100 ppb) ozone and ambient air on 4-year-old grafts of a Scots pine clone was tested in closedtop fumigation chambers. Ozone decreased the myo-inositol and inccreased the pinitol levels in the 1-year-old needles, whereas the effect in the current-year needles was less distinct. In neither case did ambient air lead to any detectable effects on these parameters compared with controls. However, there was a pronounced chamber effect in the levels of myo-inositol and pinitol compared with trees standing in the open field, where inositol was higher and pinitol lower. There is some evidence, based on visible symptoms as well as biochemical changes, that season is an important modifier of the effect of ozone on Scots pine.     

Effect of ozone exposure on polyamines in Scots pine trees

Effects of ozone exposure on polyamines in Pinus sylvestris L. were studied in a long-term experiment. Ten- to 15-year-old Scots pines were exposed to target ozone levels which began at ambient + 40 ppb in May, decreasing to ambient air only by September for 3 growing seasons. The amount of ozone applied followed the natural pattern of variation in ozone concentrations in Northern Finland. The free, soluble conjugated and insoluble conjugated polyamines were analyzed during the experiment and shortly after termination of exposure as well as at the beginning of the following growing season. A carry-over effect was observed as ozone-induced reduction of free spermidine in the oldest needle year class, which developed during the first exposure season of the experiment. This reduction was observed both after the second and the third ozone exposure season. Conversely, after termination of the experiment, levels of free polyamines increased in the following growing season, and soluble conjugated polyamines decreased in the developing needles. The post-treatment changes in polyamine concentrations are hypothesized to be caused by stress-induced injuries or delayed recovery of metabolic processes rather than protective responses. It is noteworthy that some responses in polyamines were found in the developing needles nine months after terminating the ozone exposure. This suggests that stress-induced injuries to older needles affected metabolism of new developing needles.     

Carbon fixation in Pinus halepensis submitted to ozone. Opposite response of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase

The effects of ozone exposure on carbon-fixation-related processes in Pinus halepensis Mill. needles were assessed over 3 months under controlled conditions. Ozone fumigation (200 ppb) did not induce a modification of either net CO₂ assimilation or stomatal conductance in 1-year-old needles, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activity was shown to be reduced by a half. Moreover, this ozone-induced reduction in Rubisco activity was associated with a decrease in the quantity of Rubisco, as determined by the decrease in the large subunit (LSU). On the other hand, 200-ppb ozone fumigation induced a strong increase in both activity and quantity of another carboxylating enzyme, phospho enol pyruvate carboxylase (PEPC, EC 4.1.1.31), generally considered in C₃ plants to participate in carbon catabolism processes. Ozone induced a significant decrease in the Rubisco/PEPC activity ratio which promotes the role of PEPC in trees under ozone stress. The role of this carboxylase will be discussed.     

On the response of pigments and antioxidants of Pinus halepensis seedlings to Mediterranean climatic factors and long-term ozone exposure

An experiment was carried out in open-top chambers located in eastern Spain. One-yr-old Pinus halepensis Mill. seedlings were exposed during three consecutive summers to the following ozone (O₃) treatments: charcoal-filtered air (CFA), non-filtered air (NFA) or non-filtered air plus 40 nl l⁻¹ O₃, 9 h d⁻¹, 5 d wk⁻¹ (NFA+40). Seasonal variations in Aleppo pine performance were observed since reductions in chlorophyll and cellular peroxidase levels associated with increases in superoxide dismutase activity, were recorded during the summer. Similarly, a reduction in epoxidation state was found at midday during the summer, derived from an activation of the xanthophyll cycle associated to an increment in radiation and temperature levels.
The first O₃-induced effects were recorded in previous-year needles (1991) during the first summer exposure as an increase in extracellular and total peroxidase activities and in zeaxanthin levels in the NFA+40 treatment along with a trend to a higher SOD activity in this treatment. A carry-over effect was detected since a lower winter recovery of chlorophyll levels was found in the NFA+40 seedlings along with a reduction of xanthophyll levels. A reduction in chlorophyll levels was observed in the previous-year needles (1992) from the NFA+40 treatment at the end of the second fumigation period. Realistic ozone exposures induced alterations in plant antioxidative systems and plant pigments as shown in this paper. These observations together with the reductions in stomatal conductance and net photosynthesis recorded in the same experiment, indicate that Aleppo pine is a species sensitive to ozone.     

Stand characteristics of ozone-stressed populations of Pinus jeffreyi (Pinaceae): extent,development, and physiological consequences of visible injury

The development and physiological consequences of ozone-induced visible injury was investigated in native populations of Jeffrey pine (Pinus jeffreyi) that were exposed to chronic levels of anthropogenic ozone. Stand structure analyses demonstrated that the expression of visible ozone injury symptoms within self-regenerating populations of Jeffrey pine was highly variable. Of the 975 trees surveyed, 90% exhibited some degree of visible injury and 10% were classified as resistant to ozone. Needles of the most sensitive trees developed a chlorotic mottle characteristic of ozone injury 1 year after their initiation and prematurely abscised in their third year (normal needle retention is 5–6 years). Average needle retention was not different between diameter size categories but was the most variable within the smallest size category and the least variable in the largest size category. Gas exchange measurements indicated a negative correlation between photosynthetic rate and needle surface area covered by chlorotic mottle. Chlorophyll fluorescence kinetics of the current-year needles did not differ between symptomatic and asymptomatic trees but did differ between the oldest needles, suggesting an uncontrolled physiological decline in needles about to abscise in sensitive trees. The high degree of variability of ozone-induced visible injury coupled with the reduction of physiological capacity associated with visible injury suggest that mixed conifer forests growing in polluted regions could potentially undergo shifts in community structure if sensitive Jeffrey pine individuals were to experience differential mortality as a result of ozone exposure. Intraspecific variation in ozone sensitivity may potentially lead to increased population tolerance to oxidative air pollutants, but long-term population analyses will be required to address genetic changes in response to ozone stress.     

Ameliorating effect of UV-B radiation on the response of Norway spruce and Scots pine to ambient ozone concentrations

Elevated levels of both ozone and UV-B radiation are typical for high-altitude sites. Few studies have investigated their possible interaction on plants. This study reports interactive effects of O₃ and UV-B radiation in four-year-old Norway spruce and Scots pine trees. The trees were cultivated in controlled environmental facilities under simulated climatic conditions recorded on Mt Wank, an Alpine mountain in Bavaria, and were exposed for one growing season to simulated ambient or twice-ambient ozone regimes at either near ambient or near zero UV-B radiation levels. Chlorotic mottling and yellowing of current year needles became obvious under twice-ambient O₃ in both species at the onset of a high ozone episode in July. Development of chlorotic mottling in relation to accumulated ozone concentrations over a threshold of 40 nL L^–1 was more pronounced with near zero rather than ambient UV-B radiation levels. In Norway spruce, photosynthetic parameters at ambient CO₂ concentration, measured at the end of the experiment, were reduced in trees cultivated under twice-ambient O₃, irrespective of the UV-B treatment. Effects on photosynthetic capacity and carboxylation efficiency were restricted to trees exposed to near zero levels of UV-B radiation, and twice-ambient O₃. The data indicate that UV-B radiation, applied together with O₃, ameliorates the detrimental effects of O₃. The data also demonstrate that foliar symptoms develop more rapidly in Scots pine than in Norway spruce at higher accumulated ozone concentrations. Symbols and abbreviations: LSD, least significant difference; PAS300, UV-B irradiance weighted according to the plant action spectrum of Green et al. (1974) normalized at 300 (nm); AOT40, (AOT = accumulated over threshold) reflects the sum of hourly ozone concentrations above 40 nL L^–1 during daylight hours (> 50 Wm^–2) ( Kärenlampi & Skärby 1996 ); A₃₅₀, net photosynthesis at ambient CO₂; G₃₅₀, stomatal conductance for water vapour at ambient CO₂; A₂₅₀₀, net photosynthesis at saturating CO₂ (maximal potential photosynthetic activity); CE, carboxylation efficiency; ROS, reactive oxygen species; RuBP, ribulose 1,5-bisphosphate; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; GLM, general linear model.     

Quantification of ozone influx and apoplastic ascorbate content in needles of Norway spruce trees (Picea abies L., Karst) at high altitude

The objective of the present study was to investigate whether peak concentrations of ozone can deplete the apoplastic ascorbate pool of needles from Norway spruce trees (Picea abies L. Karst.) and, thereby, contribute to damage to forest trees. Twigs of forest trees grown at high altitude (1950m above sea level; Mt Patscherkofel, Austria) were enclosed in situ in chambers and fumigated for 5-5 or 17 h with ozone concentrations ranging from 60 to 798 nmol mol^?1. Adjacent branches were fumigated with filtered air. Ozone influx into the foliage ranging from 1-7 to 17nmolm^?2s^?1 had little effect on whole-needle ascorbate or glutathione contents. However, apoplastic ascorbate decreased by about 30% when the needles were exposed to environmentally relevant ozone concentrations and increased about 3-fold at higher ozone concentrations. This response suggests the induction of ascorbate as a protective system and may also be important under field conditions. Needles of spruce trees from high altitude that were exposed to chronically increased ozone concentrations contained significantly higher apoplastic ascorbate concentrations than needles from spruce trees from lower altitudes with lower mean atmospheric ozone concentrations. The results show that peak concentrations of ozone do not act in spruce via a depletion of the apoplastic ascorbate pool.     

An open-air system for exposing forest-canopy branches to ozone pollution

We developed a chamberless system to expose branches to elevated concentrations of ozone with little alteration of micro-meteorological conditions. In a 35-year-old stand of sugar maple (Acer saccharum Marsh.), scaffolding and a platform (14 m in height) provided access to 10 branches and ten paired controls within the canopy. Ozone was delivered to the canopy through a manifold and an array of loops (38 cm in diameter) of teflon tubing individually fitted to each branch. Ozone-enriched air was discharged through numerous small holes in each loop positioned beneath the exposed foliage. A sampling system controlled by a microcomputer monitored ozone concentrations for each loop by means of composite air samples from 12 leaves, drawn through small teflon tubes (1.65 mm diameter) attached to the petioles. On average, coefficients of variation for ozone concentrations for the sample points within each branch loop were less than 50%. Between 0900 and 1700 h for 68 d of exposure, the mean hourly ozone concentrations among the branches averaged 95nmol mol⁻¹ (±13SD), about twice the ambient mean. Frequency distributions of mean hourly concentrations during exposure were unimodal and approximately log-normal, comparable to ambient ozone concentrations. The open-air loop system enables exposure of branches to gaseous pollutants under relatively natural conditions.     

The effect of ozone on the yellowing process of magnesium-deficient clonal Norway spruce grown under defined conditions

During two vegetation periods, young clonal spruce trees (Picea abies (L.) Karst.) with sufficient and poor magnesium (Mg) supply were exposed in the environmental chambers of the GSF phytotron to three levels of ozone (daily means: 18-22, 88-130, and 135-190 microg m(-3); 10% reduction at night). Previous year's needles were examined at 4-week intervals with respect to their contents of Mg, Ca, K, Mn, N, P, and chlorophyll (Chl), various parameters of Chl fluorescence, and the stability of the isolated light-harvesting Chl-a/b-protein complex LHC II. The needles of the two nutrition variants contained more than 0.53 or less than 0.27mg Mg g(-1) needle dry matter, respectively. The ratio of variable to maximal Chl-a fluorescence of the dark-adapted needles, Fv/Fm, and the photoinhibitory quenching of Fv after light treatment, SVi.v, were affected by the Mg content of the needles rather than the ozone levels. Changes of the Chl content and the behavior of the LHC II allowed differentiating between a slow process of needle yellowing occurring under Mg deficiency only, and a rapid process of needle yellowing occurring under the combined action of Mg deficiency and ozone pollution. Only the rapid yellowing process was accompanied by destabilization of the LHC II, and the degree of destabilization was correlated with the ozone concentration present in the days before sampling. The results are consistent with observations obtained at a research site in the Central Black Forest (J Plant Physiol 161 (2004) 423).     

Raman spectroscopy of pigments and oxalates in situ within epilithic lichens: Acarospora from the Antarctic and Mediterranean

The effects of ambient and elevated ozone levels on growth and photosynthesis of beech ( Fagus sylvatica ) were studied by exposing seedlings in open-top chambers for one growing season to three treatments: charcoal-filtered (CF), non-filtered (NF) and non-filtered air with addition of ozone (30 ppb ozone) on clear days for 8–10 h d⁻¹ (NF +). Ambient levels were relatively low and accumulated to an AOT40 (accumulated exposure over a threshold of 40 ppb) of 4055 ppb h (for the period 23 Apr–30 Sept). The NF + chambers received an AOT40 of 8880 ppb h. Throughout the growing season we measured growth and photosynthetic properties. The treatments did not cause strong effects: measurements of gas exchange (light-saturated assimilation rate, CO₂ and light-response curves) and chlorophyll fluorescence showed slight and mostly non-significant reductions of several parameters. No significant differences were found for growth, though in the NF + treatment (AOT40 8880 ppb h) the relative growth rate for diameter increment was at times reduced by 12% compared with the control treatment.     

The role of low-level ozone exposure and mycorrhizas in chemical quality and insect herbivore performance on Scots pine seedlings

The effects of low‐level ozone exposure and suppression of natural mycorrhizas on the above‐ground chemical quality of Scots pine (Pinus sylvestris L.) needles and insect herbivore performance were studied in a two‐year field experiment. Seedlings were fumigated with the ozone doses 1.5–1.7 times the ambient, and natural mycorrhizal infection level was about 35% reduced in roots with fungicide propiconazole. On ozone‐exposed seedlings the mean relative growth rate (MRGR) of Lygus rugulipennis Popp. nymphs was lower than on ambient ozone seedlings, but Gilpinia pallida Klug sawfly larvae grew better on elevated ozone seedlings than on ambient ozone seedlings. MRGR of Schizolachnus pineti Fabr. and Cinara pinea L. aphid nymphs or Neodiprion sertifer Geoffr. sawfly larvae or the oviposition of L. rugulipennis and N. sertifer were not affected by ozone exposure. Although ozone exposure did not affect total phenolics, total terpene, total or individual resin acid, total free amino acid, nutrient or sugar concentrations in needles, MRGR of L. rugulipennis positively correlated with total terpenes and MRGR of G. pallida positively with total amino acids. In addition, ozone exposure increased serine and proline concentration and marginally also starch concentration in needles. When mycorrhizas were reduced with fungicide, only MRGR of L. rugulipennis nymphs increased, but performance of other insect herbivores studied was not changed. However, number of L. rugulipennis eggs correlated positively with mycorrhizal infection level and also with total sugars. Reduction of mycorrhizas did not strongly affect the concentrations of analysed compounds in needles, because only phosphorus and potassium and some individual resin acids were reduced by fungicide treatment. These results suggest that low‐level ozone exposure and moderately declined mycorrhizal infection do not drastically affect either the above‐ground chemical quality of Scots pine seedlings or performance of studied insect herbivores.     

Corona discharge influences ozone concentrations near rats

Ozone can be produced by corona discharge either in dry air or when one electrode is submerged in water. Since ozone is toxic, we examined whether ozone production by corona near laboratory animals could reach levels of concern. Male rats were exposed to a corona discharge and the concentration of ozone produced was measured. The resulting concentration of ozone ranged from ambient levels to 250 ppb when animals were located 1 cm from a 10 kV source. Similar ozone concentrations were observed when a grounded water source was present. Possible explanations for, as well as concerns regarding, ozone production under these conditions are discussed.     

Acceleration of leaf senescence inFagus sylvatica L. by low levels of tropospheric ozone demonstrated by leaf colour,chlorophyll fluorescence and chloroplast ultrastructure

From April 1988 to October 1991 3-year-old seed propagated beech (Fagus sylvatica L.) trees were exposed in open-top chambers to four different levels of air pollution: (1) charcoal filtered air, (2) ambient air, (3) ambient air plus 30 nl 1^-1 ozone during the summer, and (4) ambient air plus 30 nl 1^-1 ozone during the summer and 20 nl 1^-1 SO₂ and NO₂ during the winter. Leaf colour was studied in the autumns of 1989 and 1991 and a close relationship between ozone dose and premature senescence was found. A correlation also exists between the colour groups and chlorophyll fluorescence (F_v/F_m). Ozone fumigation increases the size and speeds up the development of the plastoglobules. This is described using an index based on the volume of plastoglobules as a percentage of chloroplast volume. The index was significantly higher for ozone fumigated plants than for control plants during August to November 1989. According to all three methods it is concluded that low levels of ozone accelerate leaf senescence processes inF. sylvatica. There are indications that leaves of the first and the second flush react differently to the ozone treatment. Irrespective of the ozone treatment a special cell wall structure, probably a local suberization, is confined to the subsidiary cells in leaves of the first flush.     

Inflammatory and Repair Pathways Induced in Human Bronchoalveolar Lavage Cells with Ozone Inhalation

Background

Inhalation of ambient levels of ozone causes airway inflammation and epithelial injury.

Methods

To examine the responses of airway cells to ozone-induced oxidative injury, 19 subjects (7 with asthma) were exposed to clean air (0ppb), medium (100ppb), and high (200ppb) ambient levels of ozone for 4h on three separate occasions in a climate-controlled chamber followed by bronchoscopy with bronchoalveolar lavage (BAL) 24h later. BAL cell mRNA expression was examined using Affymetrix GeneChip Microarray. The role of a differentially expressed gene (DEG) in epithelial injury was evaluated in an in vitro model of injury [16HBE14o- cell line scratch assay].

Results

Ozone exposure caused a dose-dependent up-regulation of several biologic pathways involved in inflammation and repair including chemokine and cytokine secretion, activity, and receptor binding; metalloproteinase and endopeptidase activity; adhesion, locomotion, and migration; and cell growth and tumorigenesis regulation. Asthmatic subjects had 1.7- to 3.8-fold higher expression of many DEGs suggestive of increased proinflammatory and matrix degradation and remodeling signals. The most highly up-regulated gene was osteopontin, the protein level of which in BAL fluid increased in a dose-dependent manner after ozone exposure. Asthmatic subjects had a disproportionate increase in non-polymerized osteopontin with increasing exposure to ozone. Treatment with polymeric, but not monomeric, osteopontin enhanced the migration of epithelial cells and wound closure in an α9β1 integrin-dependent manner.

Conclusions

Expression profiling of BAL cells after ozone exposure reveals potential regulatory genes and pathways activated by oxidative stress. One DEG, osteopontin, promotes epithelial wound healing in an in vitro model of injury.     

Ozone produced by corona discharge in the presence of water

Significant levels of ozone have been detected in a reaction flask that was designed for studies using negative air ions generated from corona discharge. While diluting the gas in the vessel at the rate of 1.5 liters/min., more than 1.0 ppm O₃ was measured in the vessel when grounded water was present. Ozone levels were much less (15 ppb) in ambient air near the ion generator. Even in the absence of water, O₃ levels were only 7 ppb in the reaction flask. Without the corona discharge apparatus, levels of ozone in the gas phase (with water present) averaged 6 ppb. These results demonstrate that when negative air ions are generated from corona discharge in the presence of water, significant levels of ozone are produced. Therefore, O₃ could be directly responsible for many of the oxidizing effects that have been reportedly due to negative air ions.     

Role of ethylene in the regulation of cell death and leaf loss in ozone-exposed European beech

To test the involvement of ethylene in mediating ozone-induced cell death and leaf loss in European beech ( Fagus sylvatica L.), tree seedlings were exposed to proportionally increased or decreased field ozone levels for up to 6 months. Ozone treatment caused cell death and accelerated leaf loss at higher than ambient levels, but had only minor effects at ambient and no effects at subambient ozone levels. The emission of ethylene, the levels of its precursor, 1-aminocyclopropane-1-carboxylate (ACC), and mRNA levels of specific ACC synthase ( FS-ACS2 ) and ACC oxidase ( FS-ACO1 ) isoforms showed a persistent increase and preceded cell death by approximately 2 weeks. Inhibition of ethylene biosynthesis led to reduced lesion formation whereas application of ACC accelerated ozone-induced cell death and leaf loss. Similar results were obtained when adult beech trees were exposed to 2 × ozone by a whole tree free-air canopy exposure system. The results suggest a role of ethylene in amplifying ozone effects under field conditions in this major European broad-leaved tree species.     

Impact of four years exposure to different levels of ozone, phosphorus and drought on chlorophyll, mineral nutrients, and stem volume of Norway spruce, Picea abies

Saplings of one clone of Norway spruce, Picea abies (L.) Karst, were planted in 120 l pots in 1991 and exposed to three levels of ozone, two levels of phosphorus and two levels of water supply in 42 open-top chambers (OTCs), during 1992–1996. The effects of pots and OTCs were also tested. Nutrient concentrations of the needles were not affected by ozone, while the low phosphorus supply (LP) and drought stress (D) treatments had significant effects on several mineral nutrients, e.g. phosphorus, calcium, magnesium, manganese, sulphur and boron. Ozone reduced the chlorophyll concentration in the 2- and 3-year-old needles in 1994 and 1995. The highest ozone concentration reduced the stem volumes (− 8%), as well as the stem lengths (− 5%), of the saplings in 1993 and 1994, after two and three years of exposure. After the fourth growing season this ozone-induced reduction in stem volume disappeared which might be caused by pot limitation. LP supply and D both caused large decreases in the stem volume and length. The needles from LP treatment had as high P concentration as 1.2–1.5 mg g⁻¹, implying a need for increasing the critical value for phosphorus. The OTC enclosure stimulated the stem volume growth significantly compared to saplings growing in ambient plots. This was suggested to be attributed to the slightly higher temperature in the OTCs. The overall result is that ozone in southern Sweden is likely to have negative effects on Norway spruce trees, although much less than other environmental factors, e.g. water and phosphorus.     

Effects of air pollutants on the photosynthetic capacity of young Norway spruce trees. Response of single needle age classes during and after different treatments with O3, SO2, or NO2

Summary Potted young Norway spruce trees were exposed to different concentrations of the air pollutants ozone, sulphur dioxide and nitrogen dioxide under completely controlled environmental conditions. After the treatment, the potted trees were kept outdoors. Measurements of the maximum photosynthetic capacity (A₂₅₀₀) were performed with current-year and 1-year-old needles during and after exposure of the trees. In trees fumigated with nitrogen oxides no damage was found at the concentrations used, and the trees' ability to fix carbon dioxide was increased. Using SO₂, a rapid and marked decrease in A₂₅₀₀ was obtained within the first days of the experiment. This decrease did not continue further, but was reversed upon cessation of the fumigation. However, a clear dose-dependent decrease in A₂₅₀₀ occurred when trees were fumigated continuously with an ozone concentration of 450 nl l^–1 or more. The effect of ozone was not reversible, but continued during post-culture of the trees.     

Elevated ozone reduces methane emissions from peatland mesocosms

Although the effects of elevated ozone on aboveground carbon (C) assimilation are well understood, its effects on soil C fluxes are less certain. Mesocosms taken from a lowland raised bog in northern England were exposed in open‐top chambers for 2 years to ambient air or ambient air plus ozone elevated for 8 h day^?1 by an average of 49 ppb in summer and 10 ppb in winter. The effects of elevated ozone on methane emission and ecosystem dark respiration were measured throughout this period, along with soil and plant variables. Methane emissions were significantly reduced, by about 25%, by elevated ozone during midsummer periods of both years, but no significant effect of ozone was found during the winter periods. Dark ecosystem respiration was not significantly affected by elevated ozone. There was no evidence that effects of elevated ozone on methane emissions were mediated through changes in aboveground plant biomass or soil water dissolved organic C concentrations. Our results imply that the increased northern hemisphere background ozone concentrations over the 21st century that are predicted by most models may reduce the rate of increase in methane emissions as the region warms.     

Impact of Cabin Ozone Concentrations on Passenger Reported Symptoms in Commercial Aircraft

Due to elevated ozone concentrations at high altitudes, the adverse effect of ozone on air quality, human perception and health may be more pronounced in aircraft cabins. The association between ozone and passenger-reported symptoms has not been investigated under real conditions since smoking was banned on aircraft and ozone converters became more common. Indoor environmental parameters were measured at cruising altitude on 83 US domestic and international flights. Passengers completed a questionnaire about symptoms and satisfaction with the indoor air quality. Average ozone concentrations were relatively low (median: 9.5 ppb). On thirteen flights (16%) ozone levels exceeded 60 ppb, while the highest peak level reached 256 ppb for a single flight. The most commonly reported symptoms were dry mouth or lips (26%), dry eyes (22.1%) and nasal stuffiness (18.9%). 46% of passengers reported at least one symptom related to the eyes or mouth. A third of the passengers reported at least one upper respiratory symptom. Using multivariate logistic (individual symptoms) and linear (aggregated continuous symptom variables) regression, ozone was consistently associated with symptoms related to the eyes and certain upper respiratory endpoints. A concentration-response relationship was observed for nasal stuffiness and eye and upper respiratory symptom indicators. Average ozone levels, as opposed to peak concentrations, exhibited slightly weaker associations. Medium and long duration flights were significantly associated with more symptoms compared to short flights. The relationship between ultrafine particles and ozone on flights without meal service was indicative of ozone-initiated chemistry.