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Environmental Pollution 152 (2008) 274e284

www.elsevier.com/locate/envpol

Visible leaf injury in young trees of Fagus sylvatica L. and

Quercus robur L. in relation to ozone uptake and ozone exposure.
An Open-Top Chambers experiment in South Alpine
environmental conditions
G. Gerosa a, R. Marzuoli a,*, R. Desotgiu b, F. Bussotti b, A. Ballarin-Denti a
a
Catholic University of Brescia, Department of Mathematics and Physics, Via dei Musei 41, 25125 Brescia, Italy
b
University of Florence, Department of Plant Biology, Piazzale delle Cascine 28, 50144 Florence, Italy
Received 8 February 2007; received in revised form 12 June 2007; accepted 19 June 2007

Ozone fluxes are more accurate than AOT40 exposure index in predicting ozone
visible foliar injury onset on beech seedlings in South Alpine environmental conditions.

Abstract

An Open-Top Chambers experiment on Fagus sylvatica and Quercus robur seedlings was conducted in order to compare the performance of
an exposure-based (AOT40) and a flux-based approaches in predicting the appearance of ozone visible injuries on leaves. Three different ozone
treatments (charcoal-filtered; non-filtered; and open plots) and two soil moisture treatments (watered and non-watered plots) were performed. A
Jarvisian stomatal conductance model was drawn up and parameterised for both species and typical South Alpine environmental conditions, thus
allowing the calculation of ozone stomatal fluxes for every treatment. A critical ozone flux level for the onset of leaf visible injury in beech was
clearly identified between 32.6 and 33.6 mmol O3m2. In contrast, it was not possible to identify an exposure critical level using the AOT40
index. Water stress delayed the onset of the leaf visible injuries, but the flux-based approach was able to take it into account accurately.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Ozone flux; AOT40; Beech; Oak; Visible leaf injury

1. Introduction uptake pathways, such as cuticular uptake, appear to be negli-

The negative effects of ozone on trees have been well docu-
mented (Skärby et al., 1998; Matyssek and Innes, 1999; Krupa
et al., 2001) at biochemical, physiological, and morphological
levels, including growth reduction and the onset of typical
visible leaf injury (Innes et al., 2001).
The intensity of phytotoxic responses depends on the bio-
logically available quantity of the pollutant, i.e. the quantity
of ozone absorbed by the plant through stomata and which
cannot be neutralized by its detoxifying processes. Other
* Corresponding author. Tel.: þ390302406719; fax: þ390302406742.
E-mail address: riccardo.marzuoli@unicatt.it (R. Marzuoli).
gible (Kerstiens et al., 1992).
Ozone risk assessment, in Europe, has for a long time been
performed by means of exposure indices based exclusively on
the atmospheric concentration of ozone (AOT40) (Kärenlampi
and Skärby, 1996). This is the basis of what is known as the
UN-ECE Level I assessment, and also introduced into Com-
munity legislation (EC Directive 02/03) recently. Several
UN-ECE workshops, organized within the framework of the
Long-Range Transboundary Air Pollution Convention (http://
www.unece.org/env/lrtap/), have been dedicated to the detec-
tion of critical AOT40 thresholds for plant protection. But it
has become obvious that such a simple index is not very reli-
able for impact prediction, since it does not take into consid-
eration either the variability of environmental conditions in

0269-7491/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2007.06.045
 Author's personal copy
G. Gerosa et al. / Environmental Pollution 152 (2008) 274e284
Europe or the plant’s physiology (Karlsson et al., 2007). In
more recent workshops, the scientific community has thus sug-
gested that assessments should be performed using ozone sto-
matal flux calculations (Karlsson et al., 2003, 2007; Wieser,
2007): unlike AOT40, this parameter does not merely evaluate
atmospheric concentrations of ozone, but also considers the
stomatal conductance of vegetation.
A clear understanding of stomatal conductance behaviors
and dynamics in relation to different environmental conditions
(especially in relation to water availability), thus becomes es-
sential to quantify the amount of ozone effectively absorbed
by plants. Furthermore this information is necessary, as a pre-
liminary step, in any study that aims to define quantitative
doseeinjury relationships, and to produce an ozone risk as-
sessment according to the UN-ECE Level II approach (Fuhrer,
2000; Ashmore et al., 2004), on both local and European
scales (Emberson et al., 2000a; Mills et al., 2003; Matyssek
et al., 2007).
Parameterizations of stomatal conductance in the main tree
species of the region at South of Alps, based for the most part
on Jarvis (1976) model, are derived from studies carried out on
northern and central European ecotypes, i.e. in climate condi-
tions that are very different (Karlsson et al., 2000; Emberson
et al., 2000b; Wieser and Emberson, 2004). Many authors sus-
pect that these parameterizations may deviate significantly in
southern European conditions, but there are still too few
data available to state this with certainty (Ribas et al., 2005;
Elvira et al., 2007).
The aim of this study is to compare the effectiveness of the
flux-based approach to the exposure-based approach (AOT40),
in relation to the onset of foliar symptoms (taken as response
indicators) in the ecological conditions typical of the South
Alpine region. For this purpose, an experiment was carried
out in Open-Top Chambers (OTC) on two woody species,
very widespread in this region and displaying different degrees
of ozone sensitivity as far as visible foliar symptoms are con-
cerned (Fagus sylvatica L. and Quercus robur L.). In northern
Italy, F. sylvatica is typical of the sub-mountainous plane and
is considered moderately sensitive: ozone-induced foliar
symptoms (bronzing and stippling) have been described in
several papers (Innes et al., 2001; Vollenweider et al., 2003;
Gerosa et al., 2003; Bussotti et al., 2005). Q. robur, on the
other hand, grows in flood plain areas and no specific ozone-
induced symptomatology has yet been described. These two
species (one sensitive and one resistant) are both ecologically
important in the region South of the Alps, and were chosen in
the study to examine whether the sensitivity and/or resistance
to ozone is related to stomatal conductance, or also other
factors should be taken into consideration, thus suggesting
that ozone uptake is not the only component involved in these
mechanisms.
The use of foliar visible symptoms as response indicators
(instead of biomass increase, as suggested in UN-ECE study
groups) is due to the need to have immediate evidence of
the effects, since several studies reported significant effects
on the growth only in long-term experiments (cf., for example,
Rebbeck and Scherzer, 2002; Karnosky et al., 2005).
275
The use of biomass increase as an indicator of ozone-in-
duced injury presents serious disadvantages in the South Al-
pine and Mediterranean areas, where the growth of trees
is strongly influenced by highly variable ecological
conditions e such as edaphic fertility and changing water
availability e that can mask the effect of ozone. In this con-
text, foliar symptoms in sensitive species could be considered
as a more effective ecological indicator of the impact of ozone
on vegetation.
2. Materials and methods
2.1. Experimental set-up
The research site is located within the Forest Nursery of Curno
(Lat. 45 700 N, Long. 9 620 E, elev. 242 m asl) on the northern edge of the
Po Valley, in the foothills of the first slopes of the Alpine range in northern
Italy. The soil type is Typic Paleudalf fine-silty, mixed, mesic according to
the USDA classification, and the climate is typically continental with quite
dry and hot summers, rainy springs and autumns, and cool and dry winters.
Ozone concentration levels in this region are among the highest in Europe:
critical levels are frequently exceeded and almost all the forest areas in this
region are subjected to summer exposure levels above 20,000 ppb h. These
ozone levels have been well documented in a number of papers published
over the last 15 years (Sandroni et al., 1994; Ballarin-Denti et al., 1999; Ger-
osa et al., 1999; Gerosa and Ballarin-Denti, 2003).
Eight Open-Top Chambers and four open plots were established in summer
2003. The OTCs (3 m diameter, 2.8 m height) were constructed according to
the scheme described by Heagle et al. (1973) and already used in research pro-
grammes conducted in southern Switzerland (VanderHeyden et al., 2001;
Novak et al., 2003, 2005). The OTCs were active from 1st April to 30th Sep-
tember, in both 2004 and 2005, in the daytime, from 5.30 a.m. to 9.30 p.m.
Experiments were conducted using two controlled soil moisture regimes, wa-
tered (W) and non-watered (Dry, D), and three ozone exposure treatments:
50% ambient ozone in charcoal-filtered OTC (CF), 95% ambient ozone in
non-filtered OTC (NF), and 100% ambient ozone in open plots (OA) without
chambers. The set-up was subdivided into two blocks (W and D), separated
from each other by a steel slab sunk into the ground to a depth of about
1 m. Each of the two blocks included six plots (two OTC-CF, two OTC-NF
and two OA plots). Within each plot, 16 one-year-old bare-root seedlings
(4  Populus nigra L., 4  Fraxinus excelsior L., 4  F. sylvatica, 4  Q.
robur) were planted in autumn 2002, a year and a half before the beginning
of experimentations.
The well-watered plots (block W) received daily irrigation during the
whole season, in addition to natural precipitation, to keep the soil moisture
close to field capacity. The dry plots (block D) were irrigated only during
the first season (2003) for seedling establishment; thereafter, they received
only natural precipitation during the 2004 and 2005 seasons, except for rescue
watering. Overall, between April and September, the W plots received 928 mm
of water in 2004 and 1063 mm in 2005; whereas D plots received 487 mm of
water in 2004 and 582 mm in 2005. The two different water treatments were
applied to obtain differentiated ozone stomatal fluxes, in order to evaluate their
influence on the onset of visible foliar symptoms.
Ozone concentrations within each chamber and in the open plots were con-
tinuously monitored by an ozone automatic analyzer model 1108 RS (Dasibi
Italia s.r.l., Milano, I), via a solenoid valves switch system, which collects air
from sampling points in the centre of each plot at 90 cm above ground. The
system was managed and controlled by a personal computer equipped with
an NI-DAQ 6.9 I/O board and a Labview 6.1 programme (National Instru-
ments, Austin, Texas) devised specifically for this experimentation. At the
same time, all relevant meteorological data within six selected plots (two
OTC-CF, two OTC-NF and two OA plots) and those collected from a two-level
tower were continuously monitored and recorded (Table 1) by means of
a CR10X datalogger (Campbell Sci., Logan, Utah) equipped with an AM16/
32 relay multiplexer and an SDM-SW8A switch closure interface (Campbell
Sci., Logan, Utah).
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276 G. Gerosa et al. / Environmental Pollution 152 (2008) 274e284

Table 1
Meteorological sensors list and their measurements heights
Parameters Meteorological tower OTC (CF and NF) Open plots
Height 3m 6m 0.9 m 0.9 m
Air temperature Campbell Sci. 50Y humitters Campbell Sci. 50Y humitters Campbell Sci. 50Y Campbell Sci. 50Y
and relative humidity humitters humitters
Air pressure Druck RPT410F barometer
Wind speed EM WSS1 cup anemom. EM WSS1 cup anemom. Thermal Logic
needle anemom.
Global radiation LICOR 200SZ pyranometer
Photosynthetically LICOR 190SZ quantum LICOR 190SZ
active radiation sensor quantum sensor
Rain (wet plots) Young 52202 rain gauge
Rain (dry plots) EM ARG100 rain gauge

In the same six selected plots soil water content was monitored with TDR data-processing, however, all outlying measurements and those that displayed
sensors (CS616, Campbell Sci., Logan, Utah, one sensor for each plot). a temperature difference between the cuvette air (porometer) and leaf temper-
Climate conditions and water received by the different treatment groups ature greater than 1.0  C were discarded, in order to select more reliable
during the two growing seasons are reported in Table 2. values (Verhoef, 1997). About 500 valid measurements per species were retained.

2.2. Stomatal conductance measurements 2.3. Modelling of stomatal conductance

Water stomatal conductance was measured by means of a cycling diffusive In order to calculate stomatal fluxes, the stomatal conductance for
porometer AP4 (Delta-T Devices LTD, Cambridge, UK). T AP4 works enclos- F. sylvatica and Q. robur was modelled using the classic Jarvis multiplicative
ing the leaf in a small air chamber, equipped with a humidity sensor, and sub- approach (Jarvis, 1976):
sequent measurement of the time (called transit time) it takes for a leaf to
gw ¼ gmax ½fPHEN fT fPAR fVPD fSWC 
release sufficient water vapour to change the relative humidity in this chamber
by a fixed amount (2.3%). The cycle is then repeated to guarantee reproduc- The gmax for water value was chosen for each species by taking the 95th
ibility. This is compared with a calibration plate of known resistance in order percentile of all the valid conductance measurements, regardless of the differ-
to derive the stomatal resistance or conductance of the leaf (Monteith et al., ent water supply treatment.
1988; Bragg et al., 2004). A single gmax value for each species was used, instead of two values e
The instrument was calibrated prior to each series of measurements. Mea- gmaxdry and gmaxwet e as suggested by other authors (Grüters et al., 1995;
surements were performed once a week, from the moment leaves were fully Karlsson et al., 2000), since, in the rationale of the Jarvis model, gmax must
expanded, from May to September, at different diurnal cycles (daytime be- be understood as the maximum conductance that the plant can express in
tween 7:00 and 18:00) taking care to include different conditions of tempera- non-limiting conditions.
ture, PAR, VPD and SWC, in order to ensure that the subsequent modelling The species-specific limiting functions fT, fPAR, fVPD and fSWC, which give
would be as representative as possible. Leaf stomatal conductance was esti- a value between 0 and 1, were characterized by borderline analysis, using the
mated by taking measurements in the median portion of the lamina, on two points corresponding to the 98th percentile of grel (conductance value relative
top canopy leaves of 24 plants per species in the 12 plots. Two branches to the gmax) plotted for each class of values of environmental variable consid-
were selected on each plant; measurements were performed on the light- ered (T, VPD, SWC, PAR). It was thus possible to make the modelling less
exposed leaves from those branches, in both seasons. Overall, about 700 dependent on outlyers representing measurements of doubtful quality. Sepa-
measurements per species were performed in each of the two seasons. In the rate analysis was performed for each species.
The fPHEN functions are simple ‘‘on/off functions’’ that trigger with the leaf
Table 2 emergence and expansion for each species.
Meteorological parameters and ozone in the two growing seasons 2004e2005
Tmax Tmin O3max AOT40a Rain Rain 2.4. Calculation of ozone exposure and stomatal fluxes
( C) ( C) (ppb) (ppb h) (dry plots) (wet plots)
(mm) (mm) Ozone exposure was calculated by means of the cumulative index AOT40
2004 April 25.9 3.7 88 1938 117 117 based on the mean hourly ozone concentrations during sunlight hours:
May 27.7 6.1 101 3100 85 99.5 X 
June 33.4 13.9 153 7225 44 96.9 AOT40 ¼ ½O3 i 40 Dt
July 34.1 12.3 144 8636 81 164 ci : ½O3 i > 40 ppb
August 31.9 15.1 122 4731 124 300 ci : RadGlob > 50 W=m2
September 31.3 8.9 130 3453 35 148
In order to allow for a direct comparison with ozone stomatal fluxes, cumula-
2005 April 24.5 5.7 80 1502 118 118 tive ozone concentrations to calculate AOT40 were collected starting from the
May 31.8 9.7 146 4793 60 138 moment when leaves sprouted until 30th September. This is not the same pro-
June 35 10.7 135 8174 52 181 tocol as that contained in the EC Directive (02/03), which requires data collec-
July 34.2 14.2 118 6426 91 172 tion on forest species over a six-month period, from 1st April to 30th
August 36.4 13.6 87 3551 114 232 September, but in our opinion it has more biological sense.
September 31 12.3 115 1640 145 221 Ozone stomatal flux was calculated for a top canopy leaf according to
AOT40, accumulated ozone over a threshold of 40 ppb; O3max, maximum Emberson et al. (2000b) and Karlsson et al. (2000):
ozone concentration recorded; and T, temperature.
a
Calculated from the mean values of the open plots sampling points O3
Fst;O3 ¼
(h, 0.9 m). ðRb;O3 þ 1:68=gw Þ
 Author's personal copy
G. Gerosa et al. / Environmental Pollution 152 (2008) 274e284
where 1.68 accounts for the different diffusivity of water in air with respect to
the ozone. The sub-laminar ozone resistance Rb;O3 was calculated using the
Unsworth et al. (1984) equation:
 1=2
d rating power of the atmosphere is described by the following
Rb;O3 ¼ k
u linear function:
where the empirical coefficient k is set to a value of 132, as supported by
Thom’s (1975) measurements, d is the mean leaf width for each species,
and u is the mean wind speed at top canopy level. For the open plots the latter
was calculated with an aerodynamical approach from the wind speed profiles
recorded at the meteorological tower and the related atmospheric turbulence
and stability parameters (u*, J(L)). Inside the OTC u was set to the value mea-
sured continuously by anemometers placed at canopy level.
Atmospheric resistance Ra was not considered either in open plots or in
OTCs, since the ozone concentration was measured directly at top canopy
level.
2.5. Detection of visible leaf injuries
Visible foliar symptoms were assessed on every seedling twice a week on
selected branches, in order to detect the time of onset of the first visible symp-
tom. At the end of May of both years the selected branches displayed fully ex-
panded and mature leaves. Each assessment took into account the number of
symptomatic leaves and the percentage of leaf area affected according to a pro-
portional scale of 5%. Symptoms assessment was done with reference to avail-
able handbooks and photoguides (Innes et al., 2001).
3. Results
3.1. Stomatal conductance model for F. sylvatica and
Q. robur
Maximum stomatal conductance ( gmax) values measured in
the two species considered were: 1.16 cm s1 for F. sylvatica
and 1.87 cm s1 for Q. robur, both values referring to the Pro-
jected Leaf Area. In both species, the measured functions fPAR,
fT, fVPD and fSWC were identical in both watered and non-wa-
tered plants (Figs. 1 and 2). The function that describes the
dependence of stomatal conductance on PAR is as follows:
fPAR ¼ 1  eaPAR
where a represents a species-specific coefficient (0.007 for
F. sylvatica and 0.005 for Q. robur) and PAR represents the
photosynthetically active radiation expressed in nmol photons
m2 s1.
The fT function is described by the following equation:
 b
ðT  Tmin Þ ðTmax  TÞ
fT ¼   photochemical episodes (Fig. 4). By the end of June 2005 cu-
Topt  Tmin ðTmax  Topt Þ
where T represents the temperature of the air near the
leaf; Topt, Tmax, Tmin are three species-specific parameters
(Table 3), respectively, representing optimal, maximum and
minimum temperatures, all expressed in  C; and b is defined
as follows:
 
Tmax  Topt
b¼   Seasonal trends of ozone stomatal flux, cumulated over
Topt  Tmin
277
If T is greater than Tmax or smaller than Tmin, the function fT is
given the minimum value of 0.1.
The dependency of stomatal conductance from the evapo-
ð1  0:1Þ ðc  VPDÞ
fVPD ¼ þ 0:1
ðc  dÞ
where VPD is the water vapour pressure deficit expressed in
kPa, c represents the value of VPD at which stomatal conduc-
tance reaches its lowest value, established at 0.1, and d repre-
sents the value of VPD beyond which stomatal conductance
begins to suffer a limitation. In the case of VPD values lower
than d, fVPD is established at 1, whereas when VPD exceeds
value c fVPD is established at 0.1. Parameters c and d are spe-
cies-specific and their values are reported in Table 3.
Soil water availability (SWC) exercises its limiting action
according to the following function:

fSWC ¼ max 0:1; min 1; gSWCðh=SWCÞ
where SWC represents soil water content expressed as a per-
centage of field capacity, while g and h are two coefficients
whose values are, respectively, 1.01472 and 0.26043. The
function fSWC is identical for both species.
The function fSWC is formulated in a new way, making it
smoother than the linear functions with a sheer threshold
and steep decline that can be found in the literature.
The comparison between conductance measured in the field
and conductance modelled on the agro-meteorological param-
eters relative to the top canopy leaves enabled the authors to
evaluate the performance of the model. In both species the
model predicts stomatal conductance fairly well, without ei-
ther underestimating or overestimating it (see Fig. 3). The re-
gression curve of predicted vs measured hourly means
conductances is the following: gpredicted ¼ 1.028  gmeasured
for F. sylvatica, and gpredicted ¼ 1.090  gmeasured for Q. robur
with an R2, respectively, of 0.893 and 0.806.
3.2. Ozone exposure and uptake
During 2004, AOT40 displayed a seasonal trend of fairly
uniform increase; in 2005 a more irregular rise in exposure
was recorded, including abrupt peaks caused by very intense
mulative exposure had already reached 54% of the overall
amount for the whole season, while in 2004 the figure for
the end of June only accounted for 35% of the total.
Fig. 4 shows the dates on which the first ozone-induced leaf
symptoms appeared in F. sylvatica. No indication signalling
the onset of symptoms in Q. robur appears in Fig. 4, since
no characteristic symptom was observed in this species, al-
though some slight yellowing of leaves was noticed towards
the end of the season in NF and OA plots.
time in the two species, distinguishing between the different
 Author's personal copy

278 G. Gerosa et al. / Environmental Pollution 152 (2008) 274e284

D W fPAR D W fT
1.2 1.2

1 1

0.8 0.8
g/gmax

g/gmax
0.6 0.6

0.4 0.4

0.2 0.2

0 0
0 500 1000 1500 2000 5 10 15 20 25 30 35 40 45
T [°C]
PAR [umol m-2 s-1]

D W fVPD D W fSWC
1.2 1.2

1 1

0.8 0.8
g/gmax

g/gmax

0.6 0.6

0.4 0.4

0.2 0.2

0 0
0 1 2 3 4 5 0% 20% 40% 60% 80% 100% 120%
VPD [KPa] SWC [%FC]

Fig. 1. f limiting functions for the stomatal conductance model of Fagus sylvatica. D, non-irrigated plants; W, irrigated plants; PAR, photosynthetically active
radiation; T, temperature; VPD, vapour pressure deficit; SWC, soil water content; g, stomatal conductance; and gmax, maximum stomatal conductance.

D W fPAR D W fT
1.2 1.2

1 1

0.8 0.8
g/gmax

g/gmax

0.6 0.6

0.4 0.4

0.2 0.2

0 0
0 500 1000 1500 2000 5 10 15 20 25 30 35 40 45
PAR [umol m-2 s-1] T [°C]

D W fVPD D W fSWC
1.2 1.2

1 1

0.8 0.8
g/gmax

g/gmax

0.6 0.6

0.4 0.4

0.2 0.2

0 0
0 1 2 3 4 5 0% 20% 40% 60% 80% 100%
VPD [KPa] SWC [%FC]

Fig. 2. f limiting functions for the stomatal conductance model of Quercus robur. D, non-irrigated plants; W, irrigated plants; PAR, photosynthetically active
radiation; T, temperature; VPD, vapour pressure deficit; SWC, soil water content; g, stomatal conductance; and gmax, maximum stomatal conductance.
 Author's personal copy

G. Gerosa et al. / Environmental Pollution 152 (2008) 274e284 279

Table 3 3.3. Visible leaf symptoms in relation to ozone exposure

Summary of the coefficients’ values for the stomatal conductance model of the and uptake
two species
Parameter Fagus sylvatica Quercus robur Unit Ozone visible leaf injury occurred during the two growing
gmax (H2O) 1.16 1.87 cm/s seasons only in F. sylvatica inside non-filtered Open-Top
fPAR Chambers (NF) and those directly exposed to ambient air
a 0.007 0.005 adim.
fT
(OA). In Q. robur no characteristic symptoms were observed,
Topt 27 27 
C although some trees in NF and OA plots displayed slight leaf
Tmax 35 35 
C yellowing, at the end of the growing season. None of the plants

Tmin 12 12 C exposed to charcoal-filtered air, whether irrigated or not, dis-
b 0.5333 0.5333 adim. played any visible injury at all. Symptoms observed in F. syl-
fVPD
c 4.0 3.9 kPa
vatica were stipples and e above all e bronzing of the adaxial
d 1.8 2.1 kPa surface of top canopy leaves. In non-watered plants onset of
fSWC symptoms occurred 23 days later in 2004, and 27 days later
g 1.01472 1.01472 adim. in 2005 than onset of symptoms in watered plants. With refer-
h 0.26043 0.26043 adim. ence to ozone exposure (AOT40) in non-watered plants’ symp-
gmax, maximum stomatal conductance; fPAR, limiting function depending on toms occurred at significantly higher values (þ18.0% in 2004
photosynthetically active radiation; fT, limiting function depending on temper- and þ30.0% in 2005, see Table 4). On the other hand, the dif-
ature; fVPD, limiting function depending on vapour pressure deficit; fSWC, lim-
iting function depending on soil water content; Topt, optimal temperature;
ference between watered and non-watered plants in the level
Tmax, maximum temperature; Tmin, minimum temperature; and a, b, c, d, g, of cumulated stomatal flux ðAFstO3 Þ leading to the onset of
and h are function coefficients. symptoms was 5.7% in 2004 and 3.1% in 2005 (see Table 4).

water and ozone treatments, are illustrated in Fig. 5. As for the 4. Discussion
data reported on exposure, the arrows on the curves indicate
the date at which visible leaf injuries first appeared (in cases The model of stomatal conductance obtained from field mea-
where they occurred). The difference in the cumulative flux surements presents fairly high gmax values, if compared with
between watered and non-watered plants is not very marked; the values given in the literature for the two species (Korner
it is more evident in 2005 than in 2004 due to the climate be- et al., 1979; Aranda et al., 2000; Emberson et al., 2000b; Maty-
havior of that year. But even this small difference is sufficient ssek et al., 2004; Nunn et al., 2005). The range of gmax for beech
to cause a delay in the onset of foliar symptoms. Compared to in these studies is 100e80 mmol O3m2 s1 (Projected Leaf
AOT40, the cumulative flux ðAFstO3 Þ displays a much more Area). These values correspond to 0.42e0.76 cm/s. For oak
regular seasonal trend, including during 2005 when AOT40 this range is between 122 and 366 mmol O3m2 s1 (Projected
was very irregular. Except for the final part of the season, Leaf Area), which correspond to 0.52e1.55 cm/s. But it must be
the cumulative flux increases in an almost linear manner. At noted that our experiment used three-year-old seedlings and not
the end of the summer season e around mid-September in adult trees, as was the case in those papers: it is well-known that
2004 and in late August in 2005 e the speed of the accumu- adult trees present morphological, phenological and physio-
lation tends to decrease progressively due to the lower ozone logical differences that can exert an important influence on
concentrations in the atmosphere, a direct effect of the drop responses to stress (Kolb et al., 1997; Kolb and Matyssek,
in mean air temperature and solar radiation. 2001). Furthermore, marked differences have been observed

1.4 2
Fagus sylvatica g measured Quercus robur g measured
1.8
1.2 g predicted g predicted
1.6
1 1.4
g water [cm/s]

g water [cm/s]

0.8 1.2
1
0.6
0.8
0.4 0.6
0.4
0.2
0.2
0 0
7.30
8.00
8.30
9.00
10.00
10.30
11.00
11.30
14.00
14.30
15.00
18.00
18.30
19.00

7:30

8:00

8:30

10:00

10:30

11:30

12:00

12:30

14:00

14:30

15:00

15:30

18:00

19:00

measurment hours measurment hours

Fig. 3. Comparison between measured and modelled water stomatal conductance for Fagus sylvatica (left) and Quercus robur (right) in 2005 growing season.
Vertical bars indicate the standard error of prediction per each class of hours of measurements. g, stomatal conductance.
 Author's personal copy

280 G. Gerosa et al. / Environmental Pollution 152 (2008) 274e284

2004 2005
30000 30000
27000 NF 27000 NF
CF CF
24000 24000
21000 21000

AOT40 (ppb.h)
AOT40 (ppb.h)

18000 18000
15000 15000
12000 12000
9000 9000
6000 6000
3000 3000
0 0

01/04

01/05

01/06

01/07

01/08

01/09
01/04

01/05

01/06

01/08

01/09
01/07

date date

Fig. 4. AOT40 in 2004 and 2005 growing seasons. Downward arrows indicate the first symptom onset on irrigated plants of Fagus sylvatica. Upward arrows
indicate the first symptom onset on non-irrigated plants of F. sylvatica. No symptoms were detected for Quercus robur in both years. AOT40, accumulated
ozone over a threshold of 40 ppb; NF, non-filtered air treatment; and CF, charcoal-filtered air treatment.

Fagus sylvatica
90 90

80 NF-WET 80 NF-WET
NF-DRY NF-DRY
70 CF-WET 70 CF-WET
AFst O3 (mmol / m2)

AFst O3 (mmol / m2)

60 60

50 50

40 40

30 30

20 20

10 10

0 0
01/04/04

01/05/04

01/06/04

01/07/04

01/08/04

01/09/04

01/10/04

01/04/05

01/05/05

01/06/05

01/07/05

01/08/05

01/09/05

01/10/05
date date

Quercus robur
90 90

80 NF-WET 80 NF-WET
AFst O3 (mmol / m2)

NF-DRY NF-DRY
70 CF-WET 70 CF-WET
AFst O3 (mmol / m2)

60 60

50 50

40 40

30 30

20 20

10 10

0 0
01/04/05

01/05/05

01/06/05

01/07/05

01/08/05

01/09/05

01/10/05
01/04/04

01/05/04

01/06/04

01/07/04

01/08/04

01/09/04

01/10/04

date date

Fig. 5. Cumulative ozone dose for 2004 (left) and 2005 (right) growing seasons in Fagus sylvatica and Quercus robur. Downward arrows indicate the first symptom
onset on irrigated plants of F. sylvatica. Upward arrows indicate the first symptom onset on non-irrigated plants of F. sylvatica. AFstO3 , accumulated stomatal flux;
NF-WET, irrigated plants in non-filtered air; NF-DRY, non-irrigated plants in non-filtered air; and CF-WET, irrigated plants in charcoal-filtered air.
 Author's personal copy

G. Gerosa et al. / Environmental Pollution 152 (2008) 274e284 281

Table 4 of stomata. In our study we determined a threshold of

First symptom onset related to ozone exposure (AOT40) and stomatal flux for 1.8 kPa, almost double the level reported in the UN-ECE Map-
Fagus sylvatica grown in non-filtered OTC (2004 and 2005 growing seasons)
ping Manual (1.0 kPa; UN-ECE, 2004; Aranda et al., 2000;
Water Year Unit Kutsch et al., 2001). Perhaps this is due to the fact that the eco-
treatment 2004 2005 type of F. sylvatica used has a southern Alpine origin, likely
Date of first W 20/08/2004 24/07/2005 better adapted to the xeric and thermal conditions of Mediter-
symptom onset D 12/09/2004 20/08/2005 ranean summer climates. The same explanation can be given
Days difference 23 27 days for the differences observed in the dependence function of
between W and D
conductance on temperature. In our study, the optimum condi-
AOT40 at the first W 23,340 18,793 ppb h tion for F. sylvatica is at 27  C, 6  C higher than the value in
symptom onset D 27,552 24,446 ppb h
the European model; its maximum temperature is 37  C and
Difference of D 18.0 30.0 %
with respect to W its minimum is 12  C, much higher than the temperature of
5  C suggested in the UN-ECE Mapping Manual (UN-
Ozone dose (AFst0) W 33.633 32.604 mmol O3m2
at the first D 35.564 33.609 mmol O3m2
ECE, 2004; Emberson et al., 2000b).
symptom onset In the case of Q. robur the VPD threshold determined
Difference of D 5.7 3.1 % stands at 2.1 kPa, which is very close to the threshold given
with respect to W for Quercus ilex (2.2 kPa) by Elvira et al. (2005) or by Vitale
AFst1.6 at the first W 25.455 25.252 mmol O3m2 et al. (2005) and also proposed for inclusion in the upcoming
symptom onset D 25.972 24.266 mmol O3m2 edition of the UN-ECE Mapping Manual. But this threshold is
Difference of D 2.0 3.9 % about twice the one currently being suggested for deciduous
with respect to W
species in temperate climates (1.0 kPa).
AOT40, accumulated ozone over a threshold of 40 ppb; AFst0, accumulated The fSWC function determined is flatter and less steep than
stomatal flux; AFst1.6, accumulated stomatal flux above a threshold of
the one adopted in similar studies (e.g. Nussbaum et al., 2003).
1.6 nmol m2 s1; D, non-irrigated plants; and W, irrigated plants.
Seedlings begin to suffer the effect of water deficiency as soon
as the soil moisture falls just below field capacity, probably
due both to the soil type and to the fact that their root system
between adult trees and seedlings in several forest species as to is not fully developed, since they are still young. The correla-
their stomatal conductance (Kolb and Matyssek, 2001; Samuel- tion between decrease of water content in the soil and stomatal
son and Kelly, 2001), although we must stress that there is no conductance reduction is not linear, rather the entity of g
universally accepted interpretation of these differences which reduction tends to grow more rapidly as the lack of water
can also be caused by local site conditions and different persists (Figs. 1 and 2).
ecotypes. The comparison between the values of ozone uptake at first
In our case gmax was determined based on stomatal conduc- visible symptom onset (in the highest conductivity condition,
tance measurements carried out on seedlings grown in the best i.e. watered trees) and the values calculated over the two years
conditions known to maximize conductance (i.e. non-limiting allows us to establish an uptake critical threshold in the range
water supply for watered plants), and this definitely contrib- of 33.633e32.604 mmol O3m2. It is difficult to identify
uted to the high values outcome. a similar critical threshold for AOT40 values, since the levels
As far as the dependence of stomatal conductance on envi- vary from 18,793 (2005) to 23,340 ppb h (2004). The wide
ronmental parameters is concerned, in this study the limiting range of this critical threshold is also confirmed by other ex-
functions fPAR, fT and fVPD are of the same type as the func- perimental studies carried out in Open-Top Chambers on
tions reported in the literature (e.g. Emberson et al., 2000b; F. sylvatica (e.g. VanderHeyden et al., 2001; Gerosa et al., 2003).
Karlsson et al., 2000, 2002; Nussbaum et al., 2003; UN- Stomatal flux e as compared to AOT40 e thus clearly al-
ECE, 2004; Gerosa et al., 2004), although the values of lows us to significantly reduce the uncertainty in determining
some of the parameters differed in some cases from those de- a critical threshold for the onset of visible foliar symptoms and
termined by similar studies or given in the models used as ref- definitely appears to be capable of giving due consideration to
erence (Emberson et al., 2000a; UN-ECE, 2004). These all those environmental, biological and physiological factors
differences are due to local factors, principally the climate that play an important role in mediating plant’s response to
conditions and the plant ecotypes used. ozone-induced stress.
The coefficient of the fPAR function determined for F. sylva- Despite all this, however, in our case there is still a degree
tica (0.007, see Table 3) is slightly higher than the one re- of uncertainty, both between the two years results and between
ported in the European reference model (UN-ECE, 2004; the different soil moisture treatments in each year. In both
Nunn et al., 2005), whereas for Q. robur the same coefficient years of the study non-watered plants have higher critical
results slightly lower (0.005). It is not surprising that the sto- thresholds. This may be explained by the fact that drought
matal response curve of Q. robur to PAR is less steep than that acclimation confers enhanced oxidative stress tolerance by
of F. sylvatica, since the latter is a less heliophile species. well-coordinated induction of antioxidant defense both at
More remarkable differences were observed in the thresh- chloroplast and at mitochondrial level (Selote and Khanna-
old values of VPD which determines the progressive closure Chopra, 2006).
 Author's personal copy
282 G. Gerosa et al. / Environmental Pollution 152 (2008) 274e284
We could therefore suggest the existence of an hourly flux
threshold, below which complete ozone detoxification is en-
sured by metabolic mechanisms. Karlsson et al. (2004) have
suggested an hourly flux threshold equal to 1.6 nmol
O3m2 s1 for adult forest species based on the statistical anal-
ysis of fluxeresponse relationships for a variety of sensitive
deciduous tree species and particularly for F. sylvatica. Apply-
ing this threshold to our data, we find (Table 4) a slight de-
crease in the uncertainty for 2004, while the value of
uncertainty for 2005 remains the same, although in the oppo-
site direction. The flux thresholds that succeed in cancelling
the difference in stomatal uptake between watered and non-
watered plants are, respectively, 1.8 nmol O3m2 in 2004
and 1.0 nmol O3m2 in 2005. Nor should we rule out the
fact that the slight variability observed might be due to the
normal uncertainty pertaining to any experimental practice.
As to Q. robur, it confirms itself as a species that is partic-
ularly resistant to the photochemical stress induced by ozone:
in fact, although it has a seasonal uptake of ozone that is about
double that of F. sylvatica (67.46 mmol O3m2 in 2004,
78.40 mmol O3m2 in 2005, both in wet NF plots), it displays
no foliar injuries that can be directly related to ozone, but sim-
ply some aspecific yellowing (in the NF Chambers and in OA)
at the end of the season, due to a decrease in chlorophyll con-
tent (Bussotti et al., 2007). Bussotti et al. (2007) report mor-
phological differences between F. sylvatica and Q. robur
leaves. Leaves of Q. robur are thicker, they have a higher
leaf density, a palisade mesophyll that is more developed
than the lacunous mesophyll, and a greater quantity of chloro-
phyll per leaf surface unit. All these traits contribute to en-
hancing the photosynthesis capacity of this species, since it
is capable of feeding its detoxification processes more in-
tensely. Furthermore, Wieser et al. (2002) have stated that re-
sistance to oxidative stress depends to leaf morphology, and it
is greater in species with dense leaves.
5. Conclusions
This paper illustrates some of the findings of a research
study carried out over two consecutive years. The study used
Open-Top Chambers with filtered air and the main aim was
to compare the ozone stomatal flux approach to the AOT40-
based approach in an evaluation of ozone impact on forest
species typical of the sub-alpine region.
Water supply is reconfirmed as the most important factor in
determining the onset of ozone-induced symptoms, since
symptoms appear several days later in F. sylvatica trees kept
at a reduced water regime.
Varying water supply conditions and a high number of po-
rometric measurements performed in the field allowed for a re-
liable parametrization of the stomatal conductance model for
the two forest species considered in the study, under condi-
tions typical of the prealpine region. But it is important to
stress that the study used young trees in a non-forest context
(nursery).
The use of the stomatal flux-based approach enables us to
determine a critical threshold of ozone uptake for the onset
of visible leaf injury in F. sylvatica; whereas it was not possi-
ble to determine such a threshold using the AOT40-based
approach.
Calculation of stomatal flux is an instrument capable of
keeping in due consideration the different environmental con-
ditions that can influence responses to ozone in trees. Water
stress conditions, in fact, delay the onset of visible leaf symp-
toms, and the stomatal flux-based approach appears satisfacto-
rily to give this element due consideration. Overall, this
approach appears to provide a more accurate ozone risk eval-
uation in vegetation; it appears to be an instrument with
a greater potential than AOT40-based approach.
Some open questions persist, however. Especially, the issue
of how measurements on young trees can be transposed to for-
ests, i.e. the problem of how to upscale the findings.
On the one hand, having used seedlings instead of adult
trees may appear to limit the value of this study. On the other,
however, the findings show that ozone fluxes can be much
more intense in young trees than in adult individuals. To deter-
mine flux thresholds, based on models developed for adult
trees only, could therefore involve not giving due consider-
ation to the effects that ozone induces in younger trees, with
all the risks that this may imply in processes aimed at regen-
eration of forest margins.
Acknowledgements
The Open-Top Chamber facility at Curno, where this study
was carried out, is funded by Regione Lombardia within the
programme ‘‘Assessment of ozone air pollution on forest veg-
etation in the transalpine region of Lombardy and Canton
Ticino’’, in collaboration with the Regional Agency for Envi-
ronment Protection (A.R.P.A.), the Lombardy Foundation for
Environment (F.L.A.) and the Regional Agency for Services
to Agriculture and Forests e E.R.S.A.F.
The authors are grateful to the E.R.S.A.F. personnel for
their valuable assistance at the Curno Lombardy Region Forest
Nursery.
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