Ozone (O3) critical levels (CLe) are still poorly developed for herbaceous vegetation. They are currently based on single species responses which do not reflect the multi-species nature of semi-natural vegetation communities. Also, the potential effects of other factors like the nitrogen (N) input are not considered in their derivation, making their use uncertain under natural conditions.Exposure- and dose-response relationships were derived from two open-top chamber experiments exposing a mixture of 6 representative annual Mediterranean pasture species growing in natural soil to 4 O3 fumigation levels and 3 N inputs. The Deposition of O3 and Stomatal Exchange model (DO3SE) was modified to account for the multi-species nature of the canopy following a big-leaf approach. This new approach was used for estimating a multi-species phytotoxic O3 dose (PODy-MS). Response relationships were derived based on O3 exposure (AOT40) and flux (PODy-MS) indices.The treatment effects were similar in the two seasons: O3 reduced the aboveground biomass growth and N modulated this response. Gas exchange rates presented a high inter-specific variability and important inter-annual fluctuations as a result of varying growing conditions during the two years. The AOT40-based relationships were not statistically significant except when the highest N input was considered alone. In contrast, PODy-MS relationships were all significant but for the lowest N input level. The influence of the N input on the exposure- and dose-response relationships implies that N can modify the O3 CLe. However, this is an aspect that has not been considered so far in the methodologies for establishing O3 CLe. Averaging across N input levels, a multi-species O3 CLe (CLef-MS) is proposed POD1-MS = 7.9 mmol m⁻², accumulated over 1.5 month with a 95% confidence interval of (5.9, 9.8). Further efforts will be needed for comparing the CLef-MS with current O3 CLef based on single species responses.

In a free-air fumigation experiment with subalpine grassland, we studied long-term effects of elevated ozone (O3) and nitrogen (N) deposition on ecosystem N pools and on the fate of anthropogenic N. At three times during the seventh year of exposure, N pools and recovery of a stable isotope tracer (15N) were determined in above- and belowground plant parts, and in the soil. Plants were much better competitors for 15N than soil microorganisms. Plant N pools increased by 30–40% after N addition, while soil pools remained unaffected, suggesting that most of the extra N was taken up and stored in plant biomass, thus preventing the ecosystem from acquiring characteristics of eutrophication. Elevated O3 caused an increase of N in microbial biomass and in stabilized soil N, probably resulting from increased litter input and lower litter quality. Different from individual effects, the interaction between the pollutants remained partly unexplained.

Stomatal ozone flux and flux–response relationships were derived for winter wheat (Triticum aestivum L.) grown under fully open-air ozone fumigation. A stomatal conductance (gₛₜₒ) model developed for wheat in Europe was re-parameterized. Compared to European model parameterizations, the main changes were that the VPD and radiation response functions were made less and more restrictive, respectively, and that the temperature function was omitted. The re-parameterized gₛₜₒ model performed well with an r² value of 0.76. The slope and intercept of the regression between observed and predicted gₛₜₒ were not significantly different from 1 to 0, respectively. An ozone uptake threshold of 12 nmol m⁻² s⁻¹ was judged most reasonable for the wheat flux–response relationship in subtropical China. Judging from both flux- and concentration-based relationships, the cultivars investigated seem to be more sensitive to ozone than European cultivars. The new flux–response relationship can be applied to ozone risk assessment in subtropical regions.

The hypothesis was tested that O₃-induced changes in leaf-level photosynthetic parameters have the capacity of limiting the seasonal photosynthetic carbon gain of adult beech trees. To this end, canopy-level photosynthetic carbon gain and respiratory carbon loss were assessed in European beech (Fagus sylvatica) by using a physiologically based model, integrating environmental and photosynthetic parameters. The latter were derived from leaves at various canopy positions under the ambient O₃ regime, as prevailing at the forest site (control), or under an experimental twice-ambient O₃ regime (elevated O₃), as released through a free-air canopy O₃ fumigation system. Gross carbon gain at the canopy-level declined by 1.7%, while respiratory carbon loss increased by 4.6% under elevated O₃. As this outcome only partly accounts for the decline in stem growth, O₃-induced changes in allocation are referred to and discussed as crucial in quantitatively linking carbon gain with stem growth.

Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O₃) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O₃ fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O₃ fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O₃, determined from the mRNA levels of the major allergens. We conclude that O₃ can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase.

Recent evidence from novel phytotron and free-air ozone (O3) fumigation experiments in Europe and America on forest tree species is highlighted in relation to previous chamber studies. Differences in O3 sensitivity between pioneer and climax species are examined and viewed for trees growing at the harsh alpine timberline ecotone. As O3 apparently counteracts positive effects of elevated CO2 and mitigates productivity increases, response is governed by genotype, competitors, and ontogeny rather than species per se. Complexity in O3 responsiveness increased under the influence of pathogens and herbivores. The new evidence does not conflict in principle with previous findings that, however, pointed to a low ecological significance. This new knowledge on trees' O3 responsiveness beyond the juvenile stage in plantations and forests nevertheless implies limited predictability due to complexity in biotic and abiotic interactions. Unravelling underlying mechanisms is mandatory for assessing O3 risks as an important component of climate change scenarios.

In 2006, a controlled infection study was performed in the ‘Kranzberger Forst’ to address the following questions: (1) Will massive artificial inoculation with Apiognomonia errabunda override the previously observed inhibitory effect of chronic ozone? (2) Can biochemical or molecular markers be detected to account for the action of ozone? To this end six adult beech trees were chosen, three ozone fumigated (2× ozone) and three control trees (ambient = 1× ozone). Spore-sprayed branches of sun and shade crown positions of each of the trees, and uninoculated control branches, were enclosed in 100-L plastic bags for one night to facilitate infection initiation. Samples were taken within a five-week period after inoculation. A. errabunda infestation levels quantified by real-time PCR increased in leaves that were not fumigated with additional ozone. Cell wall components and ACC (ethylene precursor 1-amino cyclopropane-1-carboxylic acid) increased upon ozone fumigation and may in part lead to the repression of fungal infection. Chronic sublethal ozone exposure reduces both natural and artificial infestation of beech leaves by the endophytic fungus Apiognomonia errabunda.

Lichens play an important role in the biogeochemical cycling of mercury (Hg) and are commonly used as indicators of Hg enrichment in remote and anthropogenically impacted environments. To assess their capacity for Hg uptake and accumulation, we determined the concentration of gaseous elemental mercury (GEM) in air and the concentration of total Hg (THg) in transplanted thalli of two lichen species. Lichen transplants and passive air samplers (PASs) were concurrently deployed, side by side, at 10 sites within an abandoned mining area, characterized by large gradients in atmospheric Hg contamination. Highly variable time-weighted GEM concentrations determined by the PASs, ranging from 17 to 4,200 ng/m³, were mirrored by generally high Hg concentrations in transplanted thalli of both Xanthoria parietina (174–8,800 ng/g) and Evernia prunastri (143–5,500 ng/g). Hg concentrations in the two species co-varied linearly indicating about 60% greater Hg accumulation in X. parietina than in E. prunastri. Whereas Hg uptake in the fruticose E. prunastri increased linearly with GEM, a power law equation with a fractional exponent described the uptake in the foliose X. parietina. Extrapolating the relationships observed here to higher GEM levels yielded concentrations in lichen that agree very well with those measured in an earlier fumigation experiment performed under laboratory-controlled conditions. The uptake model of X. parietina was further verified by correctly estimating GEM concentrations from the THg measured in autochthonous thalli collected from the urban area adjacent to the mine site. Passive sampling can effectively provide time-weighted data of suitable spatial resolution to quantitatively describe GEM assimilation by lichens. Therefore, the combined use of passive sampling and lichen transplants can contribute to a more comprehensive understanding of the role of lichens, and potentially also of other cryptogams, in the deposition of atmospheric Hg to terrestrial ecosystems.

Air pollution has been identified as a major cause of environmental and human health damage. O₃ is an oxidative pollutant that causes leaf symptoms in sensitive plants. This study aims to adjust a multilinear model for the monitoring of O₃ in subtropical climatic conditions by associating O₃ concentrations with measurements of morphological leaf traits in tobacco plants and different environmental variables. The plants were distributed into five areas (residential, urban or industrial) in the southern region of Brazil and exposed during 14 periods, of 14 days each, during the years of 2014 and 2015. The environmental variables and leaf traits during the exposure periods were described by mean, median, standard deviation and minimum and maximum values. Spearman correlation and multiple linear regression analyses were applied on data from exposure periods. Leaf injury index, leaf area, leaf dry mass, temperature, relative humidity, global solar radiation and accumulated rainfall were used in the regression analyses to select the best models for predicting O₃ concentrations. Leaf injury characteristically caused by O₃ was verified in all areas and periods of plant exposure. Higher values of leaf injury (24.5% and 27.7%) were registered in the 13th and 12th exposure periods during spring and in areas influenced by urban and industrial clutches. The VPD, temperature, global solar radiation and O₃ were correlated to leaf injury. Environmental variables [leaf area, leaf dry mass, global solar radiation and accumulated rainfall] and primarily the VPD were fundamental to improve the adjustments done in the bioindicator model (R² ≥ 0.73). Our research shows that biomonitoring employing the tobacco “Bel-W3” can be improved by measuring morphological leaf traits and meteorological parameters. Additionally, O₃ fumigation experiment should be performed with biomonitoring as conducted in this study, which are useful in understanding the role of other environmental factors.