RESPONSES OF BIOMASS PRODUCTION AND REPRODUCTIVE

DEVELOPMENT TO OZONE EXPOSURE DIFFER BETWEEN

EUROPEAN WILD PLANT SPECIES

JÜRGEN BENDER∗ , ELKE BERGMANN and HANS-JOACHIM WEIGEL

Institute of Agroecology, Federal Agricultural Research Centre (FAL), Bundesallee 50, 38116
Braunschweig, Germany
(∗ author for correspondence, e-mail: Juergen.Bender@fal.de)

(Received 13 September 2005; accepted 21 April 2006)

Abstract. A screening study with 25 common European wild plant species were performed over
three consecutive growing seasons to investigate the effects of ozone on plant growth, reproductive
development, and resource allocation. Species were grown from seedling stage until the flowering
stage or seed maturity, respectively, in open-top chambers in different ozone-enriched atmospheres at
environmentally-relevant concentrations. Ozone treatments covered a range of concentrations from 20
to 55 ppb ozone (seasonal 8 h daily mean). The experiments revealed significant differences between
species with respect to the sensitivity of different end points toward ozone exposure. Ozone caused
a significant reduction in leaf biomass of more than 20% in six species, and a significant increase in
leaf biomass in three species. The relative ozone sensitivities of the species in terms of leaf biomass
were different from those inferred from total shoot biomass or seed production, indicating that ozone
alters resource allocation patterns in wild plants but there was considerable variation between species
in effects on the allocation to leaves, stems, flowers/fruits and seeds. Germinability of seeds was
affected by ozone such that germination rate was up to 30% lower in ozone-treated plants compared
to control plants. Based on the genotypes screened and by combining different sensitivity criteria
(vegetative growth, reproductive growth, exposure-growth response relationships) Malva sylvestris
must be regarded as the most sensitive species in this study.

Keywords: ozone, wild plants, growth, biomass, reproduction, seed germination, resource allocation

1. Introduction

Tropospheric ozone (O3 ) is regarded to be one of the most potent and widespread
phytotoxic pollutant in the atmosphere. A rise in O3 concentrations has occurred on
a large-scale over the past decades and reach their highest concentrations annualy
during the spring and summer months and thus during the period where vegetation
is most active. Concentrations of O3 are projected to increase by 0.3 to 2.0%/yr
in future years as emissions of its precursors (nitrogen oxides and volatile organic
compounds) continue to increase (Prather et al., 2003). Current ambient O3 levels
are high enough to suppress crop yields of sensitive species (Fuhrer et al., 1997) and
to affect growth and development of trees (Sandermann et al., 1997). More recently,
evidence has indicated that O3 may also have negative effects on plant species of
semi-natural (i.e. non-cultivated) vegetation (Davison and Barnes, 1998; Bender and
Water, Air, and Soil Pollution (2006) 176: 253–267
DOI: 10.1007/s11270-006-9167-1 
C Springer 2006
254 J. BENDER ET AL.

Weigel, 2003). In contrast to the amount of existent information about effects of O3

on agricultural plants and forest trees, much less is known about effects on species
from semi-natural plant communities. The few studies that have been performed un-
der realistic experimental O3 exposure conditions in terms of duration and strength
of the O3 stress provide evidence of wide variation in the O3 sensitivity between
species of semi-natural communities (Ashmore et al., 1996; Barnes et al., 1999;
Fuhrer et al., 2003), but they also suggest that many wild plant species are as sensi-
tive as the most sensitive crops (Davison and Barnes, 1998; Bergmann et al., 1999).
In these studies, the evaluation of the relative sensitivity of O3 was mostly made on
the basis of visible symptoms of injury, however, this may not be the criteria that
provides a direct evidence for an ecological impact of O3 , particularly because many
studies showed that symptoms of injury are not necessarily associated with growth
reductions (Pleijel and Daniellson, 1997; Davison and Barnes, 1998). For wild plant
species with different ecological strategies it has to be considered that traits that re-
late most directly to ecological fitness are more relevant for assessing O3 effects than
e.g. visible symptoms. The most important impact of O3 on semi-natural plant com-
munities may be through effects on growth, reproduction and resource allocation
of individual species, which may result in shifts in species composition, changes in
genetic composition, and loss of biodiversity (Barker and Tingey, 1992). Though a
number of species from various European vegetation types have been tested for their
relative growth responses to O3 exposure, exposure–growth response relationships
exist only for a small number of species (Bergmann et al., 1995; Pleijel and Daniell-
son, 1997; Bungener et al., 1999; Daniellson et al., 1999; Franzaring et al., 2000).
Research of O3 effects on the reproduction of wild plant species is particularly
scarce (Bergmann et al., 1996; Bergweiler and Manning, 1999; Black et al., 2000;
Chappelka, 2002). This is surprising since it is known that the reproductive phase
of the plant lifecycle is particular sensitive to O3 (Black et al., 2000). Alterations
in resource allocation and reproductive success of wild plants may have significant
implications for plant productivity and hence for the composition of plant com-
munities. Seed output, for example, may represent an integrated measure of plant
productivity and is definetely an important parameter of ecological significance at
least for annuals, because they particularly depend on regular seed production in
order to survive. Negative effects on the reproductive performance in response to
O3 may result from a reduction in plant growth, a decreased reproductive allocation,
or from direct effects on reproductive structures (Black et al., 2000). This paucity
of information on exposure-response relationships of O3 impacts on growth and re-
production is currently one of the major difficulties for the derivation of O3 critical
levels for semi-natural vegetation (Fuhrer et al., 2003; Bender et al., 2003).
The overall goal of this study was to assess the relative sensitivity of herbaceous
wild plant species to different levels of chronic O3 exposure on the basis of growth
and reproductive development by summarizing results from experiments performed
between 1994 and 1996 with a total of 25 native German species. Species were se-
lected to represent those typical of communities of disturbed habitats including
 RESPONSES OF BIOMASS PRODUCTION AND REPRODUCTIVE DEVELOPMENT 255

arable land, field margins or disturbed artificial habitats on fertile ground. Specific
objectives were: (i) to identify O3 sensitive species based on growth and reproduc-
tive traits, (ii) to determine the relative allocation of resources to vegetative and
reproductive growth; and (iii) to determine relationships between O3 exposure and
growth responses of selected species that may help to identify exposure thresholds.
Results of O3 effects on the development of visible symptoms of foliar injury of
these species were already presented in an earlier paper (Bergmann et al., 1999).

2. Material and Methods

2.1. PLANT MATERIAL AND CULTIVATION

The 25 species involved in this study (see Table I)are characteristic agriophytes
(ruderals), which mainly are found in a range of disturbed habitats including
TABLE I
Ozone exposure characteristics (O3 -AA2-treatment) during vegetative growth, 8h mean (10.00–
18.00), AOT40 and AOT0 are the cumulative concentrations above the thresholds of 40 ppb and
0 ppb within the individual exposure period (see days of exposure)
Days of AOT40 AOT0 24 h mean 8 h mean
Species exposure [ppb·h] [ppb·h] [ppb] [ppb]

Capsella bursa-pastoris (L.) Med. 37 2813 18560 21.2 42.1

Chenopodium album L. 28 3016 18390 26.5 49.3
Cirsium arvense (L.) Scop. 42 3995 27393 27.5 46.5
Daucus carota L. 84 7432 46089 21.8 44.0
Galinsoga parviflora Cav. 37 3530 23478 25.8 46.3
Imaptiens parviflora L. 53 3384 24999 19.9 39.9
Malva moschata L. 73 5762 38820 21.9 40.9
Malva sylvestris L. 42 3995 27393 27.5 46.5
Matricaria chamomilla L. 47 4604 30435 26.8 47.0
Matricaria discoidea DC. 55 4901 34018 25.5 44.8
Matricaria perforata Mérat 54 4463 29351 22.2 41.9
Papaver argemone L. 23 2967 14418 25.1 53.7
Papaver dubium L. 23 2967 14418 25.1 53.7
Papaver rhoeas L. 23 2967 14418 25.1 53.7
Plantago major L. 30 3528 19326 26.0 48.1
Rumex crispus L. 46 3728 23591 21.0 44.1
Rumex acetosa L. 46 3728 23591 21.0 44.1
Rumex obtusifolius L. 46 3728 23591 21.0 44.1
Senecio vulgaris L. 36 3332 22761 25.8 46.4
Solanum nigrum L. 49 4248 30769 26.0 45.0
Sonchus asper (L.) Hill. 27 6477 22478 33.8 63.8
Stellaria media (L.) Vill. 41 2658 24647 24.8 41.0
Tanacetum vulgare (L.) 68 5518 34449 21.3 43.6
Trifolium arvense L. 50 4529 33814 27.8 57.0
Urtica dioica L. 50 3256 23748 20.1 39.9
256 J. BENDER ET AL.

arable land, field margins or disturbed habitats on fertile ground. All are com-
mon members of the flora in agricultural landscapes in Germany. Seeds of all
species were obtained from a commercial source (Bornträger & Schlemmer, Off-
stein, Germany), while seeds of Senecio vulgaris and Capsella bursa-pastoris
were collected in the region of Braunschweig, Germany. Impatiens parviflora
Dc. was grown from young seedlings collected at the experimental site. Seeds
were germinated in sand or sand/soil mixtures under controlled greenhouse con-
ditions. After germination, seedlings were selected for uniform development and
transplanted into 15 l containers filled with natural soils, forming groups of five
to nine plants per container, depending on the plants’ size. Plants of the rosette-
forming Plantago major were grown singly in pots (10×10 cm). Soils were either
sandy loam, loamy sand or a loamy soil, and species were grown on the soil type
closest to that of their natural habitat. All soils were pre-fertilized with a commer-
cial NPK fertilizer corresponding to 140 kg N ha−1 . Ozone exposure (with at least
five replicate pots per species, per chamber) was started in open-top chambers 2–5
weeks after emergence (3–7 weeks after planting), when plants had developed three
or four true leaves.

2.2. OZONE TREATMENTS

During three consecutive growing seasons (1994-1996), a total of 25 plant species

were exposed to O3 in three separate season-long experiments. Plants were ex-
posed in a system of open-top chambers (for details, refer to Weigel and Jäger,
1988), at the Federal Agricultural Research Centre in Braunschweig, Germany.
A detailed description of the experiment can also be found in Bergmann et al.
(1999). Chambers were arranged in two blocks and the treatments were randomly
placed within each block. All chambers received charcoal-filtered (CF) air. All O3
treatments were achieved by the operation of two O3 generators where either a
constant addition of O3 for 8 h d−1 (only when climatic conditions were appro-
priate, i.e. when PAR > 500 μmol m−2 s−1 ) or a proportional addition of O3 to
ambient air concentrations was generated. Experimental designs differed between
the years. In 1994 and 1995, two common O3 treatments were used in order to
evaluate the relative species sensitivity based on growth and reproductive traits:
CF plus 30 ppb O3 (O3 -30; 8 h d−1 ; 10.00–18.00 hours) as a control (REF) rep-
resenting ‘background ozone’, and 60 % of ambient O3 plus constant addition of
30 ppb O3 (elevated O3 ; O3-AA2). In 1996, two additional O3 treatments were
included in order to establish exposure-growth response relationships for selected
species. The resulting O3 treatments in 1996 were: O3 -AA1 (40% of ambient air O3
concentration (AA) plus constant addition of 20 ppb); O3 -AA2 (60% of ambient
O3 plus 30 ppb); O3 -AA3 (100% of ambient O3 plus 20 ppb); O3 -30 (CF plus 30
ppb O3 ).
Ozone concentrations within the chambers and in ambient air were moni-
tored hourly using a time-share system. The following O3 exposure indices were
 RESPONSES OF BIOMASS PRODUCTION AND REPRODUCTIVE DEVELOPMENT 257

TABLE II
Ozone exposure characteristics (O3 -AA2) until plant maturity and seed production.
8 h mean (10.00–18.00), AOT40 and AOT0 are the cumulative concentrations above
the thresholds of 40 ppb and 0 ppb within an exposure period of three months
AOT40 AOT0 24 h mean 8 h mean
Species [ppb·h] [ppb·h] [ppb] [ppb]

Atriplex patula L. 13078 57949 26.1 50.6

Capsella bursa-pastoris (L.) Med 8499 49415 22.6 45.7
Chenopodium album L. 13078 57949 26.1 50.6
Daucus carota L. 8499 49415 22.6 45.7
Galinsoga parviflora Cav. 13228 60062 27.2 51.9
Malva sylvestris L. 13473 62829 28.6 53.4
Matricaria chamomilla L. 13068 62277 28.4 52.6
Matricaria discoidea DC. 13068 62277 28.4 52.6
Papaver dubium L. 9811 52715 23.7 48.0
Papaver rhoeas L. 9811 52715 23.7 48.0
Senecio vulgaris L. 13228 60062 27.2 51.9
Solanum nigrum L. 12948 62508 28.5 52.3
Sonchus asper (L.) Hill. 13607 55602 25.1 49.9
Stellaria media (L.) Vill. 12948 62508 28.5 52.3
Tanacetum vulgare (L.) 9465 51663 23.6 47.9
Urtica dioica L. 8499 49415 22.6 45.7
Urtica urens L. 13228 60062 27.2 51.9

calculated for each species: 24 h seasonal mean, 8 h seasonal mean (10.00–18.00

Central European Summer Time), total cumulative dose (AOT0), and cumulative
dose above the threshold of 40 ppb (AOT40).
Depending on the different rates of development and life cycles, exposure du-
ration varied between species. For all species listed in Table I, O3 exposure lasted
until the flowering stage, while for 17 species fumigation was continued until seed
maturity (Table II).

2.3. HARVEST OF PLANT MATERIAL AND SEED GERMINATION TESTS

Above-ground biomass of plants was harvested when species reached their flow-
ering stage, i.e. after 29 to 84 days of exposure, depending on species’ phenology
(Table I). The plants were separated into vegetative parts (leaves, stems) and flowers
or fruits and dried at 85◦ C to constant weight. For 17 species only part of the plants
per pot was harvested and the remaining plants were fumigated until seed maturity
(Table II). Number of inflorescences, seed mass per plant or thousand seed weight
were then measured.
The germinability of mature seeds was tested after a storage period of 5
month at room temperature or 5◦ C, respectively (Baskin and Baskin, 1988).
Germination tests (n = 100 seeds per plot) were carried out according to
258 J. BENDER ET AL.

International Seed Testing Association (ISTA) specifications (International Seed

Testing Association, 1993), which prescribe application of deionisied water
or 0.2% KNO3 (for A. patula, Ch. album, C. bursa-pastoris) on filter paper
and storage in a controlled environment chamber at day/night temperatures of
20/12 ◦ C and a light/dark cycle of 12/12 hours.
To test for significant O3 treatment effects, one-way analysis of variance was per-
formed for each species, followed by a LSD test if appropriate. Exposure-response
relationships were calculated for selected species by non-linear regression analysis
using chamber means from all three experimental years, i.e. the analysis included
both the data from the 1996 experiment (four O3 treatments) and, if available, data
from the 1994/1995 two-treatment comparisons.

3. Results and Discussion

3.1. OZONE EXPOSURE

On average, O3 concentrations in the chamber treatments were lower than ambient

air concentrations, with the exception of the O3 -AA3 treatment in 1996, to which
20 ppb O3 was added to ambient O3 (Figure 1). However, exposure duration varied
between species, depending on the different rates of development and life-cycles.
The length of the exposure period for individual species during vegetative growth
and the resulting exposure levels in the O3 -AA2 treatments are shown in Table I.
Exposure duration and thus cumulative exposure levels (AOT40) differed between

Figure 1. Mean diurnal O3 fumigation patterns for the different O3 treatments, compared with mean
ambient air (AA) O3 concentrations. Treatments: O3 -AA1, 40% of ambient O3 concentration plus 20
ppb O3 ; O3 -AA2, 60% of ambient O3 concentration plus 30 ppb O3 ; O3 -AA3, 100% of ambient O3
concentration plus 20 ppb O3 ; CF+30, charcoal-filtered air plus 30 ppb O3 (control treatment; REF)
(see the Materials and methods section for further details).
 RESPONSES OF BIOMASS PRODUCTION AND REPRODUCTIVE DEVELOPMENT 259

species. The calculation of the 3-month AOT40’s for species that were exposed to
O3 -AA2 until maturity and seed production (Table II) indicate that species experi-
enced about 3-4 fold higher AOT40’s than the currently proposed 3 month critical
level of 3000 ppb.h for semi-natural vegetation (UN-ECE, 2004). Charcoal filtra-
tion removed almost 100 % of ambient O3 , i.e. AOT40 values for control chambers
(O3 -30) were zero. However, 8 h seasonal mean O3 concentrations in O3 -30 were
actually lower than 30 ppb due to the aforementioned restriction in fumigation to ap-
propriate weather conditions for O3 production, i.e. when PAR > 500 μmol m−2 s−1
(8 h seasonal mean O3 levels were 20.2, 24.4, and 21.8 ppb in 1994, 1995 and 1996,
respectively).

3.2. OZONE EFFECTS ON VEGETATIVE GROWTH

There is an obvious complication in a growth-based evaluation of the relative O3

sensitivity of wild plant species with contrasting life-forms and life-cycles (Bender
et al., 2003). For example, the duration of the vegetative growth period differs
considerably between the species; hence, the relevant periods of O3 exposure for
individual species are quite different during the season. Consequently, different
times and duration for experimental exposures for different species are required.
Therefore, a comparison of the relative O3 sensitivity during the vegetative growth
period was made at a comparable developmental stage (when species reached the
flowering stage), by calculating the percentage change of biomass of plants exposed
to O3 -AA2 relative to those of the reference treatment (Table III).The species were
ranked according to their O3 responses in leaf biomass to demonstrate the large
interspecific variation in sensitivity to O3 . Ozone caused a significant reduction in
leaf biomass of more than 20% in six species, and a significant increase in leaf
biomass in three species (R. obtusifolius, M. perforata, C. album). The observed
reduction of leaf biomass in further nine species was not statistically significant,
although, for example, a 26 % reduction was found in M. chamomilla. Although a
general trend was observed towards a comparable change in both leaf and total shoot
biomass, only three species showed significant reductions in total shoot biomass.
For two of these species (S. nigrum, M. sylvestris), the extent of the reduction in
total shoot biomass (leaves, stems and flowers) was much lower than the reduction
in leaf biomass, indicating that leaf growth was more sensitive to O3 exposure than
stem growth. By contrast, the significant increase in leaf biomass in C. album was
not seen in the effect on total biomass. The weight of flowers contributed only
marginally to the total biomass and O3 had no significant influence on the onset of
flowering in the species (data not shown). In general, the relative O3 sensitivities of
the different species in terms of leaf biomass were different from those inferred from
total shoot biomass. The most sensitive species in terms of both leaf and total shoot
biomass were S. nigrum and M. sylvestris. However, there is a considerable variation
between species in effects of O3 on the allocation of resources between leaves and
stems, which may explain why O3 is sometimes found to stimulate growth or to
260 J. BENDER ET AL.

TABLE III
Effects of the O3 treatment on leaf and total shoot growth of 25 wild plant
species at the time of flowering (for time of exposure see Table I). Growth ef-
fect was expressed as the percentage change of biomass of plants treated with
O3 -AA2 relative to that of the reference treatment O3 -30 (REF, biomass in g
d.m. pl−1 ). Total shoot biomass includes leaves, stems, flowers or fruits. In-
dices indicate the results of ANOVA: ∗∗∗ = p ≤ 0.001, ∗∗ = p ≤ 0.01, ∗ =
p ≤ 0.05, and n.s. = p > 0.05. Rumex acetosa and Rumex crispus did not flower
during the experimental period
Leaves Total shoot
Species n REF O3-AA2 [%] REF O3-AA2 [%]

Solanum nigrum 18 0.809 −43.84∗∗∗ 1.831 −26.73∗

Malva sylvestris 16 1.100 −37.73∗∗ 2.541 −23.38∗
Senecio vulgaris 18 0.212 −30.73∗ 0.941 −14.98n.s.
Matricaria chamomilla 18 0.155 −26.76n.s. 0.768 −16.44n.s.
Rumex acetosa 12 2.157 −26.02∗ – –
Trifolium arvense 18 1.193 −23.50∗ 1.769 −29.35∗
Galinsoga parviflora 18 0.642 −22.63∗∗ 1.934 −20.29n.s.
Papaver dubium 18 0.812 −18.71n.s. 1.479 −13.51n.s.
Sonchus asper 18 1.331 −16.52n.s 3.141 −19.17n.s.
Urtica dioica 16 4.213 −12.68n.s. 7.346 −9.95n.s.
Tanacetum vulgare 18 1.820 −7.86n.s. 3.248 −3.90n.s.
Capsella bursa-pastoris 16 0.618 −6.79n.s 2.434 −5.46n.s.
Papaver argemone 18 0.431 −5.22n.s. 1.115 −5.92n.s.
Malva moschata 16 2.241 −3.23n.s 3.920 +1.82n.s.
Rumex crispus 18 1.254 −2.85n.s. – –
Plantago major 14 0.540 +0.45n.s. 0.968 +2.14n.s.
Cirsium arvense 18 0.714 +0.98n.s. 0.886 −4.57n.s.
Stellaria media 18 0.594 +3.54n.s. 1.503 −0.17n.s.
Papaver rhoeas 14 0.674 +3.91n.s. 1.535 −1.97n.s.
Daucus carota 20 1.748 +4.57n.s. 2.790 −18.25n.s.
Imaptiens parviflora 10 0.793 +11.49n.s. 2.643 +17.72n.s.
Matricaria discoidea 18 0.189 +17.37n.s. 0.662 −14.48n.s.
Rumex obtusifolius 18 2.140 +34.40∗ 2.331 +36.91(∗)
Matricaria perforata 14 0.577 +41.40∗∗ 1.486 +36.12∗
Chenopodium album 16 0.822 +44.77∗ 2.442 +4.65n.s.

have no effect on total shoot growth in certain species (Davison and Barnes, 1998;
Franzaring et al., 2000; Power and Ashmore, 2002; Fuhrer et al., 2003). Root growth
was not studied in the present experiment, so it cannot be determined whether root
growth was affected by O3 . There is evidence in the literature that root growth is
more affected by O3 than is shoot growth (Cooley and Manning, 1987). Ozone has
also been shown to induce changes in partitioning between shoot and root growth
in different directions in individual wild plant species (Warwick and Taylor, 1995;
Power and Ashmore, 2002). Hence any conclusions about the relative sensitivity of
the investigated species that are solely based on O3 effects on above-ground biomass
 RESPONSES OF BIOMASS PRODUCTION AND REPRODUCTIVE DEVELOPMENT 261

need to be made with caution. Nevertheless, the fact that significant adverse effects
on growth were found at exposure levels (O3 -AA2) comparable to those that species
experienced in the field suggests that many (annual) ruderals are sensitive to O3 .
There are important limitations in the aforementioned evaluation of the relative
O3 sensitivity of species based on a ‘standard’ O3 exposure (or two-treatment-
comparison), as responses may change due to different experimental treatments
or different O3 exposure profiles, respectively. In order to test this assumption,
exposure-response relationships between the percentage change in leaf biomass
and different O3 exposure regimes were established in 1996 (Figure 2) for those
species that showed a distinct reduction of leaf biomass in the standard exposure O3 -
AA2 (M. sylvestris, M chamomilla, R.acetosa. P. dubium; see Table III) or a biomass
stimulation (M. discoidea) in this treatment relative to REF plants. For these five
species the best fit to O3 -induced changes in leaf biomass was achieved when the
8-h seasonal mean was used as exposure index rather than the AOT40 (Figure 2).
Using AOT40, regression analysis generally resulted in lower R2 values and in more
linear regression curves. Similarly, Gimeno et al. (2004) reported that indices based
on averages for different periods performed better in explaining the O3 response of
flower biomass in clover species than did AOT40. However, the results shown in
Figure 2 illustrate that each species showed a negative biomass response above a
particular O3 exposure concentration. This is also true for M. discoidea, for which
higher O3 exposure levels reversed the trend of a growth stimulation observed in
the standard exposure O3 -AA2. Although AOT40 was not the best exposure index
in explaining the response in leaf biomass in this experiment the results indicate
that negative effects occurred in the range of the currently proposed O3 critical level
of 3000 ppb.h for the protection of semi-natural vegetation (UNECE, 2004).

3.3. OZONE EFFECTS ON REPRODUCTIVE GROWTH AND RESOURCE ALLOCATION

The present experiment also demonstrated striking effects of O3 on reproductive

growth, though the effects were less often significant due to very high within-
species variability in the examined reproductive parameters (Table IV). Nine species
showed a reduction in seed mass or inflorescence weight of more than 20 %, with
two species having 50 % (D. carota) and 44 % (M. sylvestris) lower seed mass in
O3 -AA2 compared with REF (P < 0.05). Similar large O3 effects are reported by
Gimeno et al. (2004) who found a reduction in flower biomass and seed output
of up to 40 % in clover species from Iberian pastures after an early-season O3
exposure period in open-top chambers. In the present experiment, seed mass was
more affected by O3 exposure than 1000-seed weight, as is reflected in the general
trend for higher percentage changes in seed mass than in 1000-seed weight. The
observed trends in O3 effects on seed production did not necessarily translate into
germination behavior. In five species (S. nigrum, U. dioica, Ch. album, S. media, U.
urens) germinability of seeds was significantly affected by O3 such that germination
rate was up to 30 % lower in O3 -AA2-treated plants compared to control plants
262 J. BENDER ET AL.

Figure 2. Exposure-response relationships between the percentage change in leaf dry matter (leaf
d.m.) relative t o the control (% of REF) and the 8 h seasonal mean and AOT40 O3 exposure index,
respectively, from five wild plant species.Data are based on chamber means and include different
separate exposure experiments.
 RESPONSES OF BIOMASS PRODUCTION AND REPRODUCTIVE DEVELOPMENT 263

TABLE IV
Effects of the ozone treatment on the production of fruits and seeds of 17 wild plant species within
one season. Effect on reproduction capacity was expressed as the percentage change in mass of seeds
(g plant−1 ), number of inflorescences per plant, thousand seed weight (th.s.w., mg) or the difference
in germination rate (%) of plants treated with Os-AA2 relative to those of the reference treatment
Os-30 (REF). Asterisks indicate the results of ANOVA:∗∗∗ = p ≤ 0.001,∗∗ = p ≤ 0.01,∗ = p ≤
0.05, n.s. = p > 0.05, – not possible. For Solanum nigrum, Stellaria media and Urtica urens ripening
seeds were collected continually and had not been referred to single individuals. Differing values
for n (number of plants) are the result of different numbers of female flowering plants.  fresh matter
of fruits; † CF as reference
Reproduction units per plant th.s.wt. Germination rate
Deviation Deviation
Species n Infl.REF SeedsREF [%] REF [%] REF Difference

Daucus 5–9 – 1.8147 −50.60∗ 0.8240 +9.39n.s. 98.3 +0.50n.s.

carota
Malva 18 – 1.9340 −44.60∗ 4.6424 −25.81∗∗ 73.5 −10.94n.s.
sylvestris
Papaver 14 – 0.1213 −37.74n.s. 0.1129 +15.24n.s. 65.9 −28.90∗
rhoeas
Solanum 18 – 8.657 −35.20− – – – –
nigrum
Matricaria 18 24.50 – −30.61∗ – – 85.6 +3.1n.s.
chamomilla †
Capsella 12 1.1937 −28.23 (∗) 0.1277 −5.62n.s. 82.2 −7.30n.s.
bursa-pastoris
Galinsoga 10 125.4 – −28.02n.s. 0.1306 +4.52n.s. 55.4 +17.30n.s.
parviflora †
Matricaria 28 14.71 – −25.12n.s. 0.1484 −4.06n.s. 99.3 −3.30n.s.
discoidea †
Senecio 18 115.4 – −20.54n.s. 0.2576 −3.45n.s. 97.5 +1.90n.s.
vulgaris †
Urtica 8–12 – 0.8716 −18.69n.s. 0.1633 −8.50n.s. 86.5 −27.53∗∗
dioica
Sonchus 18 – 0.6990 −13.84n.s. 0.3310 −5.77n.s. 96.6 −3.10n.s.
asper
Chenopodium 18 – 1.9308 −12.36n.s. 0.7300 −2.49n.s. 54.4 −26.70∗
album
Atriplex 18 – 0.7279 +4.06n.s. 0.9506 −0.97n.s. 18.7 +4.50n.s.
patula
Tanacetum 18 28.56 – +5.17n.s. – – 98.9 −0.50n.s.
vulgare †
Papaver 14 – 0.0768 +5.30n.s. 0.1405 −10.93n.s. 69.9$ −12.60n.s.
dubium
Stellaria – – – – 0.4686 −2.53n.s. 81.3 −31.45∗∗
media
Urtica – – – – 0.5832 −6.06n.s. 84.6 −31.42∗∗
urens
264 J. BENDER ET AL.

Figure 3. Effects of O3 on the relative allocation of resources to vegetative and reproductive organs
of wild plant species. Allocation is expressed as the relative change (in %) of leaf biomass and seed
mass, respectively, of plants treated with O3 -AA2 relative to those of the control treatment.

(REF). The negative impact of elevated O3 exposure on the reproductive effort of

Ch. album is particularly remarkable as this species showed a significant increase
of 44 % in leaf biomass during vegetative growth in O3 -AA2 (Table III).
The effects of O3 on the relative allocation of resources to vegetative and re-
productive organs of 13 wild plant species are shown in Figure 3. Allocation is
expressed as the relative change (in %) of leaf biomass and seed mass (or inflores-
cence weight), respectively, of plants treated with O3 -AA2 relative to those of the
control (REF) treatment. Ozone caused comparable reductions in both vegetative
and reproductive biomass in the majority of the investigated species, suggesting
that effects during vegetative growth determines reproductive development and
seed output. It is also evident that three species (Ch. album, M. discoidea, S. media)
were clear outliers, in which resources seemed to be switched to vegetative growth,
leading to an increased vegetative biomass in O3 , at the expense of reproductive
growth. These differences may reflect different priorities in resource allocation in
species having different growth and reproductive strategies, however, the mecha-
nisms of the control of resource allocation in the species are unclear (Davison et al.,
1999). Carbon allocation patterns are very complex and are controlled both by sink
demand and source control of photosynthate production (Andersen 2003). How-
ever, it cannot be determined from the present study whether O3 induced changes
in allocation were driven by changes in carbohydrate availabilty or whether these
changes are controlled by other factors.
Based on the genotypes screened and by combining the different sensitivity
criteria (vegetative growth, reproductive growth, exposure-growth response rela-
tionships) M. sylvestris must be regarded as the most sensitive species in this study.
 RESPONSES OF BIOMASS PRODUCTION AND REPRODUCTIVE DEVELOPMENT 265

In addition, Bergmann et al. (1999) have shown that M. sylvestris responded to

elevated O3 levels with characteristic visible symptoms. However, Bergmann et al.
(1999) performed a detailed analysis of the species’ responses considering the total
extent of visible injury, O3 threshold doses for the incidence of symptoms, and
modelled exposure-response relationships and identified C. arvense and S. asper
as the most sensitive species exhibiting O3 -specific symptoms. These species were
less affected by O3 in terms of vegetative and reproductive growth in the present
experiment, confirming results from other studies which showed that symptoms
of injury do not always coincide with measurable effects on growth or biomass
(Davison and Barnes 1998).

4. Conclusions

The experiments revealed significant differences between species with respect to the
sensitivity of different end points toward O3 exposure. The relative O3 sensitivities
of the species in terms of leaf biomass were different from those inferred from total
shoot biomass, seed production and seed germinability, indicating that O3 alters
resource allocation patterns in wild plants but there was considerable variation
between species in effects on the allocation to leaves, stems, flowers/fruits and
seeds. However, significant adverse effects of O3 on vegetative and reproductive
traits, respectively, were found on a number of the investigated species at low,
environmentally-relevant, O3 exposure levels. Hence, our study suggests that many
wild plant species are similar sensitive to O3 in the field. Many studies of O3
impacts on semi-natural vegetation have not reported effects on reproduction. The
observed adverse effects on the reproductive performance of species (seed yield
and quality) could have serious consequences for the establishment and survial of
the progeny, which may result in alterations of the productivity and composition of
plant communities.

Acknowledgements

We would like to thank Carina Trenkler, Peter Braunisch and Wilfried Woyde for
technical assistance. This work was partly supported by the German Umweltbun-
desamt.

References

Andersen, C.P. (2003). Source-sink balance and carbon allocation below ground in plants exposed to
ozone. New Phytologist, 157, 213–228.
266 J. BENDER ET AL.

Ashmore, M.R., Power, S.A., Cousins, D.A., & Ainsworth, N. (1996). Effects of ozone on native grass
and forb species: a comparison of responses of individual plants and artificial communities. In:
Kärenlampi, L., Skärby, L. (Eds.), Critical levels for ozone: testing and finalizing the concepts.
UNECE workshop report. University of Kuopio, Kuopio, Finland, pp. 104–108.
Barker, J.R., & Tingey, D.T. (1992). Air pollution effects on biodiversity. Van Nostrand Reinhold,
New York, 322 pp.
Barnes, J., Bender, J., Lyons, T., & Borland, A. (1999). Natural and man-made selection for air
pollution resistance. Journal of Experimental Botany, 50, 1423–1435.
Baskin, C.C., & Baskin, J.M. (1998). Germination ecophysiology of herbaceous plant species in
temperate region. American Journal of Botany, 75, 286–305
Bender, J., & Weigel, H.J. (2003). Ozone stress impacts on plant life. In: Ambasht, R.S., & Ambasht,
N.K. (Eds.), Modern Trends in Applied Terrestrial Ecology. Kluwer Academic/Plenum Publ.,
New York, pp. 165–182.
Bender, J., Bergmann, E., & Weigel, H.J. (2003). Multi-year experiments on ozone ef-
fects on semi-natural vegetation: implications for the development of critical levels. In:
Karlsson, P.E., Selden, G., & Pleijel, H. (Eds.), Establishing ozone critical levels II.
UNECE Workshop Report. IVL Report B 1523. IVL Swedish Environmental Research
Institute, Gothenburg, Sweden, pp. 211–217.
Bergmann, E., Bender, J., & Weigel, H.J. (1995). Growth response and foliar sensitivity of herbaceous
species to ozone exposures. Water Air Soil Pollution, 85, 1437–1442.
Bergmann, E., Bender, J., & Weigel, H.J. (1999). Ozone threshold doses and exposure-response
relationships for the development of ozone injury symptoms in wild plant species. New Phytologist,
144, 423–435.
Bergweiler, C.J., & Manning, W.J. (1999). Inhibition of flowering and reproductive success in spread-
ing dogbane (Apocynum androsaemifolium) by exposure to ambient ozone. Environmental Pol-
lution, 105, 333-339.
Black, V.J., Black, C.R., Roberts, J.A., & Stewart, C.A. (2000). Impact of ozone on the reproductive
development of plants. New Phytologist, 147, 421–447.
Bungener, P., Nussbaum, S., Grub, A., & Fuhrer, J. (1999). Growth response of grassland species to
ozone in relation to soil moisture condition and plant strategy. New Phytologist, 142, 283–293.
Chappelka, A.H. (2002). Reproductive development of blackberry (Rubus cuneifolius), as influenced
by ozone. New Phytologist, 155, 249–255.
Cooley, D.R. and Manning, W.J. (1987). The impact of ozone on assimilate partitioning in plants: a
review. Environmental Pollution, 47, 95–113.
Daniellson, H., Gelang, J., & Pleijel, H. (1999). Ozone sensitivity, growth and flower development
in Phleum genotypes of different geographic origin in the Nordic countries. Environmental and
Experimental Botany, 42, 41–49.
Davison, A.W. and Barnes, J.D. (1998). Effects of ozone on wild plants. New Phytologist, 139,
135–151.
Davison, A.W., Ashmore, M.R., Bender, J., Chappelka, A.H., & Weigel, H.J. (1999). Critical levels
for semi-natural vegetation. In. Fuhrer, J., Achermann, B. (Eds.), Critical levels for ozone: Level
II. Environmental Documentation 15, 73–77.
Franzaring, J., Tonneijck, A.W.N., Kooijman, A.W.N., & Dueck, T.A. (2000). Growth responses to
ozone in plant species from wetlands. Environmental and Experimental Botany, 44, 39–48.
Fuhrer, J., Skärby, L., & Ashmore,M.R. (1997). Critical levels for ozone effects on vegetation in
Europe. Environmental Pollution, 97, 91–106.
Fuhrer; J., Ashmore, M.R., Mills, G., Hayes, F., & Davison, A.W. (2003). Ozone critical levels for
semi-natural vegetation. In: Karlsson, P.E., Selden G., & Pleijel, H. (Eds.), Establishing ozone
critical levels II. UNECE Workshop Report. IVL Report B 1523. IVL Swedish Environmental
Research Institute, Gothenburg, Sweden, pp. 183–198.
 RESPONSES OF BIOMASS PRODUCTION AND REPRODUCTIVE DEVELOPMENT 267

Gimeno, B.S., Bermejo, V., Sanz, J., de la Torre, D. and Gil, J.M. (2004). Assessment of the effects
of ozone exposure and plant competition on the reproductive ability of three therophytic clover
species from Iberian pastures. Atmospheric Environment, 38, 2295–2303.
International Seed Testing Association: 1993, International rules for seed testing. Rules 1993. Seed
Science & Technology, 21 (Suppl.2), 1–321.
Pleijel, H., & Daniellson, H. (1997). Growth of 27 herbs and grasses in relation to ozone exposure
and plant strategy. New Phytologist, 135, 361–367.
Power, S.A., & Ashmore, M.R. (2002). Responses of fen and fen-meadow communities to ozone.
New Phytologist, 156, 399–408.
Prather, M., Gauss, M., Berntsen, T., Isaksen, I., Sundet, J., Bey, I., Brasseur, G., Dentener, F., Derwent,
R., Stevenson, D., Grenfell, L., Hauglustaine, D., Horowitz, L., Jacob, D., Mickley, L., Lawrence,
M., von Kuhlmann, R., Muller, J., Pitari, G., Rogers, H., Johnson, M., Pyle, J., Law, K., van Weele,
M., & Wild, O. (2003). Fresh air in the 21st century. Geophysical Research Letters 30, 72-1–72-4.
Sandermann, H., Wellburn, A.R., & Heath, R.L. (1997). Forest decline and ozone. A comparison of
controlled chamber and field experiments. Ecological Studies 127, Springer, Berlin, 400 pp.
UNECE: 2004, Mapping Manual 2004. Manual on methodologies and criteria for modelling and
mapping critical loads & levels and air pollution effects, risks and trends. www.icpmapping.org.
Warwick, K.R., & Taylor, G. (1995). Contrasting effects of tropospheric ozone on five
native herbs which coexist in calcareous grassland. Global Change Biology, 1, 143–
151.
Weigel, H.J., & Jäger, H.J. (1988). Zur Ökotoxikologie von Luftschadstoffen. II. Aufbau und Funk-
tionsweise einer Expositionsanlage aus open-top Kammern zur Untersuchung von Immission-
swirkungen auf Pflanzen. Landbauforschung Völkenrode, 38, 182–195.