One of the most important threats is tropospheric ozone (O3), the third greenhouse gas (after CO2 and CH4) and a significant phytotoxic air pollutant being of most concern to European forests.

Despite the considerable reduction in precursor emission in Europe and the evidence that mean summer concentrations have leveled off or decreased slightly since the year 2000, ozone exposures in many rural and remote sites continue to be potentially harmful for forest vegetation (Schaub et al., 2018, Vazquez et al, 2024).

Current O3 levels are high enough (annual mean >40ppb) to negatively affect trees by inducing e.g. visible leaf symptoms, decreasing foliar chlorophyll content, accelerating leaf senescence, decreasing photosynthesis, growth and carbon sequestration (Sicard et al, 2019). In 2021 and 2022, all EU Member States recorded levels of ground-level ozone and nitrogen dioxide above the recommended levels of the World Health Organization. The highest levels of ground-level ozone were recorded in the Mediterranean and Central Europe (MZOZT, 2024). Ozone concentrations exceeded 40 ppb on 37.3 % of the Euroepan forest sites and were significantly higher in the Alpine and the Mediterranean regions (Ferretti et al, 2024). The recently revised National Emission Ceilings Directive of the European Union (2016/2284/EU, hereafter NEC Directive) targets a 30% reduction in air pollutant emissions by 2030, compared with 2005 levels (Moura et al., 2022). O3 is the most damaging air pollutant for forests however, impacts cannot be easily monitor in forests. The ozone-induced visible injury on foliage is considered to be the only readily detectable, direct indicator of ozone impact on vegetation (Ferretti el al, 2024). It is usually the first clear and unequivocal sign of the presence of phytotoxic levels of O3 and is a reliable biomarker of monitoring the effects of O3 air pollution in forests as recommended by EU ICP-Forests (International Co-operative Programme on Assessment and Monitoring of Air Pollution Effects on Forests). Ozone visible injuries can provide insight into areas where ozone concentrations are high and help track changes over time. Given that plant sensitivity to ozone is species-specific, sensitive species may decline, altering biodiversity and ecosystem functions like nutrient cycling and habitat provision. For the foliar O3 injury occurrence, the plant must be: 1) a species genetically predisposed to be sensitive to O3 ; 2) exposed to ambient levels that exceed the threshold required for injury, and 3) experience environmental conditions (e.g. temperature, solar radiation, soil water content) that facilitate the uptake of O3 (Hoshika et al, 2018, Sicard  et al 2020, Moura et al, 2022).

Furthermore, the recently developed stomatal flux-based approach estimates the amount of O3 that is absorbed into the leaves or needles through stomata and integrates the effects of multiple climatic factors, vegetation characteristics and local and phenological inputs on O3 uptake or flux. Phytotoxic Ozone Dose above a threshold Y of uptake (PODY) is the accumulated stomatal O3 flux and can be modelled using the Deposition of Ozone and Stomatal Exchange model (DO3SE). Currently, PODY is considered promising as new European legislative standard (Paoletti et al, 2019). However, a validation of the threshold Y under different field conditions is still missing.  Epidemiological studies, where large-scale biological responses are compared with field data, may provide useful information for establishing the best standards and thresholds for forest protection from O3 and deriving new stomatal flux-based critical levels (CLef) for forest protection. As highlighted in a latest EU directive on ambient air quality and cleaner air (2024/2881/EU), these variables are also needed to improve monitoring of the effects of air pollutants on ecosystems and to assess the effectiveness of air pollution control strategies in EU. Furthermore, an excess of O3 is currently stressing plant ecosystems and may negatively affect the nutrient use of plants. Plants may modify leaf turnover rates and nutrient allocation at the organ level to counteract O3 damage (Paoletti et al, 2024). Ozone affects the foliar chemical composition. Climatic scenarios for 2100 suggest that regions with high endemic richness, e.g., the Mediterranean Basin, are most threatened by high levels of O3 (Agathokleous et. al, 2020). Given the fact, that high ozone concentrations are observed in the Mediterranean region, it is crucial to extend our knowledge on the potential ozone impacts on forest health with in situ studies.

Mediterranean forest ecosystems cover more than 30% of the land area of Croatia and are probably the most endangered forest ecosystems, due to peculiar climatic Mediterranean condition. They are under the combined influence of local, regional, and long-distance pollution sources (Jakovljević et al, 2019). Therefore, in the prinical investigator’s previous HRZZ research project “Assessment of Atmospheric Deposition and Ozone levels in Mediterranean Forest ecosystems (DepOMedFor), a team from Croatian Forest Research institute (CFRI) and Instiute for Adriatic crops and karst reclamaltion (IAC) focused on the estimatation of atmospheric inputs to forest ecosystems and identification of the possible impact of acid deposition and O3 on crown condition, tree nutrition, growth, soil and water availability, estimation of the present deposition loads and identification of cause-effect relationships between physiological and chemical parameters and vitality indicators in Mediterranean forests (Jakovljević et al, 2019). The project included passive ozone measurements and exposures over the two reagions along Adriatic coast and  calculation of different O3 metrics provided from the literature, i.e., accumulated exposure AOT40 and stomatal O3 fluxes with an hourly threshold of uptake(Y) to represent the detoxification capacity of trees used for forest protection. (Jakovljević et al, 2021). Passive ozone measurements and monitoring of forest health indicators, namely growth and crown defoliation, were performed in Quercus ilex, Quercus pubescens, Pinus halepensis and Pinus nigra forests. For all the analysed species, ozone levels were close to reach the upper plausibility limits for passive monitoring of air quality at forest sites (100 ppb). O3 metrics based on exposure were found to be higher in pine plots than in oak plots, while the highest values of uptake-based metrics were found on P. nigra. Regarding relationships between environmental variables and forest-health response indicators, the crown defoliation was significantly correlated with the soil water content at various depth while the tree growth was correlated with the different O3 metrics (Jakovljević et al., 2021). For the first time ozone concentrations and  O3 metrics had been calculated in the typical Mediterranean forest ecosystems of the Eastern Adriatic coast using in-situ measurements. Nevertheless, two Mediterranean species were selected (Quercus pubescens and Pinus nigra) that had not been investigated before (according to our knowledge). In the study we highlighted the importance of further epidemiological studies under Mediterranean field conditions that combine ozone monitoring with forest-health indicators for a deeper understanding of its impact on Mediterranean species over time (Jakovljević et al, 2021). Passive sampler monitoring was good alternative for measuring ozone concentations in forests sites at that time. However, it is characterized by uncertainties that reduce their reliability and low temporal resolution, from one week to one month, while POD1 and AOT40 require hourly data. Therefore, in our research we needed to apply functions to estimate hourly concentrations, starting from weekly or biweekly data. Meteo data were not measured directly into the field but all the meteorological variables required for the DO3SE model to compute stomatal conductance were derived from ERA5 (Jakovljević et al, 2021). The impact of O3 on forest-health indicators of adult trees in the Mediterranean is still poorly investigated under field conditions, therefore, further research is warranted.  In this context,  we want to highlight the importance of further investigation of the epidemiological studies under Mediterranean field conditions. that combine active ozone monitoring with forest-health indicators for a deeper understanding of its impact on Mediterranean forest ecosystems.  As the ozone may induce injuries that are of biological significance over time, symptoms suggesting oxidative stress caused by high ground-level ozone concentrations along Adriatic coast should be investigated. Besides ozone concentration, the expression of visibal foliar symptoms (VFS) at a given site is known to be modulated by several factors related to vegetation (species composition, inter- and intra-specific variability), atmosphere (e.g., Vapour Pressure Deficit) and soil (e.g., soil moisture). As for vegetation, species diversity, plant traits, morphological characteristics, detoxification capacity, and microclimate can all play a considerable role in modulating the expression of VFS even for the same species at a given site ( Moura et al., 2022).  It  can be therefore expected that the same species exposed to similar ozone concentrations at different sites may not always display the same frequency of VFS. In the same line, different species exposed at similar ozone concentrations at the same site may also have differentiated VFS responses depending on their functional traits. Under this perspective, the expression of VFS can integrate the various factors mentioned above, and – therefore – the occurrence of VFS can be considered as a sort of “fingerprint”, a useful and ecologically meaningful indicator for detecting the risk that ozone may pose to the forest vegetation at a given site (Ferretti el al, 2024). At forest sites, tropospheric O3 can be monitored with either passive, cumulative, total exposure samplers (like in DepOMedFor project) or continuously operating, mechanical, real-time active monitors (Carrai et al, 2021). At remote sites, the availability of power supply is often limited, and this has been a major  limitation to the use of the active O3 monitoring so far. However, recent studies provided and insite of using solar panels that have relatively low cost (Carrai et al, 2021). Their use, supported by batteries, is a widely adopted solution in remote meteorological stations. In addition, less trips to the forests are required for data collection thanks to remote connection via GPRS for the active monitoring system than for the passive approach (Carrari et al, 2021). These innovative results support active O3 monitoring (AM) rather than passive monitoring (PM). The advantage of AM is greater in the longer monitoring windows, e.g., for the evergreen Mediterranean forests requiring all-year-long monitoring and unfortunately suffer of low temporal resolution in PM (2 weeks to a month) so that it is impossible to detect a high peak concentration of O3 during daytime when plants are photosynthetically active and thus absorbing much O3. The advantage of AM compared to PM is also driven by the lower number of trips from the research center to forest sites. In particular, the reduced global warming potential of active monitoring leads to social benefits regarding carbon reduction. These results indicated an advantage of AM from an environmental, economic, and social point of view. In addition, the active system supplies reliable hourly data that are suitable for stomatal O3 flux estimation, supporting policymakers in assessing ozone  impacts on forests (Carrari et al, 2021).

Considering facts mentioned above, monitoring O3 concentrations at remote forest sites is challenging, however, new technologies are developed that can permit affordable active real-time ozone measurements  as well as the measurements of many others environmental variables. These should be considered for securing technological readiness while keeping attention to data comparability (Ferretti et al, 2024). Therfore, we want to improve monitoring by using a new approach with active O3 monitors (1h time resolution with a realtime data registration, energy supply achieved by solar panels) and sensors for meteorological parametars that will be instaled close to previously investigated P. nigra and Q. pubescens sites where we obtained the most significant results. Nevretheless, these Mediteranean species are still rarely investigated. Propoesed research will give us an answer how plants species in our Mediterranean forest ecosystems respond to ground-level O3 pollution. Furthermore, our team also incorporates  reseracher from Italy, Dr Yasutomo Hoshika from CNR IRET. His institute established the free-air O3 eXposure (FO3X), a unique facility in Mediterranean Europe to assess the effects of O3 on vegetation within the framework of the European AnaEE (Analysis and Experimentation on Ecosystems) Research Infrastructure.  O3-like VFIs can be verified in free-air O3 eXposure (FO3X). Experiments by free-air O3 enrichment are considered as the best approach to provide realistic estimates of tree and plant responses under real-world conditions (Paoletti et al. 2017). Symptoms of O3 VFIs were reported in several tree species at the FO3 X (Hoshika et al. 2018, 2020). However, there is a need for validation studies to confirm whether the O3-like VFIs observed in actual forests are similar enough to real O3 VFIs produced in the FO3X (Moura et al, 2022). A new generation free-air O3 fumigation facility such as FO3X is an appropriate tool for the validation activities of field-observed O3 VFI. As Meditearnean forests are considerable areas of the world terrestrial biodiversity, the ozone FACE (Free Air Controlled Exposure) experiments need to be expanded to more species to realize a proper and representative assessment of forest health under O 3 pollution (Moura et al, 2022). Accordingly, O3MedFor project aims to combine comprehensive datasets  of newly instaled active monitoring stations with forest-health indicators for a deeper understanding of ozone impacts on Mediterranean forest ecosystems. Moreover, an innovative free-air ozone facility will be used to calculate ozone-exposure and fluxbased standards and to estimate the maximum levels of ozone that  Mediterranean species can tolerate.

To coup with these the project will face five main scientific objectives:

1) to set up and test an innovative integrated monitoring system  for the continuous measurement of parameters affecting forest ecosystems

2) to estimate ozone impacts to forest ecosystems and to identify plant-response indicators (e.g. radial and height growth, crown condition, nutrients, visible foliar O3 injury, vegetation diversity)

3) to validate and characterize ozone visible injury and to parametrize stomatal ozone flux model for selected species using an innovative free-air ozone facility

4) to identify cause-effect relationships between physiological and chemical parameters of trees, soil,vegetation, meteorological parameters and ozone and to derive new flux-based critical levels for selected species

5) to translate acquired knowledge on Mediterranean forest ecosystems to interested stakeholders (public institutions, forest management, scientific communities etc.)

Considering our previous research experience in Mediteranean forest ecosystem,  the expertise gained also as a national experts of ICP Forests panels and the engagement of a team members from Italy, Dr Alessandra De Marco from ENEA (a team member also in DepOMedFor) and Dr Yasutomo Hoshika from CNR IRET, we are confident that multidisciplinary team with different skills in  forest ecology, air pollution monitoring, modelling and impacts, ecophysiology, biodiversity, silviculture, plant biology and forest management will succeed in the realization of this project. The team will work on spreading scientific expertise and exchange on innovative research practices regarding Mediterranean forest ecosystems.

The processing of data resulting from monitoring in selected Mediterranean forests, will give the necessary information on the status of the ecosystem and the reaction of these forests to ozone and climate change. The results will also give an opportunity to extend the research to other forest plots since such extensive research with active monitoring stations has not been carried out in Croatia so far.  Also, with acquired results and collaboration with Italian scientists we want to complete the picture of typical Mediterranean forests alon the Adriatic coast, and thus translate acquired knowledge to the scientific community and public institutions involved in forest management, natural resource protection and air quality assessment (e.g. Croatian Forests Ltd., Ministry of enviromental protection and green transition). Moreover, these achievements will contribute to strengthening cooperation and advancement to the ICP-Forests monitoring framework. Project objectives are also strictly related to Article 9 of the NEC Directive (Directive (EU) 2016/2284) that requires Member States to monitor the impact of air pollution on the ecosystems in their territory.