A number of species from various European vegetation types have also been included in long-
term experiments to date. Bergmann et al. 1996, 1998 and Pleijel and Danielsson 1997 studied
the ozone sensitivity of a number of annual and biennial ruderals. Ashmore et al. 1996 concen-
trated on a range of dry calcareous grassland species while Grub et al. 1997, Bungener et al.
1999 fumigated perennial species from managed pastures. No published information so far exists
on the ozone sensitivity of species from wetlands including fen meadows and mires although rem-
nants of such ecosystems are under special protec- tion throughout Europe. Changes in hydrology
and eutrophication have already had negative ef- fects in the past decades Joyce and Wade, 1998,
but are there any effects known of the long-term impact of ozone on wetlands? The uptake of
ozone and other air pollutants is inherently cou- pled to the gas exchange of a plant Reich, 1987
and in studies on crops it has been shown that readily transpiring plants grown under moist soil
conditions are more susceptible to ozone than plants grown under a slight drought stress. It can
be hypothesised that vegetations from perma- nently wet environments are at a greater risk to
adverse ozone concentrations than plants and vegetations growing in a dry habitat. Further-
more, the importance of relative growth rates and leaf morphology in relation to the ozone sensitiv-
ity of plant species requires testing.
To study the ozone sensitivity of wetland spe- cies, an experiment was performed using common
taxa from the Dutch flora. Ten perennial herbs and grasses from extensively managed wet grass-
lands were used in a fumigation experiment with open-top chambers to investigate the ozone sensi-
tivity in terms of growth responses. The results of the experiment will be presented and discussed in
this paper.
2. Material and methods
2
.
1
. Culti6ation and fumigation Seeds of ten plant species Table 1 were
collected in the institute garden at Wageningen in 1996. Plants in this garden originated from
remnants of fen-meadows Cirsio-Molinietum
plant community, Schamine´e et al., 1996 in the Eastern Netherlands. Seeds were germinated in
washed sand in a greenhouse in spring 1997. After
3 weeks
one seedling
per pot
was transplanted into 3 l pots for the intermediate
harvest and 5 l pots for the final harvest. These pots were filled with a sand:potting
mixture 1:2.
The commercially
available potting mixture Lentse 3 had a pH of 5.5 and
consisted of 70 peat and 30 river clay, to which 1.5 kg m
− 3
slow release NPK 12:14:24 was added at the start of the experiment. No
additional nutrients were supplied during the experiments.
The open-top
chambers OTCs
and the
fumigation system were previously described by Dueck 1990. Four ozone concentrations were
used in duplicate: charcoal-filtered air CF, non-filtered air NF, non-filtered air plus 25 nl
l
− 1
O
3
NF + and non-filtered air plus 50 nl l
− 1
O
3
NF + + . Chambers were arranged in two blocks and the treatments were randomly
placed within each block. Ozone was generated from pure oxygen via electric discharge Sorbios
generator and added to the NF air from 10:00 to
19:00 CET
using massflow
controllers. Ozone-levels were measured sequentially in the
OTCs with an ozone analyser Monitor Labs model
8810. Ozone
exposure levels
are presented as seasonal daytime mean values and
as accumulated exposures over a threshold of 40 nl l
− 1
AOT40. The AOT40 is expressed as nl l
− 1
h and is calculated as the sum of differences between the hourly ozone concentrations and
the cut-off threshold of 40 nl l
− 1
when the global radiation exceeds 50 Wm
− 2
Ka¨renla¨mpi and Ska¨rby, 1996. The experiment commenced
on 20 May 1997 with ten pots five 5 l and five 3 l pots per species randomly arranged in each
of the eight chambers. The OTCs were 6 m
2
large and mutual shading effects of the growing plants were avoided by re-mixing the remaining
pots after the intermediate harvest, which was performed after 4 weeks. An automatic watering
system was used to supply water to the pots.
2
.
2
. Visual assessment and har6ests of plants During the course of the experiment visual
assessments of the plants were made once a week. Numbers of flowers and senescent leaves were
counted and the plants were observed for visible injuries. After 28 days intermediate harvest, 16
June five plants per species and treatment were harvested to determine leaf numbers, leaf area
and dry weights of leaves, stems, roots and flow- ers. Dry weights were determined by drying the
plant material at 80°C for 48 h. Relative growth rates RGR and specific leaf areas SLA were
determined according to Hendry and Grime 1993.
A final harvest was performed with the other five plants per species at a time depending on the
species’ phenology Table 1. For the seven spe- cies developing flowers, harvests were performed
when seed ripening had begun. Succisa pratensis, Carex nigra and Danthonia decumbens did not
produce flowers and were harvested between 10 September and 8 October. Areas of green leaves
were measured in all species except C. nigra and D. decumbens. Dry weights and numbers of green,
senescent and dead leaves were determined in all species, except the numbers of leaves in Achillea
ptarmica, C. nigra and D. decumbens. The propor- tions of dead and senescent leaves in relation to
total leaves number and biomass were consid- ered as senescence parameters. Dry weights of
stems and flowers of all plants were determined.
2
.
3
. Data processing and statistics Data were processed separately for the two
harvests. To test for significant ozone treatment effects, one-way analyses of variance ANOVA
were performed on the untransformed data for each species. Data on percentage senescence and
root:shoot ratios RSR was arc-sin transformed prior to the analysis according to Sokal and Rohlf
1981. Analyses followed a randomised block-de- sign with the four ozone treatments placed at
random within each of the two blocks.
Table 1 Accumulated exposures over a threshold of 40 nl l
− 1
AOT40, in ml l
− 1
h
a
and seasonal daytime mean ozone concentrations 10:00–19:00 CET, in nl l
− 1
for exposures of 4 weeks intermediate harvest and a growing season final harvest of ten wet grassland species
Exposure until
b
Treatment
c
CF NF
NF+ NF++
Intermediate harvest 7.5
16 June All species
3.3 0.8
Final harvest 2.7
A. ptarmica L. 11.0
19 August 25.1
2.9 C. nigra L. Reichard
12.5 30 September
29.3 26.3
11.5 2.9
C. dissecturm L. Hill 09 September
D. decumbens L. DC. 2.9
12.5 29.6
08 October E. connabinum L.
26.1 11.4
2.9 26 August
7.2 1.3
17.7 29 July
L. flos-cuculi L. 2.3
9.7 22.9
L. salicaria L. 13 August
15 September M. caerulea L. Moench
2.9 11.8
27.2 P. lanceolata L.
30 July 1.3
7.3 18.1
26.4 10 September
11.5 S. pratensis Moench
2.9 Seasonal 9 h daily means nl l
− 1
Intermediate harvest 16 June
4.5 35.5
58 77.5
8 October Whole season 1997
3 33.5
53.5 77
a
Identical to AOT40 in ppm h
− 1
.
b
The experiment commenced on 20 May 1997.
c
CF, charcoal-filtered air; NF+, non-filtered air plus 25 nl l
− 1
O
3
; NF++ non-filtered air plus 50 nl l
− 1
O
3
.
Fig. 1. Contribution of senescent leaves to total leaf biomass in five plant species exposed to four levels of ozone
for a growing season. Species are from left to right A. ptarmica black bars, C. nigra striped bars, E. cannabinum grey bars,
M. caerulea dotted bars and P. lanceolata white bars. For significance levels see Table 2.
nl l
− 1
O
3
in the second week of August. During the whole season, AOT40 in the NF treatment
remained below the 3 month critical level of 3 ml l
− 1
h, which was proposed by the UN-ECE Ka¨renla¨mpi and Ska¨rby, 1996. The summer of
1997 thus represents a summer with low ozone exposure.
In the NF + + treatments ozone concentra- tions occasionally reached 120 nl l
− 1
in early June and 150 nl l
− 1
in August. There were no signifi- cant differences between the treatment replicates.
The AOT40 and exposure duration at the final harvest differed between species Table 1 and the
AOT40 varied between 17.7 ml l
− 1
h for Lychnis flos-cuculi and 29.6 ml l
− 1
h for D. decumbens in the NF + + treatments.
3
.
2
. Visible injury and senescence Foliar injury was first observed in Eupatorium
cannabinum in the NF + + chambers 4 weeks after the onset of the fumigation. The ozone-re-
lated spots appeared on the upper surface of the first order leaves. Leaves that were produced later
in the season did not show foliar injury. In the middle of the season, small whitish spots were
observed in the centre of leaves of L. flos-cuculi. These rather un-specific symptoms occurred only
in some plants from the NF + + treatment and were accompanied by a structural change of the
mesophyll, which appeared to be water-soaked.
Until the intermediate harvest no signs of ozone-enhanced senescence were observed at the
weekly visual assessments. At the final harvest senescence appeared to be significantly affected by
ozone in five species Table 2, Fig. 1. Ozone-en- hanced senescence was paralleled by a significant
reduction of green leaf area in A. ptarmica, E. cannabinum and Plantago lanceolata Fig. 2.
3
.
3
. Growth responses ANOVAs were calculated separately for the
two harvests for each of the ten species Table 2. Results of significant ozone treatment effects are
