Quantifying ozone uptake and its effects on the stand level of common beech (Fagus sylvatica L.) in Southern Germany

Stand level O(3) fluxes were calculated using water balance calculations for 21 Common beech (Fagus sylvatica L.) stands and O(3) data from 20 monitoring stations in Southern Germany. For this intention, the daily loss of water by evapotranspiration per stand area was set against the daily O(3) uptake. During the last 30 years, O(3) uptake ranges between 0 and 187 mmol ha(-1) d(-1) per stand area. Cumulative O(3) uptake (CUO(3)), ranging between 0.1 and 0.7 mmol m(-2) yr(-1) per stand area, shows increasing trends since 1971 with considerably greater values at high altitudes. Effects in radial growth were used to derive an initial approximate critical threshold value for O(3) impacts on the vitality and growth of mature beech stands in Southern Germany. It is concluded that this concept of O(3) flux estimation in combination with dendroecological analyses offers both a site specific and regional applicable approach to derive new critical levels for O(3).     

Spatial variability and temporal stability of throughfall deposition under beech (Fagus sylvatica L.) in relationship to canopy structure

Although the spatial variability of throughfall (TF) in forest ecosystems can have important ecological implications, little is known about the driving factors of within-stand TF variability, particularly in deciduous forests. While the spatial variability of TF water amount and H+ deposition under a dominant beech (Fagus sylvatica L.) tree was significantly higher in the leafed period than in the leafless period, the spatial TF deposition patterns of most major ions were similar in both periods. The semiannual TF depositions of all ions other than H+ were significantly positively correlated (r=0.68-0.90, p<0.05) with canopy structure above sample locations throughout the entire year. The amounts of TF water and H+ deposition during the leafed period were negatively correlated with branch cover. We conclude that the spatial heterogeneity of ion deposition under beech was significantly affected by leaves in the growing period and by branches in non-foliated conditions.     

Ozone exposure thresholds and foliar injury on forest plants in Switzerland

Canton Ticino in southern Switzerland is exposed to some of the highest concentrations of tropospheric ozone in Europe. During recent field surveys in Canton Ticino, foliar symptoms identical to those caused by ozone have been documented on native tree and shrub species. In Europe, the critical ozone level for forest trees has been defined at an AOT40 of 10 ppm.h O3 (10 ppm.h accumulated exposure of ozone over a threshold of 40 ppb) during daylight hours over a six-month growing season. The objective of this study was to determine the amount of ambient ozone required to induce visible foliar symptoms on various forest plant species in southern Switzerland. Species were grown within eight open-top chambers and four open plots at the Vivaio Lattecaldo Cantonal Forest Nursery in Ticino, Switzerland. Species differed significantly in terms of the ppb.h exposures needed to cause visible symptoms. The most to least symptomatic species grown within open-plots in this study rank as Prunus serotina, Salix viminalis, Vibrnum lantana, Rhamnus cathartica, Betula pendula, Rumex obtusifolius, Sambucus racemosa, Morus nigra, Prunus avium, Fraxinus excelsior, Rhamnus frangula, Alnus viridis, Fagus sylvatica and Acer pseudoplatanus. Similar rankings were obtained in the non-filtered chamber plots. The ranking of species sensitivity closely follows AOT values for the occurrence of initial symptoms and symptom progression across the remainder of the exposure season. Species that first showed evidence of foliar injury also demonstrated the most sensitivity throughout the growing season, with symptoms rapidly advancing over ca. 25-30% of the total plant leaf surfaces by the end of the observation period. Conversely, those species that developed symptoms later in the season had far less total injury to plant foliage by the end of the observation period (1.5 to < 5% total leaf area injured). The current European ambient ozone standard may be insufficient to protect native plant species from visible foliar injury, and many more native species may be sensitive to ozone-induced foliar injury than are currently known.     

Ozone air pollution and foliar injury development on native plants of Switzerland

The objectives of this study were to examine the foliar sensitivity to ozone exposure of 12 tree, shrub, and herbaceous species native to southern Switzerland and determine the seasonal cumulative ozone exposures required to induce visible foliar injury. The study was conducted from the beginning of May through the end of August during 2000 and 2001 using an open-top chamber research facility located within the Lattecaldo Cantonal Forest Nursery in Canton Ticino, southern Switzerland (600 m asl). Plants were examined daily and dates of initial foliar injury were recorded in order to determine the cumulative AOT40 ppb h ozone exposure required to cause visible foliar injury. Plant responses to ozone varied significantly among species; 11 species exhibited visible symptoms typical of exposures to ambient ozone. The symptomatic species (from most to least sensitive) were Populus nigra, Viburnum lantana, Salix alba, Crataegus monogyna, Viburnum opulus, Tilia platyphyllos, Cornus alba, Prunus avium, Fraxinus excelsior, Ribes alpinum, and Tilia cordata; Clematis spp. did not show foliar symptoms. Of the 11 symptomatic species, five showed initial injury below the critical level AOT40 10 ppmh O3 in the 2001 season.     

Preliminary results of modeled ozone uptake for Fagus sylvatica L. trees at selected EU/UN-ECE intensive monitoring plots

The objective of this study was to establish whether EU and UN-ECE/ICP-Forests monitoring data (i) provide the variables necessary to apply the flux-based modeling methods and (ii) meet the quality criteria necessary to apply the flux-based critical level concept. Application of this model has been possible using environmental data collected from the EU and UN-ECE/ICP-Forests monitoring network in Switzerland and Italy for 2000-2002. The test for data completeness and plausibility resulted in 6 out of a possible total of 20 Fagus sylvatica L. plots being identified as suitable from Switzerland, Italy, Spain, and France. The results show that the collected data allow the identification of different spatial and temporal areas and periods as having higher risk to ozone than those identified using the AOT40 approach. However, it was also apparent that the quality and completeness of the available data may severely limit a complete risk assessment across Europe.     

Ozone sensitivity of Fagus sylvatica and Fraxinus excelsior young trees in relation to leaf structure and foliar ozone uptake

During the summer of 2001, 2-year-old Fraxinus excelsior and Fagus sylvatica plants were subjected to ozone-rich environmental conditions at the Regional Forest Nursery at Curno (Northern Italy). Atmospheric ozone concentrations and stomatal conductance were measured, in order to calculate the foliar fluxes by means of a one-dimensional model. The foliar structure of both species was examined (thickness of the lamina and of the individual tissues, leaf mass per area, leaf density) and chlorophyll a fluorescence was determined as a response parameter. Stomatal conductance was always greater in Fraxinus excelsior, as was ozone uptake, although the highest absorption peaks did not match the peaks of ozone concentration in the atmosphere. The foliar structure can help explain this phenomenon: Fraxinus excelsior has a thicker mesophyll than Fagus sylvatica (indicating a greater photosynthesis potential) and a reduced foliar density. This last parameter, related to the apoplastic fraction, suggests a greater ability to disseminate the gases within the leaf as well as a greater potential detoxifying capacity. As foliar symptoms spread, the parameters relating to chlorophyll a fluorescence also change. PI (Performance Index, Strasser, A., Srivastava, A., Tsimilli-Michael, M., 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. In: Yunus, M., Pathre, U., Mohanty, P., (Eds.) Probing Photosynthesis: Mechanisms, Regulation and Adaptation. Taylor & Francis, London, UK, pp. 445-483.) has proved to be a more suitable index than Fv/Fm (Quantum Yield Efficiency) to record the onset of stress conditions.     

Effects of ozone exposure in open-top chambers on poplar (Populus nigra) and beech (Fagus sylvatica): a comparison

Rooted cuttings of poplar (Populus nigra) and seedlings of beech (Fagus sylvatica) were exposed to ozone in open-top chambers for one growing season. Three treatments were applied: charcoal-filtered (CF), non-filtered (NF) and non-filtered air plus 30 ppb (nl l(-1)) ozone (NF+). Extra ozone was only added on clear days, from 09:00 until 17:00-20:00. The AOT40s (accumulated exposure over a threshold of 40 ppb), calculated from April to September were 4055 ppb.h for the NF and 8880 ppb.h for the NF+ treatments. For poplar ozone exposure caused highly significant reductions in growth rate, light-saturated net CO(2) assimilation rate, stomatal conductance, F(v)/F(m) and chlorophyll content. The largest effects were observed in August at which time ozone concentrations were elevated. A reduction was noticed in new leaf production, while accelerated ageing and visible damage to leaves caused high leaf losses. For beech the responses were similar but less pronounced: ozone exposure resulted in non-significant growth reductions, slight changes in light-saturated photosynthesis and accelerated leaf abscission. The chlorophyll content of beech leaves was not affected by the ozone treatments. The results confirmed previous observations that fast-growing tree species, such as most poplar species and hybrids, are more sensitive and responsive to tropospheric ozone than slower-growing species, such as beech. The growth reductions observed and reported here for beech were within the range of those reported in relationship to the AOT40 (accumulated exposure over a threshold of 40 ppb) critical level for ozone.     

Determination of herbicides in stemflow and throughfall of beeches (Fagus sylvatica L.) and in rainfall

The pesticide contamination of water samples collected in and nearby a beech forest in northern Germany was evaluated. For this purpose, a method for the collection of water samples from stemflow and throughfall of beeches (Fagus sylvatica L.) and rainfall was developed in response to the demands for the analysis of organic contaminants in water samples. Furthermore a sensitive and selective multiresidue method was developed to determine 18 pesticides, frequently used in Germany, in aqueous samples. The samples collected were taken from the stemflow, the crown throughfall and the rainfall between May and November 2001. Analysis based on reversed-phase liquid chromatography with a pneumatically assisted electrospray mass spectrometer followed a solid phase extraction using C-18 extraction cartridges. Isoproturon, metolachlor, prosulfocarb and terbuthylazine were found during and shortly after their application period. In rainfall metolachlor, terbuthylazine and prosulfocarb were detectable in concentrations between 5 and 65 ng l(-1) and isoproturon in concentrations between 20 and 360 ng l(-1) respectively. In most of the samples, concentrations of those four pesticides were higher and detectable for a longer time in stemflow than in rainfall. Crown throughfall samples were collected from the end of August to November. Absolute deposition of isoproturon to forest soil were up to 70 times higher in comparison to rainwater samples.     

Effects of simultaneous ozone exposure and nitrogen loads on carbohydrate concentrations, biomass, growth, and nutrient concentrations of young beech trees (Fagus sylvatica)

Beech seedlings were grown under different nitrogen fertilisation regimes (0, 20, 40, and 80 kg Nha(-1)yr(-1)) for three years and were fumigated with either charcoal-filtered (F) or ambient air (O3). Nitrogen fertilisation increased leaf necroses, aphid infestations, and nutrient ratios in the leaves (N:P and N:K), as a result of decreased phosphorus and potassium concentrations. For plant growth, biomass accumulation, and starch concentrations, a positive nitrogen effect was found, but only for fertilisations of up to 40 kg Nha(-1) yr(-1). The highest nitrogen load, however, reduced leaf area, leaf water content, growth, biomass accumulation, and starch concentrations, whereas soluble carbohydrate concentrations were enhanced. The ozone fumigation resulted in reduced leaf area, leaf water content, shoot growth, root biomass accumulation, and decreased starch, phosphorus, and potassium concentrations, increasing the N:P and N:K ratios. A combined effect of the two pollutants was detected for the leaf area and the shoot elongation, where ozone fumigation amplified the nitrogen effects.     

The importance of atmospheric deposition, charge and atomic mass to the dynamics of minor and rare elements in developing, ageing, and wilted leaves of beech (Fagus sylvatica L.)

The amounts of sixty elements in developing, maturing, senescent and wilting leaves, and in the wintering dead leaves attached to the branches, are reported for a beech (Fagus sylvatica) forest on mor Podzol in south Sweden, a site with no local sources of pollution or geological anomalies. The amounts (contents per leaf) of K (potassium), Rb (rubidium), Cs (caesium), Cu (copper) and P (phosphorus) were highest in young leaves, decreasing throughout the growing season and usually in the subsequent winter. The entirely opposite pattern with a continuous, mostly even increase of the amounts was measured with Be (beryllium), Ba (barium), Hg (mercury), Al (aluminium), Tl (thallium), Pb (lead), Bi (bismuth), V (vanadium), W (tungsten), As (arsenic), Sb (antimony), and Se (selenium). Amounts of rare-earth elements and some transition metals, such as Co (cobalt), Ti (titanium), and the actinides Th (thorium) and U (uranium) were more stable during the growing season, after an initial increase in early summer, but increased greatly in the winter. This winter increase in dead attached leaves has to be accounted for by uptake from long-distance transported constituents in dry and wet deposition. It was similar to deposition rate estimates using moss carpets from the same locality. A passive uptake was positively related to ionic charge and atomic mass. However, the amounts of several, mainly non-essential elements, such as Ni (nickel), Sc (scandium), Zr (zirconium), Cr (chromium), Ag (silver), and Cd (cadmium) were not much lower in the young or maturing leaves than in the wintered dead leaves of this deciduous (hardwood) forest and a proportion apparently originated from internal translocation in the trees. Seasonal fluxes or cycling of many of the scarce or rare elements reported here have never been studied before in forest ecosystems.     

Effects of chronic elevated ozone exposure on gas exchange responses of adult beech trees (Fagus sylvatica) as related to the within-canopy light gradient

The effects of elevated O₃ on photosynthetic properties in adult beech trees (Fagus sylvatica) were investigated in relation to leaf mass per area as a measure of the gradually changing, within-canopy light availability. Leaves under elevated O₃ showed decreased stomatal conductance at unchanged carboxylation capacity of Rubisco, which was consistent with enhanced δ¹³C of leaf organic matter, regardless of the light environment during growth. In parallel, increased energy demand for O₃ detoxification and repair was suggested under elevated O₃ owing to enhanced dark respiration. Only in shade-grown leaves, light-limited photosynthesis was reduced under elevated O₃, this effect being accompanied by lowered F_v/F_m. These results suggest that chronic O₃ exposure primarily caused stomatal closure to adult beech trees in the field regardless of the within-canopy light gradient. However, light limitation apparently raised the O₃ sensitivity of photosynthesis and accelerated senescence in shade leaves.     

Comparison of ozone uptake and sensitivity between a phytotron study with young beech and a field experiment with adult beech (Fagus sylvatica)

Chamber experiments on juvenile trees have resulted in severe injury and accelerated loss of leaves along with reduced biomass production under chronically enhanced O3 levels. In contrast, the few studies conducted on adult forest trees in the field have reported low O3 sensitivity. In the present study, young beech in phytotrons was more sensitive to O3 than adult beech in the field, although employed O3 regimes were similar. The hypotheses tested were that: (1) differences in O3 uptake were caused by the ontogenetically higher stomatal conductance of young compared to adult trees, (2) the experimental settings in the phytotrons enhanced O3 uptake compared to field conditions, and (3) a low detoxification capacity contributes to the higher O3 sensitivity of the young trees. The higher O3 sensitivity of juvenile beech in the phytotrons is demonstrated to relate to both the experimental conditions and the physiological responsiveness inherent to tree age.     

Ozone exposure-response relationships for biomass and root/shoot ratio of beech (Fagus sylvatica), ash (Fraxinus excelsior), Norway spruce (Picea abies) and Scots pine (Pinus sylvestris)

Current-year seedlings of beech, ash, Norway spruce and Scots pine were exposed during one growing season to different, but moderate, ozone (O(3)) scenarios representative for Switzerland (50, 85, 100% ambient, 50% ambient+30 nl l(-1)) in open-top chambers (OTCs) and to ambient O(3) concentrations in the field. Biomass significantly decreased with increasing O(3) dose in all species except for spruce. Losses of 25.5% (ash), 17.4% (beech), 9.9% (Scots pine) were found per 10 microl l(-1) h accumulated O(3) exposure over a threshold concentration of 40 nl l(-1) during daylight hours (AOT40). Ratios of root/shoot biomass (RSR) also significantly decreased with increasing AOT40 levels in beech and ash, but not in Norway spruce and Scots pine. The data show that the deciduous species beech and ash were more susceptible to O(3) with respect to RSR and biomass than the coniferous species Norway spruce and Scots pine.     

History effect of light and temperature on monoterpenoid emissions from Fagus sylvatica L.

Monoterpenoid emissions from Fagus sylvatica L. trees have been measured at light- and temperature-controlled conditions in a growth chamber, using Proton Transfer Reaction Mass Spectrometry (PTR-MS) and the dynamic branch enclosure technique.De novo synthesized monoterpenoid Standard Emission Factors, obtained by applying the G97 algorithm (Guenther, 1997), varied between 2 and 32 μg g_DW^?1 h^?1 and showed a strong decline in late August and September, probably due to senescence.The response of monoterpenoid emissions to temperature variations at a constant daily light pattern could be well reproduced with a modified version of the MEGAN algorithm (Guenther et al., 2006), with a typical dependence on the average temperature over the past five days.The diurnal emissions at constant temperature showed a typical hysteretic behaviour, which could also be adequately described with the modified MEGAN algorithm by taking into account a dependence on the average light levels experienced by the trees during the past 10–13 h.The impact of the past light and temperature conditions on the monoterpenoid emissions from F. sylvatica L. was found to be much stronger than assumed in previous algorithms.Since our experiments were conducted under low light intensity, future studies should aim at confirming and completing the proposed algorithm updates in sunny conditions and natural environments.     

Sources of errors in assessing ozone visible symptoms on native vegetation

This paper aims to identify the problems regarding the evaluation of ozone (and ozone-like) symptoms, by examining the results of the 4th UN/ECE ICP-Forests Intercalibration Course for the assessment of ozone-induced visible symptoms. Trees, shrubs and herbaceous species were evaluated in a tree nursery, at Lattecaldo (Switzerland) and under open field conditions at Moggio (Italy). The main findings were: (i) the most expert surveyors tended to be grouped in the same cluster and, during the field exercises, they tended to assess in a more conservative manner compared to the less trained participants; (ii) the agreement was greater in assessing the absence rather than the presence of symptoms; (iii) typical interveinal stippling on the upper leaf surface was more accurately evaluated than discoloration; (iv) uncertainties resulted mainly for species which showed greater variability in their symptom manifestation, and for certain herbaceous species.     

Temporal patterns of foliar ozone symptoms on tall milkweed (Asclepias exaltata L.) in Great Smoky Mountains National Park

Incidence and severity of ozone-induced foliar symptoms on tall milkweed (Asclepias exaltata L.) along selected trails in Great Smoky Mountains National Park (GRSM) were determined by two surveys/season conducted from 1992 through 1996. Overall incidence was 73%, and was 84%, 44%, 90%, 58%, and 82% for 1992-1996, respectively for the same clusters. Average incidence was 61% and 84% for the 1st and 2nd surveys, respectively. Seasonal comparisons showed two distinct injury groupings regarding incidence and severity of injury: 1992, 1994 and 1996 (high injury); 1993 and 1995 (low injury). No discernible patterns were observed between symptomatic and asymptomatic plants regarding height, herbivory or flowering. Regression analyses indicated no differentiation in foliar symptoms regarding topographic position, aspect, slope or elevation over the 5-year study period. Our findings indicate other micro-site or genetic factors may control ozone sensitivity of tall milkweed in GRSM.     

Seasonal ozone response of mature beech trees (Fagus sylvatica) at high altitude in the Bavarian forest (Germany) in comparison with young beech grown in the field and in phytotrons

Mature beech trees (Fagus sylvatica) grown at two different altitudes in the Bavarian forest were compared with young beech trees grown at nearby field sites or in phytotrons for their macroscopic and physiological responses to different ozone (O(3)) exposures. Cumulative O(3) exposure expressed as the sum of hourly mean concentrations above the canopy ranged between 100 and 150 microl l(-1) h, with the vertical O(3) profiles at the higher altitude site being enhanced by 30%. O(3) profiles at all sites were reduced by up to 20% with increasing depth within and beneath the canopy. The leaf discoloration that developed in the absence of premature leaf loss was similar in the sun foliage of mature and young trees (including plant grown in the phytotron). Injury became apparent at low O(3) exposures, expressed as accumulated hourly means over a threshold of 40 nl l(-1) (AOT40 <3.5 microl l(-1) h) at the lower site in both the mature trees and the young beech at the field site, but only occurred when AOT40 values reached 7 microl l(-1) h at the upper site, and 6 microl l(-1) h in the phytotrons. However, the association between injury and O(3) exposure was improved when cumulative ozone uptake to sun leaves was the ozone index, used with values of about 3 mmol m(-2) resulting in visible injury in both mature and young beech growing in phytotrons. Under high ozone exposure levels of inositol were lowered, whilst concentrations of lignin-like materials were enhanced in mature beech. Similar responses were observed in young beech grown in phytotrons. As the sun foliage was affected by only a small and variable extent each year, the seasonal O(3) impact at high altitude did not appear to pose an acute risk to mature beech trees.     

Ozone uptake (flux) as it relates to ozone-induced foliar symptoms of Prunus serotina and Populus maximowiziixtrichocarpa

Field studies were conducted during 2003 and 2004 from early June to the end of August, at 20 sites of lower or higher elevation within north-central Pennsylvania, using seedlings of black cherry (Prunus serotina, Ehrh.) and ramets of hybrid poplar (Populus maximowiziixtrichocarpa). A linear model was developed to estimate the influence of local environmental conditions on stomatal conductance. The most significant factors explaining stomatal variance were tree species, air temperature, leaf vapor pressure deficit, elevation, and time of day. Overall, environmental factors explained less than 35% of the variation in stomatal conductance. Ozone did not affect gas exchange rates in either poplar or cherry. Ozone-induced foliar injury was positively correlated with cumulative ozone exposures, expressed as SUM40. Overall, the amount of foliar injury was better correlated to a flux-based approach rather than to an exposure-based approach. More severe foliar injuries were observed on plants growing at higher elevations.     

Ozone-induced nitrate formation in needles and leaves of Picea abies, Fagus sylvatica and Quercus robur

Much attention has been paid to ozone as a major cause of novel forest decline in Europe. In combination with acidic mist, O(3) has been observed to increase ion leaching. Besides cations lake Mg(2+), Ca(2+), K(+), NH(4)(+), considerable amounts of nitrate were found to be leached by acidic mist from needles of Norway spruce. Controlled fumigation experiments, with 100, 300, and 600 microg O(3)m(-3) over 22 days continuously, have led to a nitrate accumulation of 94.1 +/- 14.8, 119.4 +/- 28.7 and 198.9 +/- 14.9 microg NO(3)(-1) g(-1) FW, respectively, in leaves of Quercus robur. Similar values were found in leaves of Fagus sylvatica and current and previous year needles of Picea abies. Nitrate levels of controls receiving charcoal filtered air were well below 40 microg NO(3)(-) g (-1) FW. Statistically significant elevated nitrate levels were observed after only 48 h of continuous fumigation with 600 microg O(3)m(-3), in all tree species tested, and after 144 h in the 100 microg O(3)m(-3) treatment. In another experiment, trees of Picea abies were kept in two charcoal (C) and two Purafil plus charcoal (P/C) ventilated chambers, and fumigated with O and 500 microg O(3)m(-3) in cabinets of each filter-type in order to eliminate NO(x) from chamber air. After 29 days of continuous ozone fumigation, NO(3)(-) accumulation in needles amounted to 102.0 +/- 37.7 and 137.4 +/- 40.5 microg g(-1) FW in P/C and C-filtered chambers, respectively. Nitrate contents of controls were below 30 microg NO(3)(-)g(-1) FW at the end of the experiment. No significant differences in NO(3)(-) accumulation between filter treatments were observed. Since NO(x) was reduced by more than 95% in the Purafil/charcoal versus the charcoal treatment, NO(3)(-) accumulation in needles can be attributed predominantly to the influence of ozone and not to direct NO(2) uptake of needles by the possible oxidation of NO to NO(2) in the presence of ozone.     

Tree age dependence and within-canopy variation of leaf gas exchange and antioxidative defence in Fagus sylvatica under experimental free-air ozone exposure

We characterized leaf gas exchange and antioxidative defence of two-year-old seedlings and 60-year-old trees of Fagus sylvatica exposed to ambient (1 x O3) or two-fold ambient (2 x O3) O3 concentrations (maximum of 150 ppb) in a free-air canopy exposure system throughout the growing season. Decline in photosynthesis from sun-exposed to shaded conditions was more pronounced in adult than juvenile trees. Seedling leaves and leaves in the sun-exposed canopy had higher stomatal conductance and higher internal CO2 concentrations relative to leaves of adult trees and leaves in shaded conditions. There was a weak overall depression of photosynthesis in the 2 x O3 variants across age classes and canopy positions. Pigment and tocopherol concentrations of leaves were significantly affected by canopy position and tree age, whereas differences between 1 x O3 and 2 x O3 regimes were not observed. Glutathione concentrations were significantly increased under 2 x O3 across both age classes and canopy levels. Seedlings differed from adult trees in relevant physiological and biochemical traits in ozone response. The water-soluble antioxidative systems responded most sensitively to 2 x O3 without regard of tree age or canopy position.