In search for evidence: combining ad hoc survey, monitoring, and modeling to estimate the potential and actual impact of ground level ozone on forests in Trentino (Northern Italy)

Environ Sci Pollut Res (2018) 25:8206–8216 DOI 10.1007/s11356-017-9998-x OZONE AND PLANT LIFE: THE ITALIAN STATE-OF-THE-ART In search for evidence: combining ad hoc survey, monitoring, and modeling to estimate the potential and actual impact of ground level ozone on forests in Trentino (Northern Italy) Elena Gottardini 1 & Fabiana Cristofolini 1 & Antonella Cristofori 1 & Marco Ferretti 2,3 Received: 7 April 2017 / Accepted: 22 August 2017 / Published online: 27 September 2017 # Springer-Verlag GmbH Germany 2017 Abstract A 5-year project was carried out over the period 2007–2011 to estimate the potential and actual ozone effect on forests in Trentino, Northern Italy (6207 km2) (Ozone EFFORT). The objective was to provide explicit answers to three main questions: (i) is there a potential risk placed by ozone to vegetation? (ii) are there specific ozone symptoms on vegetation, and are they related to ozone levels? (iii) are there ozone-related effects on forest health and growth? Different methods and techniques were adopted as follows: monitoring ozone levels, ad hoc field survey for symptoms on vegetation and chlorophyll-related measurements, modeling to upscale ozone measurements, ozone flux estimation, statistical analysis, and modeling to detect whether a significant effect attributable to ozone exists. Ozone effects were assessed on an ad hoc-introduced bioindicator, on spontaneous woody species, and on forest trees. As for question (i), the different Responsible editor: Philippe Garrigues * Elena Gottardini elena.gottardini@fmach.it Fabiana Cristofolini fabiana.cristofolini@fmach.it Antonella Cristofori antonella.cristofori@fmach.it Marco Ferretti marco.ferretti@wsl.ch 1 Research and Innovation Centre, Fondazione Edmund Mach (FEM), Via E. Mach, 1 38010 San Michele all’Adige, Trento, Italy 2 Swiss Federal Institute for Forests, Snow and Landscape Research WSL, Zürcherstrasse 111, 8903 Birmensdorf, Switzerland 3 TerraData environmetrics, Via L. Bardelloni 19, 58025 Monterotondo M.mo, Grosseto, Italy ozone-risk critical levels for both exposure and stomatal flux were largely exceeded in Trentino, evidencing a potentially critical situation for vegetation. As for question (ii), specific ozone foliar symptoms related to ozone exposure levels were observed on the introduced supersensitive Nicotiana tabacum L. cv Bel-W3 and on the spontaneous, ozone-sensitive Viburnum lantana L., but not on other 33 species surveyed in the field studies. Regarding question (iii), statistical analyses on forest health (in terms of defoliation) and growth (in terms of basal area increment) measured at 15 forest monitoring plots and tree rings (at one site) revealed no significant relationship with ozone exposure and flux. Instead, a set of factors related to biotic and abiotic causes, foliar nutrients, age, and site were identified as the main drivers of forest health and growth. In conclusion, while ozone levels and fluxes in the investigated region were much higher than current critical levels, evidence of impact on vegetation—and on forest trees in particular—was limited. Keywords Ozone . Forests . Risk . Foliar symptoms . Growth . Defoliation Introduction Tropospheric ozone plays a double role in global change scenarios: on one hand it contributes to global warming with its positive radiative force (Myhre et al. 2013); on the other hand, its potential effect on forest vitality and growth may impact the C sink potential of vegetation. Wittig et al. (2009), through a meta-analysis study, quantified the impact of tropospheric ozone concentrations in northern hemisphere temperate and boreal forests. Specifically, authors report that current concentrations lead to a reduction of total biomass of trees by 7%, while expected concentrations for 2050 and 2100 are Environ Sci Pollut Res (2018) 25:8206–8216 predicted to cause a decrease of 11 and 17% respectively. Modeling studies estimate 1 to 16% reduction of net primary productivity (NPP) in temperate forests (Ainsworth et al. 2012). By reducing the C sink strength of global forest, ozone may have a negative feedback on global warming scenarios and on costs of related remedial actions (Felzer et al. 2004; Sitch et al. 2007). The subject of ozone effects on forest vegetation received an extensive coverage over the past 20 years (Lindroth 2010), with different investigation approaches (open-top chambers— OTC, free-air fumigations, field observational studies), different responses (visible symptoms, physiology, growth, health, diversity), and different vegetation targets (potted and/or planted juvenile plants, spontaneous herbs and shrubs, mature forest trees) being considered. Much evidence for ozone impact derived from fumigation and OTC experiments with juvenile trees (Baldantoni et al. 2011), branch fumigation (Pinelli and Tricoli 2008), open-air fumigation carried out on ad hoc planting, highly responsive broadleaves (Karnosky et al. 2007), or on few native mature trees (Matyssek et al. 2010). On the other hand, evidences reported on the basis of field observational studies are much less clear (e.g., Braun et al. 2014; Bussotti and Ferretti 2009). Although information on physiological responses of trees to ozone is important as they may identify early warning signals of possible effects on tree condition and performance, managers of forest resources are mostly concerned with questions about the actual impact on forest health and growth, e.g., BIs there a problem with ozone?^, BHow big is this problem?^, BAre there measurable ozone-related effects on vegetation?^, BIs ozone causing a deterioration of forest health and growth?^. Answers to these questions are essential in two respects: (i) to promote management actions (e.g., air quality policy, forest management) that can be carried out locally to limit the level and impact of ozone and (ii) to support air quality and policy negotiations that can be promoted at regional, European, and global level to limit air pollution by ozone. The importance of the above set of questions and answers varies with the ecological, economical and societal value of forests in a given area, and the likelihood of ozone impact. Here, we present a synthesis of the results obtained by the project BOzone EFfects on FORests in Trentino^ (Ozone EFFORT). Trentino is an alpine region in Northern Italy, where forest vegetation is central in terms of coverage (347,200 ha, 56.0% of the entire region) and growing stock, with 71.9 ± 5.2 Tg C stocked in the aboveground biomass (43.2%), litter (2.6%), soil organic matter (44.6%), and belowground biomass (9.6%) (Rodeghiero et al. 2010; Tonolli and Salvagni 2007). The growth of forests in Trentino has been estimated to result into 0.54 Mt. of C sequestered per year. At the same time, ozone concentration measured in two remote sites (Passo Lavazè and Monte Gaza) has been reported to be high (Mangoni and Buffoni 2005; http://www.appa.provincia. 8207 tn.it/pianificazione/Piano_tutela_aria/-Piano_tutela_aria/) and to cause exceedances of protection limits for vegetation (Gerosa et al. 2003). With this background, Ozone EFFORT was undertaken in 2007 to provide answers to some practical questions (see below). Designed over a 5-year basis, the project aims at combining monitoring, ad hoc-field surveys, and modeling to estimate the potential and actual impact of ground level ozone on forests in Trentino (Northern Italy). The project includes several components (Fig. 1; Table 1): & & & Ozone measurements and modeling. A series of ozone measurement campaigns were carried out at monitoring sites and systematically spread across the region. Subsequently, a modeling approach was applied to estimate (i) ozone concentration and exposure at 1 × 1 km grid resolution and (ii) ozone flux at one selected site over a 14-year period Direct assessment of specific effects (e.g., visible foliar symptoms) on vegetation, with ad hoc-designed investigations on introduced bioindicators and native species Assessment of non-specific effects (tree growth and health) based on existing forest monitoring data Detailed results of individual components of the project have been published elsewhere (Cristofolini et al. 2011; Ferretti et al. In this issue; Gottardini et al. 2010a, 2010b; Gottardini et al. 2012; Gottardini et al. 2014a, 2014b; Cristofori et al. 2015) (see Table 3). In this paper, we summarize concept, organization, and main results obtained. It is worth noting that, although organized and developed on a local basis, the concept proposed and the results obtained could be of relevance for studies at larger scales. Materials and methods Study area The project was carried out in Trentino, a 6207 km2 alpine region of Northern Italy, extending between 45° 44′ and 46° 28′ north latitude and between 10° 32′ and 11° 53′ east longitude (Fig. 2). The elevation ranges from 66 m (Lago di Garda) to 3769 m a.s.l. (Monte Cevedale), with a 70% of the total area lying above 1000 m a.s.l. Based on corine land cover data (CLC Corine Land Cover 2000), 66% of the province area is covered by forest and seminatural areas, with 28% of conifers and 23% of broad-leaved and mixed forests; agriculture covers 9% of the area, and artificial surfaces only 2%. The study area is characterized by three distinct climatic zones: sub-Mediterranean (annual mean temperature, T mean: 12 °C; total annual precipitation, P tot: 800–1000 mm), oceanic-temperate (T mean: 10– 8208 Environ Sci Pollut Res (2018) 25:8206–8216 Fig. 1 The scheme of the Ozone EFFORT project. Three questions of increasing complexity were identified (left y axis) and addressed along the project lifespan (bottom x axis). The questions targeted different plant populations/community (top x axis) and addressed different assessment endpoints (right y axis). Specific investigations are reported within textboxes. Width and location of textboxes correspond to timespan, question, and target of the relevant investigations. For example, Q1 BIs there a potential risk for vegetation due to ozone?^ is addressed by means of measuring and modeling ozone concentration and exposure over the 2007–2011 period across all the target vegetation with the aim to evaluate the extent of area where the risk thresholds were exceeded. In 2011 such an investigation was supplemented by flux modeling at one forest plot 13 °C; P tot: 900–1000 mm), and continental-alpine (T mean: 8–9 °C; P tot: 1000–1500 mm) (Eccel and Saibanti 2005). symptoms, and the significance of its relationship with ozone levels. Question 3 (BAre there effects of O3 on tree health and growth?^) is about the impact of ozone on growth and health of forest trees. Forest is an essential ecological resource (Fig. 2) and a valuable economical asset in Trentino. It is therefore of great interest to answer this question. Here, the endpoint is to evaluate the role and significance of ozone in explaining defoliation and growth. Study concept, approaches, methods, and data sources The project is designed around three increasingly complex questions (Fig. 1). These questions were agreed upon between resource managers and scientists, address different related endpoints, and have been investigated across the entire lifespan of the project. While a number of new ad hoc investigations have been undertaken, the project also builds upon existing data collected over the study area (Table 1). Question 1 (BIs there a potential risk for vegetation due to O3?^) is about the potential risk due to ozone and has been evaluated on the basis of data on ozone concentration, exposure, and flux and with reference to international standards set by the UNECE (CLRTAP 2014) and EU Directive (2008). For the most part, this question has been covered all along the project lifespan, 2007–2011. Question 1 is linked to a defined endpoint, i.e., quantification of the area where risk thresholds in terms of critical levels (CLs) are exceeded. Question 2 (BAre there symptoms on vegetation related to O3?^) is about the occurrence of specific visible foliar symptoms (VFS), used as an indicator of ozone impact on vegetation, either on specifically standardized bioindicator plants or native vegetation. The relevant endpoint is the frequency of symptomatic plants and/or severity of ozone Approach and methods for question 1: assessment of potential risk Such an assessment is based on different steps: i. Measurement of ozone levels on a network of sites over the forest area in Trentino. The transnational Level I systematic grid of the UNECE ICP Forests was used to allocate 15 sites for passive samplers supplemented by additional 5 sites in 2009. Measurements were carried out weekly from May to July in 2007–2011 (Cristofori et al. 2015). ii. Modeling and mapping of ozone across the whole region. Geostatistical linear models were evaluated to predict the mean ozone concentration in 1 × 1 km grid cells across the entire study domain. A generalized linear model was built, and universal kriging (Krige 1976) was then applied in order to predict mean ozone concentrations in each 1 × 1 km grid cell across the whole Trento province for The Ozone EFFORT project. Data sources, sites, studied period, survey method, and statistical approach adopted Question Data sources Sites Data coverage, year Time resolution Survey method/statistical approach Is there a potential risk for vegetation due to O3? Existing conventional monitors Ad hoc-installed passive sampling Existing passive sampling from other monitoring programs Existing meteorological data from other monitoring programs Ad hoc survey on Nicotiana tabacum Ad hoc survey on native vegetation Ad hoc survey on Viburnum lantana Existing defoliation and growth data from national monitoring programs Existing meteorological data from other monitoring programs Existing foliar data (one composite, pooled sample from five trees per site) Existing soil data 6(1) 20 1(2.b) 2007–2011 2007–2011 1996–2009 hourly weekly weekly Direct ozone concentration measurements/ geostatistical modeling; stomatal flux modeling 1(2.b); 113(3) 1996–2009; 2007–2011 2007 2008–2009 2009–2010 1996–2009; 2007–2011 1996–2009; 2007–2011 1997–2009; 1995–2001 1995; 1995 and 2000 1996–2009 hourly Are there symptoms on vegetation related to O3? Are there effects of O3 on tree health and growth? Existing site data Existing passive sampling form other monitoring programs 9 6 6–30 1(2.b); 15(2.a) 1(2.b); 15(2.a) 1(2.b); 15(2.a) 1(2.b); 15(2.a) 1(2.b); 15(2.a) 1(2.b) weekly twice (2008); once (2009) bi-weekly (2009); once (2010) yearly (defoliation); every 5 years (growth) hourly Environ Sci Pollut Res (2018) 25:8206–8216 Table 1 Direct observation of visible symptoms/ PERMANOVA statistical analysis Direct observation of defoliation and growth/ multiple regression model; linear mixed model every 2 years; every 2 years una tantum una tantum weekly (1) Local Environmental Protection Agency (APPA TN) (2) Local network of the UNECE ICP Forests managed by Autonomous Province of Trento (PAT) and within the National Forest Monitoring program: (2.a) level I sites; (2.b) level II site (3) Local meteorological network (Meteotrentino) 8209 8210 Environ Sci Pollut Res (2018) 25:8206–8216 Fig. 2 Map of the study area (Trentino, Northern Italy). Gray: urban fabric (CORINE land cover code 111, 112); yellow: agricultural areas (211–3, 221–2, 231, 241–4); light green: broadleaved forest (311); dark green: coniferous forest (312) and mixed forest (313); and light blue: water bodies (512) the considered period (2007–2011) (see Cristofori et al. 2015 for details). AOT40 was subsequently estimated for each 1 × 1 cell across the whole area by a linear model (Ferretti et al. 2012) and areas for different levels of exceedance and for different land-use classes were calculated (Cristofori et al. 2015). iii. Estimation of ozone stomatal flux. Estimation of ozone stomatal flux was performed at Passo Lavazè, a Picea abies (L.) Karst. Level II plot belonging to the national forest intensive monitoring network (Ferretti et al. 2000). Hourly ozone concentrations were calculated on the basis of weekly ozone concentrations measured by passive samplers, applying the methodology of Gerosa et al. (2007). Despite other methods exist (e.g., Krupa et al. 2001; Tuovinen et al. 2009), the method by Gerosa et al. (2007) was the only one based on actual measurements from passive sampling and at the same time (i) validated across large geographical areas, (ii) with no need of additional data, and (iii) previously and successfully applied for the same site (Gerosa et al. 2003). Subsequently, the ozone stomatal flux for the upper canopy leaves was calculated by the application of the DO3SE model, fully described by Emberson et al. (2007). Then, the phytotoxic ozone dose (PODY) was estimated by integrating the stomatal fluxes above the instantaneous non-effect threshold Y over the measuring period. A Y threshold of 0 nmol m−2 s−1 was set for this study (POD0) (CLRTAP 2014) (for details, see Ferretti et al. In this issue). Approach and methods for question 2: visible symptoms on vegetation Three investigations have been carried out on this topic and these are as follows: (i) Investigation by means of Nicotiana tabacum L. cv BelW3. The rationale was to use a worldwide-known specific bioindicator to document—on a standardized basis—the possible occurrence of a direct impact of ozone on vegetation. Full details about this investigations are in Cristofolini et al. (2011). Nine sites were selected in relation to two ranges (low and high) of expected ozone levels. Within each range, the site selection was on a random basis. Each site was equipped with six tobacco plants, ozone passive samplers, and a temperature/ relative humidity datalogger. Leaf injury index (LII) and plant height were recorded on a weekly basis during the period 30th of May–27th of June 2007. (ii) Investigation on Viburnum lantana L. The study was performed under field conditions and designed in two consecutive steps. Firstly, May–September temporal development of specific foliar symptoms was investigated in 2009 at two different ozone exposure levels on native plants selected following a fully randomized design. Ozone concentrations were measured on a weekly basis, while foliar injuries were assessed every 2 weeks (Gottardini et al. 2010a). Chlorophyll-related variables were concurrently measured (Gottardini et al. 2014a). Secondly, the relationship between O3 exposure and the frequency of symptomatic plants was investigated. Observations were performed once in August Environ Sci Pollut Res (2018) 25:8206–8216 2010 on 10 (min)–30 (max) plants in n = 30 1 x 1 km cells, randomly selected over the entire region following a stratified design, with elevation (< 700 m a.s.l.; ≥ 700 m a.s.l.) and AOT40 levels (≤ 4.5 ppm h; 4.5–9 ppm h; ≥ 9 ppm h) identifying the strata boundaries (Gottardini et al. 2014b). (iii) Investigation on native vegetation at forest plots. Assessments were carried out on 6 out of 15 UNECE ICP Forests Level I plots in 2008 and 2009 and at the Level II plot of Passo Lavazè in 2008–2011. Visible ozone symptoms were assessed on woody species along the lightexposed forest edge closer to the ozone measurement site (see 2.2.1) following the method described by Schaub et al. (2010) (Gottardini et al. 2012). Approach and methods for question 3: impact of ozone on health and growth Question 3 has been investigated by means of existing defoliation and growth data collected as part of the UNECE ICP Forests Level I and Level II programs, and subsequently processed by different statistical techniques (see details in Ferretti et al. In this issue). According to the present knowledge, ozone exposure recorded in Trentino was enough to cause growth reduction on Norway spruce trees (e.g., CLRTAP 2014), and instances of reduced growth were reported for adult Norway spruce in Sweden (Karlsson et al. 2006). The different studies were carried out: & & & Periodical (2007–2011) mean defoliation of n = 520 trees and mean relative basal area increment (BAI) from n = 354 trees from 15 Level I plots and one Level II plot were processed by means of multiple linear regression with site, climate, soil and foliar chemistry, ozone, and frequency of damage symptoms (recorded as mean number of biotic and biotic-damaging agents observed on trees in the plot) used as predictors. Assessed trees were mostly Norway spruce (Picea abies (L.) H. Karst., n = 186, 36%) and larch (Larix decidua Mill., n = 160, 31%), the remaining 33% being composed by several assorted species (Fagus sylvatica L., Quercus pubescens Willd., Robinia pseudoacacia L., Abies alba Mill., Pinus sylvestris L., Pinus cembra L.). Annual defoliation data from the same trees and period were processed by means of linear mixed models (LMM) with the same set of predictors as above. Annual tree rings width data collected from 23 trees (two cores each) for the period 1996–2009 at one Level II plot (Passo Lavazè), for which ozone exposure and stomatal flux have been estimated, were detrended to account for age and autocorrelation and processed by means of correlation analysis (Spearman Rho). 8211 Quality assurance Quality assurance procedures covered the entire project from survey design to data processing (Elzinga et al. 2001). Accuracy of environmental measurements was tested for both ozone concentrations and meteorological parameters; data were checked for completeness and, when necessary, discarded and/or imputed according to defined procedures (Cristofolini et al. 2011; Gottardini et al. 2010b). Assessment of response variables was performed by adopting standard operating procedures (SOPs) (Schaub et al. 2010), training for the personnel through the attendance to specific courses (UNECE ICP Forests intercalibration course on the assessment of ozone visible injury) and using pictorial atlases as reference standards (Innes et al. 2001; VDI 3957 2003). Independent field checks, objective measurements of visually-assessed leaf injuries (Francini et al. 2009), and microscopic observations were used to further ensure the quality and reliability of data collected in the field. Results and discussion Is there a potential risk for vegetation due to ozone? After geo-statistical modeling of ozone concentration, AOT40 was estimated over the entire study area for a 13-week period between end of April and beginning of August. Figure 3 shows the mean 2007–2011 AOT40 values mapped for the forest and seminatural areas in Trentino, aggregated according to four categories of risk based on the UNECE concentrationbased CL for forest trees (5 ppm h; CLRTAP 2014): no exceedance (< 5 ppm h), exceedance from one to two times the CL (5–10 ppb h), exceedance from two to four times the CL (10–20 ppb h), and exceedance of more than four times the CL. The areas exceeding twice or more the UNECE CL resulted to be 3448 km2, i.e., 90% of the entire forest and seminatural vegetation area. It is worth noting that these figures were estimated for a period of ca. 13 weeks, i.e., ca. 50% of the April–September accumulation period set for forest vegetation. At the Passo Lavazè Level II site, the estimated ozone flux values ranged from 31.0 to 61.4 mmol m−2 s−1 over the entire 1996–2009 period. POD1 values, ranging from 20.1 to 47.7 mmol m−2 s−1, exceeded frequently and largely the critical level of 9.2 mmol m−2 recommended for Norway spruce (CLRTAP 2014) (Ferretti et al. In this issue). Overall, the different CLs thresholds were largely exceeded in Trentino, evidencing a potentially critical situation for vegetation. These results match the findings at European scale, where—despite a general decreasing trend in summer ozone concentration observed in the recent years (Tørseth et al. 2012; Schaub et al. 2015; EEA Report No 28 2016)—most 8212 Environ Sci Pollut Res (2018) 25:8206–8216 Fig. 3 Map of AOT40 values (in ppbh) for all 1 × 1 km cells on forest and semi natural areas in Trentino, corresponding to CORINE land cover codes 311, 312, 313, 321, 322, 324 of the forests are still exposed to ozone values above the limit value (EEA Report No 28 2016). Are there specific ozone symptoms on vegetation related to ozone? The supersensitive Bel-W3 tobacco cultivar developed visible injuries (Fig. 4a) and reduction in height increment significantly correlated to ozone, as well as to air temperature and humidity. When taking into account the relative weight of different predictors and the strong covariation of environmental drivers, biological responses of tobacco proved to reflect the complex interaction between plant and environment more Fig. 4 Foliar ozone symptoms on native vegetation: sharply defined, dot-like lesions on Nicotiana tabacum L. cv Bel-W3 (a), red-dark stipples on Viburnum lantana L. (b), and Rhamnus cathartica L. (c) than the simple effect of ozone concentration (Cristofolini et al. 2011). When considering native species, a consistent response in relation to ozone exposure was displayed by the ozonesensitive V. lantana (Fig. 4b). V. lantana showed a temporal development of specific foliar symptoms consistent with ozone exposure trend throughout the entire growing season (Gottardini et al. 2010a). Ozone symptoms were confirmed by optical microscopy on leaf specimens in order to avoid misclassification and confusion with unspecific anthocyanin accumulation that may occur besides typical ozone symptoms. Concurrently with—or even before—the onset and time development of foliar symptoms, plants showed a decrease in chlorophyll content (ChlSPAD) and vitality (evaluated by Environ Sci Pollut Res (2018) 25:8206–8216 Table 2 Summary results of the statistic applied to verify the significance of ozone in explaining forest tree defoliation and growth; P values are in brackets 8213 Response Mean periodical defoliation Annual defoliation Periodical relative growth Plots, n Trees, n Statistical method Significant predictors 15 520 Multiple linear regression Frequency of damage 15 520 Linear mixed model Frequency of damage 15 354 Multiple linear regression Foliar N:Mg (0.002), symptoms (0.0002), symptoms (0.024), DBH (0.011), foliar N:K (0.0255), foliar N:K (0.02) available water (0.039), precipitation (0.0372), aspect (0.046) aspect (0.0345) means of chlorophyll a fluorescence variables) (Gottardini et al. 2014b). At the regional scale, a significant higher frequency of symptomatic plants and symptomatic leaves per plant occurred at the sites with higher ozone exposure (AOT40 > 9 ppm h), especially at high elevations (> 700 m a.s.l.) (Gottardini et al. 2014a). On the other hand, a survey carried out on n = 281 1 x 1 m quadrates nearby Level I and II forest monitoring sites revealed that none of the 33 woody species observed showed ozone foliar symptoms. Ozone symptoms were instead observed occasionally off-network on V. lantana plants and on other five woody species (Gottardini et al. 2012): Acer campestre L., Cornus mas L. (ozone sensitive, Innes et al. 2001), Cornus sanguinea L. (idem, ibidem), Prunus mahaleb L. and Rhamnus cathartica L. (idem, ibidem) (Fig. 4c). Symptoms on these species were identified by experts during the 9th UNECE ICP Forests intercalibration course on the assessment of ozone visible injuries, 2008 (Gottardini et al. 2012). Given the high ozone exposure measured in Trentino, a higher frequency of symptoms on local vegetation may have been expected. Other studies, however, provided similar, controversial results: in some cases, although ozone thresholds defined for the vegetation protection were exceeded, no correlation was found with leaf symptoms (Ferretti et al. 2007; Baumgarten et al. 2009; Hůnová et al. 2011); in other cases, evident relationship between symptom severity and ozone concentration occurred, but only when environmental conditions were favorable for stomatal conductance and also for ozone uptake (Diaz-de-Quijano et al. 2016). Visible foliar symptom is the only indicator of ozone impact easily detectable in the field (Schaub and Calatayud 2013) and it is considered a valuable tool for the assessment of the actual risk posed by ozone to native vegetation under real field condition (Novak et al. 2003; Smith 2012). In this respect, the results of this project are supportive of the use of a native species (i.e., V. lantana) as an in situ bioindicator to assess the potential harmful effect of ozone on vegetation in forest areas. These results were confirmed by a follow-up study (Gottardini et al. 2017). Table 3 Summary of the Ozone EFFORT project starting questions, expected endpoints, and related answers obtained after the 5-year (2007–2011) project implementation; bibliographic references are reported in association to the three project questions, where results have been published Question Endpoint Answer Is there a potential risk for Extent in space and levels of 55% of Trentino forests is exposed to ozone vegetation due to O3? exceedance of O3 risk thresholds values (AOT40) twice the EU critical level for the protection of vegetation (9000 ppbh). This portion increases to 98% if the EU long term value (3000 ppbh) is taken in account. Also considering a time series of stomatal flux at one forest site, exceedances of CL are very frequent. Are there symptoms on Occurrence of visible injury and Visible ozone injury related to ozone levels vegetation related to O3? relationship with O3 levels occurrences on the bioindicator Nicotiana tabacum L. cv Bel-W3 and on the sensitive Viburnum lantana L. Other few native off-plot species shows specific ozone symptoms on leaves. No symptoms were evident on woody species at level I and II forest monitoring sites. Are there effects of Significance of O3 in explaining Tree health and growth are not in relationship O3 on tree health and defoliation and growth with ozone exposure but with biotic and abiotic damage and foliar nutrition. growth? Reference Cristofori et al. 2015 Gottardini et al. 2010b Cristofolini et al. 2011 Gottardini et al. 2010a Gottardini et al. 2012 Gottardini et al. 2014a Gottardini et al. 2014b Ferretti et al. 2017, In this issue 8214 Are there effects of ozone on tree health and growth? Table 2 provides a synthesis of the main results obtained with annual and periodical defoliation and periodical BAI at the entire set of Level I and II sites (Ferretti et al. In this issue). As for annual and periodical defoliation, the mean number of observed biotic and abiotic damaging agents on individual trees and N:K foliar ratio were the most significant predictors. As for periodical relative BAI, foliar N:Mg, and DBH (a proxy for tree age) were the most significant predictors. In both cases, other site factors (water availability, annual precipitation, and aspect) were significant, while ozone concentration was not. Annual data for defoliation (1998–2009) and growth (1996–2009) of Norway spruce for the Passo Lavazè site were processed in relation to annual AOT40 and stomatal flux for the same period. Without the possible confounding factor of spatial variability, these data permit to evaluate whether time trends in ozone levels (exposure and fluxes) can be related to defoliation and tree growth. Even in this case, however, significant relation with ozone was found neither for defoliation (Spearman r = − 0.426 and − 0.608 for POD0 and AOT40, respectively; P > 0.05), nor for radial growth (Spearman r = 0.028 and r = − 0.063 for POD0 and AOT40, respectively; P > 0.05). Our findings contrast with those obtained from open-top chambers with juvenile trees (e.g., CLRTAP 2014), but are close to what was reported for Norway spruce in Swiss forests (where estimated ozone effects on growth were much less clear than for beech, Braun et al. 2014), and are in line with other studies carried out in Italy (Ferretti et al. 2003; Ferretti et al. 2014). Elsewhere, results were somewhat contradictory; for example, a slight effect on growth of Norway spruce was found in Sweden (Karlsson et al. 2006), but not in Czechia, both on defoliation (Hůnová et al. 2010) and growth (Sramek et al. 2012). In the latter, a significant effect on defoliation was Fig. 5 Synthesis of the evidences arising from Ozone EFFORT project, regarding the potential risk and actual impact of ozone along an increasing complexity of target vegetation Environ Sci Pollut Res (2018) 25:8206–8216 found, although predictors related to biotic-abiotic damage and nutrients were apparently not included in the analysis. Conclusions Ozone EFFORT was planned to fill the knowledge gaps about potential risk and actual impact posed by ozone on vegetation in Trentino (Northern Italy). Designed as a series of modular and complementary studies carried out from 2007 to 2011, Ozone EFFORT permitted to obtain data and information on ozone concentrations and exposure levels in forest sites, on the presence of ozone-specific foliar symptoms, and on the effect of ozone on forest health and growth. The answers to the three main Ozone EFFORT project questions are summarized in Table 3. We conclude that, although ozone concentration, exposure, and stomatal fluxes exceed by far the CLs set to protect vegetation and may create a concern, the actual measurable impact on forests vegetation is limited. While evidence of foliar symptoms has been documented, such an evidence decreases when moving from introduced, potted, and standardized bioindicators to native species growing at the very site. When considering the impact on mature forest trees, evidence of measurable effects on recent tree health and growth is not significant. This pattern of decreasing evidence when moving from theoretical to practical effects can be conceptually synthesized as in Fig. 5. Our findings contrast results obtained from open-top chambers with juvenile trees, and confirm that evidence of ozone impact on tree growth and health under actual field condition is weak. We conclude that the concern about ozone effects on forests arising from experimental results with juvenile trees is not supported by our study, and that ozone effects on mature forests were somewhat overestimated, at least in Italy, and perhaps across Europe. Our study, however, explores only a limited time window and therefore cannot rule out the possibility of a previous impact, that may have occurred decades ago, when ozone levels increased across Europe. If this was the case, we may speculate that plants grown for decades under oxidative pressure (a typical situation in the alpine environment, even without ozone) may have developed functional traits able to support defense and repair process. In this line, together with increased atmospheric carbon dioxide (CO2) and deposition of nitrogen (N) that may also play a positive role in limiting ozone effects, an acclimation of Trentino forests to oxidative (and ozone) stress may have occurred over time. 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