Air Quality and Ecosystems Research Centre (C.R.IN.ES)

CRINES was established in 2007 by the Lombardy Regional Council, FLA and ERSAF (Regional Office for Agricultural and Forest Research) to serve as a permanent pilot structure to support the development of research projects and promote partnerships between academic and non-academic research units. CRINES pilot centre studies the real impact of air pollutants on cultivations, forests and plant ecosystems in Lombardy. CRINES is a science lab working on the assessment of risks that threaten Lombardy’s ecosystems due to environmental conditions.

The origins of CRINES

In December 2002, the Lombardy Regional Council, FLA, ERSAF and ARPA established the pilot study centre CRINES to be opened at Curno (a little town in the province of Bergamo) within the ERSAF Regional centre for the Enhancement of Forest Biodiversity (a former forest wood lot). In that research area, an integrated system of 16 open-top chambers (OTC) and 6 open-top controlled-fumigation rings was placed, able to simulate several different air pollution exposure patterns on plant ecosystems. The agreement between all parties included the start-up of the TOP (Transboundary Ozone Pollution) 3-year study (2003-2005) on the impact of ozone on some of most diffused forest species in the pre-Alps.

This research project, named “The impact of ozone transboundary pollution on plant ecosystems of the transalpine area between Lombardy and the Canton Ticino” was included in the “Supplementary Act of the Framework Program on environmental protection and energy between the Italian government and the Lombardy Regional Council”, signed by the parties on September 5, 2002, article 8 “Monitoring of the impact of ozone”. This Act has later been adopted by DGR no. VII/11433 of November 29, 2002, while on December 16, 2002 the Agreement Protocol was signed by the Lombardy Regional Council, ARPA, ERSAF and FLA, and provided with a technical annex, the Project Feasibility Study. During research activities, the following research units have also been included in the project: Università Cattolica del Sacro Cuore in Brescia (Division of Mathematics and Physics), Università degli Studi di Firenze (Division of Vegetal Biology), Università degli Studi di Milano Bicocca (DISAT) and Università degli Studi di Milano (Division of Vegetal Physiopathology). In October 2006, a further 3-year Agreement was signed by the Lombardy Regional Council, ERSAF and FLA, to update CRINES’s original scientific purpose, set out its current technical implementation and approve its funding up to 2008.

CRINES premiered during the international meeting on the “Impacts of air pollution on plant ecosystems: assessment, perspectives and policies”, held at Curno on May 25, 2007. At the end of 2008, the 3-year Agreement was refreshed to ensure the centre received financial, logistic and managerial support for the next three years.

OZONE and health risks

Tropospheric ozone is a “secondary” pollutant which is created by a chemical reaction between volatile organic compounds (VOC) and oxides of nitrogen (NOx) in the presence of sunlight, mostly during the summer months: in fact, it is known as a summertime air pollutant. Motor vehicle exhaust, industrial emissions and natural sources emit NOx and VOC that help form ozone. Together with PM10, ozone is a pollutant that is a significant health risk; it also damages crops, trees and other vegetation, most significantly in the “Lake areas” within the provinces of Varese, Como, Lecco and Bergamo.

Harmful ground-level ozone concentrations lead to reduced agricultural crop (30%) and commercial forest yields, and to reduced survivability of delicate seedlings such as wheat, bean, lucerne, soybean, clover and barley. High concentrations result also in increased susceptibility of plant ecosystems to diseases, pests and other stresses such as defoliation, leaf curling, chlorosis and necrosis, and also reduced radial growth. Badly enough, “bad” ozone concentrations in the Padan plain have been slowly though constantly increasing in the last 15 years, while other air pollutants, such as sulfur dioxide, haven’t.

The need to study and fight the harmful effects of tropospheric ozone – which occurs in the layer closest to the Earth’s surface, the troposphere – emerged back in 1979, when 40 countries from all over the world met in Geneva and signed the Convention on Long Range Transboundary Air Pollution (CLRTAP), intended to gradually reduce and prevent long-range transboundary air pollution.

CLRTAP has been extended by eight protocols that identify specific measures to be taken by Parties, including the Protocol to Abate Acidification, Eutrophication and Ground-level Ozone, signed by 25 Parties in Gothenburg in 1999 and adopted by the EU.

Recent studies show that ozone is probably the best yardstick to consider in the prevention and abatement of biological, ecological and, mostly, economic damages, since – as we said before – it damages and reduces crop yields.

The European Union has issued a set of landmark directives on air quality, and Italy is expected to adopt them: directive no. 96/72/EC and related directive no. 2003/02/EC, aimed at setting protective health and plant protection-based standards for ozone in the air, and also directive no. 2008/50/EC “on ambient air quality and cleaner air for Europe”, setting limit values for pollutants and deadlines for attaining half and long-term compliance with those values.

What is being done about ozone?

Two research projects are currently being implemented, POP-STRESS and MEDOZ; they have been started at CRINES by several university and other research units.


POP-STRESS PROJECT – Assessment of the impact of stress factors from environmental modifications on biomass cultivations

Coordinamento: Prof. Filippo Bussotti, Università degli Studi di Firenze

Chief Researcher: Filippo Bussotti, Università degli Studi di Firenze Partners:
- Division of Vegetal Biology, Università degli Studi di Firenze
- Division of Mahematics and Physics, Università Cattolica del S.C. in Brescia
- Fondazione Lombardia per l’Ambiente
- ERSAF Lombardia

Partnerships on specific subjects have been signed with:
- Division of Bioenergy, Geneva University (CH)
- WSL, the Swiss Federal Research Institute on forests, snow and landscape, Birmensdorf, ZH (CH)

Basic information

Increased tropospheric ozone concentrations, temperature and water stress conditions are strongly influenced by global change and are likely to affect photosynthesis and vegetation’s net primary production (NPP). Today, strikingly high ozone concentrations are registered in Italy and specifically in the Padan plain, and possible near-future climate change scenarios will register even higher concentrations, together with rising temperatures and a decrease in precipitations. These factors have detrimental effects on photosynthesis and vegetation’s NPP, and can significantly damage the necessary environmental conditions for safe vegetal biomasses’ production processes. This situation requires European governments to implement shared policies for biomass-generated energy production.

The reduced forest growth is a response indicator of plants damaged by high concentrations of tropospheric ozone. Plants have several defense mechanisms against this kind of stress and the effect of high ozone concentrations on forest growth interferes on a biochemical, physiological and morphological basis, negatively impacting photosynthesis.

Plants growth and biomass production are strictly connected to their photosynthetic capacity. We do not know much about the relationship between plants growth and major photosynthetic standards such as net photosynthesis and others, determined through the analysis of chlorophyll fluorescence in differently restrictive environmental conditions, due to one or more stress factors. Knowing this kind of relations and defining plants’ stressed physiological behavior indicators can help researchers spot early risk conditions for the exposed vegetation in restrictive environmental conditions.

Goals and actions

The project's aim is to:

- trace specific stress indicators (ozone and water stress) in rapid-growing plant species (poplars);
- quantify the effects of stress on plants, by studying the relations between stress condition, photosynthetic standards and growth;
- assess the plants’ response precocity to stress and to environmental conditions change, and study compensation and stress acclimatization mechanisms.

Methodology and state-of-the-art report

Research started April 1, 2008 and continued throughout 2009. 6 open-top chambers (OTC) have been set out, among which 3 charcoal-filtered chambers, equipped with active carbon filters reducing by 50% the concentration of ozone if compared to the surrounding environment, and 3 non-filtered chambers, containing about 95% of ozone. Two poplar clones are currently under study, Populus maximowiczii Henry x P x berolinensis Dippel (Oxford Clone), an hybrid plant used in Central-Eastern Europe to build fences and perform short rotation forestry (in fact, it is a key plant for biomass productions), and non-hybrid Populus nigra (Jean Pourtet Clone).

Plants have been produced by cutting, encouraged to growth independently in pots and exposed to two different ozone levels (filtered and non-filtered chambers) and to two different water systems (irrigated and non-irrigated).

Starting June 2009, research units have performed the following:
• net photosynthesis measurements by gas analyzers (ADC, BioScientific Ltd., and CIRAS, PPSystems),
• stomatic conductance measurements (by gas analyzers and porometer AP-4 Delta-T, Devices) to assess gas exchange,
• direct fluorescence (HandyPea, Hansatech) and modulated fluorescence (by FMS2, Hansatech) measurements to evaluate plants photosynthetic efficiency in terms of radiant energy dissipation.

These measurements have been performed in 5 different day hours and repeated every fifteen days. Starting from July, measurements are repeated on a weekly basis.

The stomatic conductance periodical measurement and the water potential measurement (by means of a Scholander pressure chamber), performed at pre-dawn and midday, and the Relative Water Content (RWC) measurement have helped researchers quantify water stress. Measurements of stems’ height and diameter and the “pinning method” (based on the periodical injuring of plant stems to monitor the intra-annual change dynamics on a high time resolution and to anatomically [no. of vessels and windpipes] quantify the stem’s diametral growth) have been used on a similar time schedule to assess the impact of these stress factors, both independently and integratedly, on plants growth. Moreover, epigean and hypogean biomass measurements have been performed. More evidence on photosynthetic efficiency, i.e. on the growth potential of plants exposed to stress conditions, will be obtained through the analysis of carbon (δ13C) and oxygen (δ18O) isotopic discriminations.
Analyses on measurement campaigns data are currently being performed and will be discussed in the forthcoming state-of-the-art report.


MEDOZ PROJECT- Synergic effect of abiotic stresses in species living in the Macchia shrubland biome by means of field and simulated environments surveys

Chief Researcher: Fausto Manes, Università di Roma “La Sapienza”

- Division of Vegetal Biology, Università di Roma “La Sapienza”
- Division of Mathematics and Physics, Università Cattolica del S.C. in Brescia
- Division of Forest Meteorology, Università degli Studi di Sassari
- Fondazione Lombardia per l’Ambiente

Basic information

Coastal ecosystems are key ecological and economic areas, have great species diversity and, as recently stated in IPCC’s 2007 annual report, are likely to suffer from climate change effects, which can make their now-restrictive environmental conditions harsher. They’re poorly layered soil, have remarkably variable water resources distribution and availability and also suffer from a constant blow-in of marine aerosols, which play a key role in the distribution of vegetation and influence both structure and physiology. Aerosol has mechanical and chemical effects on vegetation. The mechanical effect is more intense during the winter season and is due to big particles, mostly based on minerals (sands and particulate), while the marine component (NaCl) triggers functional alterations.

Scientific works on the subject prove that aerosol is taken in little consideration in researches aiming at quantifying Macchia shrubland biome woody species functional processes, and we have very few studies on the effects of chronic exposure to aerosols on water balance and pollutants flows. In fact, almost all studies focus on surfactants, which are significantly responsible for sea water pollution, while studies on the interactions between aerosol-derived and other air pollutants-derived (such as ozone) damages are still few. The exposure of coastal vegetation to this pollutant might prove significant, since breezes flow patterns expose coastal vegetation to both marine aerosols and pollutants from inland.

In fact, coastal areas are densely populated in Italy as well as all over the world; for this reason, any study covering the vegetation of these areas should consider the ecological footprint of urban areas. More attention is now being paid to increased emissions mainly due to motor vehicle exhaust, which reduce air quality and make up for the main cause of secondary polluted precursors like ozone. Ozone reaches peak concentrations during the summer months, when vegetation in the Mediterranean area suffers from dryness, which can become severe due to changed seasonal precipitations patterns.

To fully understand how increased pollutants concentrations and dryness can negatively affect water response and vegetal production rates, researchers have to quantify and study the impact of these factors on the functionality of each single plant organ (leaves, branches, roots) and on its stomatic structures, which play a key role in balancing the exchanges between vegetation and the atmosphere. In fact, the detrimental effect of ozone on vegetation is proportional to the atmospheric absorption, which in turn depends on ozone environmental levels and on stomatic conductance, gs, a key factor in the assessment of ozone flow patterns, used to measure the exposure of vegetation to this pollutant.

Consequently, each factor influencing the stoma opening/closing mechanisms can also influence the impact of ozone on vegetation.

Another key element is the study of interaction mechanisms (both synergic and antagonistic) between different kinds of stress (aerosols – water stress), which can change plant response to environmental factors, thus reducing gas exchanges and the flow of ozone, which can thus be absorbed mostly non-stomatally.

Evergreen plant species form the Mediterranean climax vegetation, and the study on how these species can optimize CO2 absorption and water loss in different climatic conditions under different stress types has a striking relevance in the wide-ranging study of coastal shrubland species physiology.

Goal and actions

Since coastal ecosystems are complex systems, researchers should develop specific research projects aimed at establishing more specific cause-effect relations among single natural stress factors through different theoretical and methodological approaches, thus matching monitoring patterns in natural conditions with controlled conditions experiments. This is the background to the Medoz project, which includes two different experiment formats performed at study sites located in the Presidential Estate in Castelporziano (RM), the Oristano forest wood lot and CRINES at the ERSAF Regional forest wood lot in Curno (BG).

Methodology and state-of-the-art report

The abovementioned research programs will be performed thanks to an integrated approach which includes field surveys and controlled-conditions experiments. Results will then be stored into a single database which will help researchers study widely and exhaustively the functionality of species and the processes occurring under different ecological conditions.

The first scheduled experiment is currently framing out the physiology of Quercus ilex L. (Holm Oak) and Phillirea latifolia L. (Phyllirea), located inside the Presidential Estate in Castelporziano at varying distance from the coastal line (200mts, 500mts, 1000mts). The experiment aims at assessing how exposure to marine aerosol influences water and species pollutants flows in the Mediterranean region.

At the same time, another experiment has been started in April 2009 under controlled conditions at the CRINES centre in Curno on Holm Oak and Strawberry Tree potted seedlings, placed inside 6 Open-Top Chambers, two for each experimental theory.

20 plants in each OTC are exposed to two stress factors, ozone and salty spray (two sets, salty-spray atomized plants and non-atomized plants). Ozone treatment develops on three levels: about +30% higher-than-environment, filtered (-50%), and non-filtered with ozone levels similar to environmental levels (about 95%). Experiments have been started in May and comprise a 3-day physiological measurements session, including: 1 gas exchange daily cycle (at 8, 10 am and 12, 14, 16 and 18 pm), “a” chlorophyll fluorescence measurements, pre-dawn and midday water potential measurements and structural measurements of such standards as SLA (Specific leaf area), plant diameters and height, plant density and stomatic damage detected under a scanning electron microscope (SEM). During the test final phase, from July 20 to August 10, plant response to forced stress and the potential interaction between these two factors will be assessed through a water stress test, performed after two months of treatment. The possible different response to water stress will then be used to highlight the impact of and interaction between ozone and marine aerosol. In this phase, abovementioned measurements will be performed twice a week, while photosynthesis response curves to CO2 and night measurements will be added to spot night transpiration.

The second test is performed simultaneously in the three study sites. Fifteen individuals for each species (Holm Oak, Phyllirea and Strawberry Tree) belonging to two different populations (of Sardinian and Latial origin) have been potted in the study sites. This test aims to assess how the three species’ phenotypical plasticity influences the plant water frame and in what degree the latter changes the use of water in each of the three species; and to assess in what degree the studied species offset structural changes with physiology changes. The test was started October 2008 with the allocation of the vegetal material and its schedule, started on May 2009, required researchers to perform several repeated defoliations on each plant (once a month, starting from May, 40% or 70%); in the future, plants will be forced to water stress, and this will allow researchers to assess the different response and resistance to stress of plants grown in the three study sites, and the impact of defoliations. Plant response to water stress will be assessed in terms of both foliar water potential evolution patterns and stomatic conductance in increased dryness conditions. This response also results from physiological and structural readjustments performed by plants during a 2-year growth span, and therefore the individual total growth, the relation between leaf area and conductance area, the stomatic density, the leaf shape factor, the maximum conductance rate and light response curves will also be considered to assess the maximum quantum yield. Moreover, the replication of the test in geographical sites belonging to three different phytoclimatic areas will help researchers prove that a possible divergence in the response to defoliation derives from climatic factors.
This whole set of tests will also allow to fully understand to which signals stomatic conductance and stomatic density respond, i.e. whether to such standards as soil water concentrations and environmental humidity, or uniquely to the actual water availability to each leaf surface unit.

Learn more about C.R.IN.ES

C.R.IN.ES Advisory Committee
Lombardy Regional Council: Gian Luca Gurrieri
ERSAF: Sauro Coffani
Fondazione Lombardia per l'Ambiente: Antonio Ballarin Denti

Science Advisor
Giacomo Gerosa,  Università Cattolica del Sacro Cuore di Brescia giacomo.gerosa@unicatt.it
Technical Advisor
Riccardo Marzuoli,  Fondazione Lombardia per l'Ambiente riccardo.marzuoli@flanet.org
Regione Lombardia 
Fondazione Lombardia per l'Ambiente