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Detection of effects of ozone on birch, Betula pendula Roth., by chemometrical evaluation of concentrations of lipid components in leaves
ANALmcA CHIMICA
ACW ELSEVIER
Analytica
Chimica Acta 304 (1995) 209-216
Detection of effects of ozone on birch, Betula penduZa Roth., by chemometrical evaluation of concentrations of lipid components in leaves Rune Slimestad a,*, Svein Gjelsvik b, Otto Grahl-Nielsen a Department
a
of Chemistry, University of Bergen, N-5007 Bergen, Norway
h Norwegian Forest Research Institute. N-5047 Fana, Norway
Received 3 June 1994; revised 17 October 1994; accepted 23 October
1994
Abstract Birch trees were exposed to ozone-free air and air containing 150 and 300 pg 0,/m” in open-top chambers, as well as ambient air. Lipid components, mainly fatty acids, in the leaves were determined by methanolysis, gas chromatography and principal component analysis of the chromatographic data. A decrease in the absolute amounts and a change in the relative amounts of the components was observed after three months of exposure. These effects occurred before visible damage of the leaves could be observed, and may be used as biomarker for ozone stress on trees. Keyw0rd.c: Chemometrics;
Chromatography;
Principal component
analysis;
1. Introduction In investigations on forest decline there is a need for parameters which can be used to detect effects of air pollution on trees before visible damage can be observed. With ozone as a major air pollutant, it is reasonable to search for effects on membrane lipids which contain double bonds susceptible to oxidation. On this aspect fatty acids have been analysed in needles and leaves of exposed trees. Kyburz et al. [l] were not able to detect effects on the fatty acids in needles of pine, Pinus syloestris, and leaves of poplar, Populus euramericana, grown in controlled ozone fumigations, while effects were detected in field-grown spruce, Picea abies L., showing needle
* Corresponding 0003-2670/95/$09.50
author. 0 1995 Elsevier Science B.V. All rights reserved
SSDl 0003-2670(94)00542-7
Birch trees; Ozone
loss. Fangmeier et al. [2] demonstrated ozone effects on needles of loblolly pine, Pinus taeda L., fumigated with ozone. They suggest that fatty acid composition may prove to be a useful biomarker to detect ozone stress. During the last years we have established a chemometric method for the analysis of fatty acids. In addition to multiple marine applications [3-51, it has also been used in distinguishing between two species of spruce, Picea abies and P. obollata [6], and to compare populations of Artemisia noroegica [71.
This method is simpler than the conventional methods for fatty acid analysis, in that methanolysis is carried out directly on the sample material, without previous extraction and fractionation. It i, &SO more powerful than conventional methods in that information is withdrawn from all the analysed fatty
210
R. Slimestad et al. /Analytica
acids simultaneously by the way of multivariate statistics. The purpose with the present investigation was to see if effects on leaves of birch, Bet&a pendula Roth., trees grown under controlled ozone fumigation in open top chambers could be detected by chemometrical evaluation of analytical data of fatty acids before visible damage occurred to them. Hereby the possibility of using a change in absolute or relative amounts of fatty acids in early detection of effects on birch trees will be elucidated.
2. Experimental
2.1. Plant material As test material was used one year old plants of Bet&a pendula Roth., provenance Stange, Southeast Norway. The plants were raised for one year in trays with 125-ml pots in Ulvik nursery, West Norway. One month before the experiment started (April 1991) they were transferred to 2-l plastic containers with fertilized peat (Floralux). Throughout the experiment the plants were added nutrient solution equivalent to 10 g N per square meter per year (Superba blue, added NH,NO, and Ca(NO,),). The plants were watered to maintain field capacity. When the ozone exposure started in early May, the plants were about 25 cm high and the apical bud was just about to burst. By the end of the experiment in October the plants were 90-100 cm high, and most of the leaves had been dropped. Six open top chambers (OTC) were used for the ozone exposure, 3.25 m in diameter, and 3.5 m high, made of galvanized steel profiles and coated with transparent Filmtex plastic sheet (quality 201 BOl, colour 001). According to the manufacturer about 90% of the visible light penetrates the sheet. One plant was placed in each OTC. In addition, two plants were placed in field outside the chambers and given the same fertilizing and watering treatment as the plants inside the chambers. Filtered air, 300 m3/h, was added to the OTC. Natural ozone was removed first by passing the air through a filter with active carbon layer. The concentration of ozone in the filtered air was less than 10 pg 03/m3. Ozone was produced in a Fischer ozone generator 502 from
Chimica Acta 304 (1995) 209-216
oxygen of quality 4.8 (purity 99.998%), and added to the filtered air. The exposure time was 8 h daily (from 08.00 to 16.00). The ozone concentration was 300 pg 03/m3 in two chambers, 150 pg 0,/m3 in two chambers, while no ozone was added to the filtered, ozone-free air which was led to the last two chambers. In ambient air the ozone concentration generally varied between 30 pg /m3 and 70 pg/m3 at day time, but higher and lower concentrations also often occurred. Night values were always lower than day values. The ozone concentration was monitored by a Monitor Labs ozone analyser, Model 8810 UV. Twelve sampling points were connected by methanol washed PTFE tubes via an automatic switcher to the monitor. The sampling points were: 1 and 2 m above the ground in the four ozone chambers, 2 m above the ground in the two ozone-free chambers and two sampling points for ambient air. The air was sucked by a pump with 1000 m3/h capacity through the switcher into the monitor for 5 min for each channel at the time, i.e., once every hour. The results were recorded by a Campell Scientific 21X Micrologger. Two leaves were collected from each of the eight trees on July 10, August 18, and September 11, 1991. Fully developed leaves, as low as possible on the trees, but without any signs of visual damage, were carefully selected. The leaves sampled in July and August were of approximately the same age, but in September younger leaves, higher up on the trees, had to be selected from the ozone-exposed trees to avoid the damaged ones. The leaves were stored at -30°C until analysis in June 1993. 2.2. Extraction
and methanolysis
A small strip of each leaf (8-20 mg) was accurately weighed and transferred to a thickwalled glass tube with PTFE-lined screw-cap. The dry weight of the material was determined from remaining pieces of leaf. As internal standard, an accurately determined amount of heneicosanoic acid, 21:0, in chloroform solution had been added to the tube and thereafter the chloroform had been evaporated before the leaf sample was transferred to the tube. The leaf material was treated with 600 ~1 anhydrous methanol containing 2 M HCl for 15 h at 100°C in tightly capped tubes. Approximately half of the methanolic
R. Slimestad et al. /Analytica
solution was evaporated under a stream of N, gas and replaced with water. The mixture was extracted twice with 1.5 ml hexane by strong mixing on a whirl mixer. The two extracts were combined and evaporated down to 0.5 ml by passing N, gas over the solution. 2.3. Gas chromatography Gas chromatography was performed on a 30 m X 0.32 mm i.d. fused silica column, DB-23 from J&W Scientific, with a 0.25 pm thick layer of (50% cyanopropyl) methylpolysiloxane as stationary phase and helium as mobile phase with a flow rate of 2.35 ml/min. Aliquots of 1 ~1 were injected by an HP 7673 autoinjector. The column was kept at 60°C for 2 min after injection, thereafter the temperature was raised to 145°C at a rate of 30”C/min and further to 220°C at a rate of 2.8”C/min where it was kept for 3 min. The eluting components were detected on a flame ionization detector, whose output was coupled to a VG Multichrom lab-data system for storage and treatment of the chromatograms. To identify the components in the methanolysate, a mixture of 20 fatty acids methyl esters, from 14:0 to 24:l (from NuCheckPrep, Elysian, USA) was chromatographed under identical conditions as the
Chimica Acta 304 (1995) 209-216
211
samples. In this manner the 13 fatty acids presented in Table 1 and Fig. 1 were identified. To verify this, and to attempt to characterize the other components present in the methanolysate, mass spectra of the peaks were recorded from samples and a reference mixture on a HP5993C quadropole mass spectrometer connected to a gas chromatograph. The chromatograph was equipped with the same column as used earlier and run under the same conditions. 2.4. M&variate
data treatment
The amounts of the 23 most prominent components in the samples were obtained by integration of their peaks in the chromatograms (Fig. l), assuming that all had the same response factor. The amounts were expressed as percentage of the sum of the components. The average values of the four samples for each ozone concentration at each sampling time are given in Table 1. The chromatographic data, i.e., the areas of the peaks representing the 23 components, were fed into the computer for principal component analysis based on the program SIMCA [8], using the program package SIRIUS [9]. The data were first normalized to percentage of total area for each sample and then weighted by taking their logarithm, thereby giving
Fig. 1. Gas chromatogram of lipid extract from Be&a pendula Roth. In the shorthand nomenclature, A:BnC, for fatty acids, A refers to the total number of carbon atoms, B to the number of double bonds and C to the number of carbon atoms from the center of the double bond farthest removed from the carboxyl group to, and including, the therminal methyl group. When an acid contains more than one double bond, there arc always two single bonds between each double bond. By mass spectrometric analysis the 10 minor peaks, X1-X10, which could not be identified by help of fatty acid reference mixture, were tentatively characterized as: Xl: unidentified, X2: methyl 16.methylheptadecanoate [ll], X3: betuligenol derivative (121, X4: contains an aromatic moiety, X5: unidentified, X6: methyl branched eicosanoate, X7: betuligenol derivative, X8: fatty acid methyl ester, X9: unidentified and X10: fatty acid methyl ester.
10.4* 1.6 27.9+0.3
rated Unknown Total
ll.&+ 1.6 27. I + 2.9
11.6& 1.7 27.7 f 2.2
44.8k4.5
45.5 + 2.8
rated Polyunsatu-
44.7+2.0
1.0~0.2 0.2 & 0.2 27.Ok6.0 3.1kO.8 2.1kO.2 2.8kO.3 1.2+0.2 15.Ok3.3 29.8k3.1 1.4kO.3 0.2 * 0.0 2.7k 1.6 1.9kO.3 0.3 & 0.2 0.8kO.2 1.3+0.6 0.7 * 0.4 1.5 +0.4 0.2kO.l 1.2+0.7 1.7* 1.1 3.4t0.1 0.5 * 0.2 36.1+ 6.2 7.5 +0.9
l.lkO.3 0.1 kO.0 26.6 f 2.3 3.lkO.5 2.0 + 0.3 2.7 * 0.4 1.0 * 0.2 12.0+ 1.8 32.7 f 0.8 1.5 5 0.2 0.2 + 0.0 3.0* 1.7 2.0+ 0.3 0.2+0.1 0.9iO.l 1.2* 0.2 0.8 + 0.6 1.7kO.4 0.2+0.1 1.5 * 0.4 2.3+0.9 2.6* 1.0 0.4+0.1 36.2 f 2.9 7.1+0.7
0.9+0.1 0.1 f 0.0 27.1+ 1.6 3.7 + 0.2 2.5 f 0.1 2.1 kO.1 1.7+0.5 12.8k2.4 32.7 + 1.5 1.6k0.0 0.2 + 0.0 2.2+0.2 1.9*0.3 0.2LO.O 0.9*0.1 1.5 f 0.2 okJ+o.1 1.8 f 0.5 0.2kO.l 0.9 f 0.2 1.5 + 1.0 2.5 f 1.1 0.3 f 0.0 36.2k 1.6 7.8 k 0.5
14:o 14:ln5 16:0 16:ln7 18:0 18:ln9 18:ln7 18:2n6 18:3n3 20:o 20:ln9 22:o 24:0 Xl X2 X3 x4 X5 X6 x7 X8 x9 x10 Saturated Monounsatu-
11.7*1.7 21.4k7.4
40.5 + 2.8
16.9 +2.0 22.7 + 1.4
40.5 + 1.8
l.lkO.2 0.1 +o.o 26.1 k 1.6 2.6kO.2 2.8 + 0.3 2.lkO.l 1.3kO.2 8.4f0.4 32.1 k 1.8 1.6kO.l 0.2+0.1 2.6k 1.4 2.1 kO.4 1.4kO.5 0.6 + 0.0 1.3+0.2 0.9+0.3 1.7kO.5 0.5+0.1 2.OkO.4 2.8+0.6 3.6kO.8 2.1+1.5 36.3 +2.2 6.3 + 0.3
Ambient
l.OkO.2 0.1+0.1 28.2+3.7 4.0 + 1.3 2.3 f 0.3 2.8 + 0.5 l.lkO.4 16.0+ 1.4 24.5 f 2.4 1.7kO.5 0.2+0.1 3.8 + 1.7 2.7kO.9 0.4kO.l 0.5kO.l 1.5+0.6 1.1+0.6 2.1+ 1.0 0.3+0.1 1.3 + 0.6 1.5+0.4 2.6 + 0.7 0.4+0.2 39.7 + 4.2 8.2 + 1.5
August 300
150
0
Ambient
Component
July
300 pg ozone per m3 air
16.4 + 4.4 26.6 + 2.6
41.Ok3.6
1.0+0.2 0.1 f 0.0 25.3 f 1.9 2.6 + 0.7 2.3 f 0.2 2.3 f 0.2 0.9 + 0.5 8.3 f 0.5 32.7k3.1 1.3+0.1 0.2 f 0.0 2.1+ 0.3 2.1 f 0.4 0.7 f 0.3 0.9 + 0.2 1.2kO.4 l.lkO.5 1.9 f 0.6 0.4 k 0.2 2.5 f 0.4 2.7 + 0.5 3.4+0.7 1.6kO.6 34.1+ 3.1 6.1& 1.4
0
18.9k2.1 21.6 f 1.9
35.7 + 3.6
1.3 f 0.3 0.1 f 0.0 28.2 + 2.5 2.3 f 0.6 3.OkO.3 3.2 + 0.4 1.1 kO.4 11.2+0.7 24.5 f 3.5 1.7 f 0.2 0.2 + 0.0 2.2 f 0.2 2.2 f 0.2 1.0+0.2 0.3 f 0.2 1.0 + 0.2 0.3+0.1 1.4 f 0.2 0.3 + 0.0 3.3 f 0.7 3.9 f 1.2 5.2* 1.3 2.2 + 0.9 38.6 f 2.6 6.9 + 0.8
150
13.4 f 2.0 15.4 k 2.6
44.9k6.9
1.9kO.8 0.lj;O.l 23.7 + 1.4 1.9kO.4 3.4+0.5 3.3 f 0.9 0.4kO.l 16.0+2.3 28.9 + 6.5 2.3 + 0.3 0.2+0.1 3.3 f 0.4 1.2kO.l 0.9+0.2 0.3 f 0.2 1.3+0.3 0.3kO.l 1.4kO.3 0.3+0.1 2.6 + 0.5 2.2* 1.1 3.1* 1.4 1.0+0.5 35.8+ 1.8 5.9* 1.0
300
18.7L2.7 25.3*4.4
41.7k2.7
1.7kO.7 0.1 +o.o 24.9k3.1 2.OkO.4 2.4 + 0.2 2.0+0.2 0.6kO.2 9.2 + 1.0 33.5 f 2.5 1.5kO.2 0.2+0.1 2.7kO.6 1.3+0.6 1.5 +0.6 0.8-tO.2 2.OkO.6 0.5 + 0.2 1.6+0.7 0.5kO.l 2.7+ 1.4 2.2kl.l 3.0+ 1.0 3.9* 1.3 34.5 + 3.3 4.9 + 0.5
Ambient
September
18.7 + 2.0 22.4 + 1.2
21.0+2.7 16.6 + 1.6
29.7 + 1.8
1.6kO.4 0.2 f 0.1 31.1 k4.9 2.0 + 0.3 3.OkO.3 2.4 + 0.4 0.8 f 0.2 11.6+0.2 18.1+ 1.8 2.0+0.5 0.2 f 0.0 2.8 + 0.3 3.4kO.5 0.7kO.3 0.1 +o.o 2.0 k 0.6 0.3 kO.1 2.lkO.8 0.3 +0.1 2.7kO.8 3.4+ 1.8 5.9+ 1.3 3.5 +0.8 43.9 * 5.0 5.6 kO.5
1.5 +0.3 0.1 +o.o 25.7k2.1 2.4kO.3 2.7+0.3 1.9kO.3 l.OkO.2 7.8+0.6 32.0 f 1.7 1.7+0.3 0.2kO.O 2.OkO.3 2.3 + 0.6 1.9kO.5 0.6+0.1 1.3 *0.1 0.5 f 0.2 0.8 + 0.4 0.4kO.2 2.6kO.4 3.2kO.9 4.3 + 1.3 2.3kl.O 35.9 + 2.3 5.6kO.5 39.8 * 1.8
150
0
14.3 f 1.1 16.5 + 1.5
41.5 + 5.8
1.5 * 0.5 0.3 + 0.4 27.8 + 6.3 1.7+0.2 2.8 f 1.0 2.3 + 0.2 0.4 + 0.2 16.6+3.2 24.9 + 4.8 1.7+0.4 0.2+0.0 4.2+1.3 1.4 f 0.4 0.6 + 0.0 0.2 f 0.0 2.0 f 0.4 0.3kO.l 1.6+0.2 0.3+0.1 2.6 + 0.6 2.5 & 0.6 3.6 k 0.5 0.6+0.1 39.4 I) 6.8 4.9 * 0.5
300
Table 1 Relative, as percent of sum + SD., and total amounts, in rig/g dry weight, of methanolysable components, in leaves of birch exposed to ambient air, ozone-free air and 150 and
R. Slimestad et al. /Analytica
the components, or variables, present in small amounts relatively higher importance. The computer then positioned the samples in a coordinate system with one coordinate for each of the 23 variables. New coordinates in the direction of the largest and second largest variance among the samples, were computed. The larger part of the systematic variance between the samples will be described by these new coordinates, or principal components. The principal components were computed for various combinations of the samples, and the results are reported as biplots of samples and variables in the coordinate system of the first and second principal component (Fig. 2).
3. Results and discussion 3.1. Visual effects on the trees Ten days after the exposure started on May 10, the first leaves were fully developed, with normal green colour. During the summer new leaves were formed regularly from the apical bud on all trees, and leaf growth continued during the whole experimental period. The newly developed leaves looked normal in all cases. In the chambers with 300 pg 0,/m3 the lowest leaves started to turn yellow in the middle of June, while yellowing of the lowest leaves started in the first week of July on the trees exposed to 150 pg 0,/m3. Necrotic spots developed along the leaf edge and between the veins about two weeks later on the yellow leaves on the trees exposed to both concentrations. By the end of July the leaves had fallen off the lower half of the trees in the chambers with highest ozone concentrations. The same fate occurred to the trees exposed to 150 /*g 0,/m’ approximately three weeks later. The leaves on the trees in the chambers with ozone-free air and on those in the open air kept the green colour, and were attached to the plants until the time of normal leaf abscission in fall. 3.2. Chemical effects on the leaues The methanolysate of the leaves was dominated by the three fatty acids, 16:0, 18:2n6 and 18:3n3,
Chimica Acta 304 (1995) 209-216
213
which made up about 70% of the total amount of the recorded components (Table 1). Saturated fatty acids with even number of carbon atoms, from 14:0 to 24:0, make up about 36% of the total, monoenic acids make up 6% while the two polyunsaturated acids, 18:2n6 and 18:3n3, constitute about 40% of the total. The exposure to ozone caused a decrease in total amount of components in the methanolysate of the leaves (Table 1). Although the standard deviation was large, a decrease in the total amount of the leaves exposed to the highest concentration of ozone was apparent in the July samples, i.e., after 60 days of exposure. For the leaves harvested in September the decrease in total amounts was obvious for those exposed to both 150 and 300 pg 0,/m’. These variations could probably be due to the differences in stress situations. Healthy plants have a greater possibility to recover, while plants acting upon stress have got their tresholds for recovery raised [IO]. An evaluation of the relative amounts of the components indicated that there had been a change caused both by ozone exposure and by ageing of the leaves (Table 1). Of the three fatty acids present in highest amounts, 16:0 did not appear to change, while 18:2n6 was higher and 18:3n3 was lower in exposed leaves than in the unexposed and in those from trees grown in ambient air. A change with ageing of the leaves was also manifested in the unidentified and tentatively identified components (Table 1). The unidentified component Xl, the betuligenol derivative, X7, and the unidentified fatty acid, X10, increased from July to September, while there was a tendency to decrease in the 16-methyldecanoic acid, X2, and in the aromatic component, X4. To obtain a more complete picture of the changes in the fatty acid composition caused by ageing and exposure to ozone, principal component analyses (PCA) were performed on the data. Computation of all samples simultaneously did not give any clearcut pattern among the samples. The samples from each month were then treated separately. No effect was seen on the leaves sampled in July. The leaves from August showed distinct different lipid content due to the difference in ozone-fumigation. This becomes visible in Fig. 2(I) where the samples from the trees grown in untreated and ambient air formed one
R. Slimestad et al. /Analytica
214
FC2 20%
Chimica Acta 304 (1995) 209-216
X8
x2
-
e PC1 41%
PC1 36%
III
PC2 21%
Iv x4 X6
X8
J J
1 X8 16:l
\
J
Xl
18: x9
240
c PC1 62%
P PC1 56%
V
cl?J”,‘, 16:l n7 18:l n7 2O:l n9 l6:O 1833 n3 18:l nj 18:2 n6 22:o
X8
PC1 42%
R. Slimestad et al. /Analyrica Chimica Acta 304 (1995) 209-216
group in the PCA-plot, whereas those from the trees exposed to 1.50 and 300 pg 0,/m3 formed the second and third groups. This distinction was even more pronounced for the September samples, Fig. 2(II). The plots in Fig. 2(I) and Fig. 2(H) are biplots, which show the importance of the variables, fatty acids and unidentified components, for the distinction between the samples. In both plots 18:3n3, 18: ln7 and 16:ln7 lie on the left side of the origin together with the unidentified components X2, X4, X6 and X8. This indicated that these components have higher relative amounts in the unexposed leaves. On the other hand, 18:2n6, 14:ln5, 18:ln9 and the saturated acids 16:0, l&O, 20:0 and 22:0 occur in higher relative amounts in the exposed leaves. Since the total amount of components decreased in the exposed leaves, the latter group of components, i.e., 18:2n6, etc., did not increase in absolute amounts, but decreased less than the former group of components. Of the two polyunsaturated fatty acids present in high amounts in the leaves, 18:3n3 was more susceptible to attack by ozone than 18:2n6. The effects of ozone on the fatty acids of the needles of loblolly pine was found to be similar [2]. In that case the absolute amounts of 18:3n3 decreased while the 18:2n6 increased. The composition of the methanolysable components in the leaves also changed with age. When the samples from each of the four categories of exposure were computed separately, the samples from July and September were distinctly different in all cases, with the August samples falling in between, Fig. 2(111-IV). However, the age-effect on the various components appeared to be dependent on the degree of ozone exposure. In the zero ozone and ambient-air grown leaves the relative amounts of fatty acids
21s
decreased with age, while the unidentified components increased (Fig. 2(111-IV)). In the leaves exposed to increased levels of ozone, the two unidentified components X2 and X4 decreased with age (Fig. 2(V-VI)). The relative position of the fatty acids in these two plots was different from their position in the PC plots in Fig. 2(111-IV).
4. Conclusion We have here demonstrated that it is possible with our chemometric method to detect effects on Beth pendula Roth. from exposure to ozone in concentrations of 150 pg/m3 and higher for three months. No visible damage on the analyzed leaves were observed, but the leaves exposed at 300 pg 03/m7 contained a slightly, grey edge. Of the major fatty acids, the relative amounts of 18:3n3 decreases and 18:2n6. increases upon ozone exposure. The total amount of lipid components in the leaves decreases. Simultaneously an effect of ageing was observed. Therefore, when the method is applied in the search for non-visible effects of exposure to ozone, it is necessary to use reference leaves of the same age as exposed leaves.
References [l] S. Kyburz, W. Eichenberger, T. Keller, H. Rennenberg and P. SchrGder, Eur. _I. For. Path., 21 (1991) 49. [2] A. Fangmeier, L.W. Kress, P. Lepper and W.W. Heck, New Phytol., 115 (1990) 639. [3] 0. Grahl-Nielsen and T.N. Barnung, Mar. Environ. Res., 17 (1985) 218. 141 0. Grahl-Nielsen and 0. Mjaavatten, Mar. Biol., 110 (1991) 59. [5] A. Viga and 0. Grahl-Nielsen, Comp. Biochem. Physiol.. 96B (1990) 721.
Fig. 2. Principal component plots of leaves from Bet&a pendula and of the fatty acids in the leaves. The principal components, PC 1 and PC 2, are computed on the basis of the 23 fatty acids. The variance which is described by the two PCs is given as percent of the total variance among the samples. The positions of the fatty acids in the plots indicate their influence on the PCs, and thereby the influence on the position of the samples in the plots; i.e., the further away from the origin a fatty acid lies in the horizontal direction, the higher is its influence on PC I. Each letter indicate one leaf. Plots I and II show leaves collected in August and September, respectively. A are leaves from trees grown in ambient air, B leaves from trees grown in ozone-free air, C leaves from trees grown in air with 150 pg 0,/m” and D leaves from trees grown in air with 300 pg 0,/m’. Plot III shows leaves from trees grown in ambient air; J are leaves sampled in July, A leaves sampled in August and S leaves sampled in September. In a similar manner, plots IV to VI show leaves from trees grown in 0, 150 and 300 Fg 0,/m”,
respectively.
216
R. Slimestad et al./Analytica
[6] 0. Grahl-Nielsen, 0. Mjaavatten and D.O. (avstedal, Nor. J. Bot., 11 (1991) 613. [7] D.O. OvstedaI and 0. Mjaavatten, PI. Syst. EvoI., 181 (1992) 21. [8] C. AIbano et al., Proc. Symp. On Appl. Stat., Copenhagen, (1981). [9] O.M. Kvalheim and T. Karstang, Chemom. Intell. Lab. Syst., 2 (1987) 235.
Chimica Acta 304 (1995) 209-216 [lo] H. Sandermann, C. Langebartels and W. Heller, Z. Umweltchem. ijkotox., 2 (1990) 14. [ll] W.M.N. Ratnayahe, A. Timmins, T. Ohshima and R.G. Ackman, Lipids, 21 (1986) 518. [12] R. Hegnauer, Chemotaxonomie der Planzen, Vol. 3, Birkhluser Verlag, Basel, Stuttgart, 1964, p. 259.
ACW ELSEVIER
Analytica
Chimica Acta 304 (1995) 209-216
Detection of effects of ozone on birch, Betula penduZa Roth., by chemometrical evaluation of concentrations of lipid components in leaves Rune Slimestad a,*, Svein Gjelsvik b, Otto Grahl-Nielsen a Department
a
of Chemistry, University of Bergen, N-5007 Bergen, Norway
h Norwegian Forest Research Institute. N-5047 Fana, Norway
Received 3 June 1994; revised 17 October 1994; accepted 23 October
1994
Abstract Birch trees were exposed to ozone-free air and air containing 150 and 300 pg 0,/m” in open-top chambers, as well as ambient air. Lipid components, mainly fatty acids, in the leaves were determined by methanolysis, gas chromatography and principal component analysis of the chromatographic data. A decrease in the absolute amounts and a change in the relative amounts of the components was observed after three months of exposure. These effects occurred before visible damage of the leaves could be observed, and may be used as biomarker for ozone stress on trees. Keyw0rd.c: Chemometrics;
Chromatography;
Principal component
analysis;
1. Introduction In investigations on forest decline there is a need for parameters which can be used to detect effects of air pollution on trees before visible damage can be observed. With ozone as a major air pollutant, it is reasonable to search for effects on membrane lipids which contain double bonds susceptible to oxidation. On this aspect fatty acids have been analysed in needles and leaves of exposed trees. Kyburz et al. [l] were not able to detect effects on the fatty acids in needles of pine, Pinus syloestris, and leaves of poplar, Populus euramericana, grown in controlled ozone fumigations, while effects were detected in field-grown spruce, Picea abies L., showing needle
* Corresponding 0003-2670/95/$09.50
author. 0 1995 Elsevier Science B.V. All rights reserved
SSDl 0003-2670(94)00542-7
Birch trees; Ozone
loss. Fangmeier et al. [2] demonstrated ozone effects on needles of loblolly pine, Pinus taeda L., fumigated with ozone. They suggest that fatty acid composition may prove to be a useful biomarker to detect ozone stress. During the last years we have established a chemometric method for the analysis of fatty acids. In addition to multiple marine applications [3-51, it has also been used in distinguishing between two species of spruce, Picea abies and P. obollata [6], and to compare populations of Artemisia noroegica [71.
This method is simpler than the conventional methods for fatty acid analysis, in that methanolysis is carried out directly on the sample material, without previous extraction and fractionation. It i, &SO more powerful than conventional methods in that information is withdrawn from all the analysed fatty
210
R. Slimestad et al. /Analytica
acids simultaneously by the way of multivariate statistics. The purpose with the present investigation was to see if effects on leaves of birch, Bet&a pendula Roth., trees grown under controlled ozone fumigation in open top chambers could be detected by chemometrical evaluation of analytical data of fatty acids before visible damage occurred to them. Hereby the possibility of using a change in absolute or relative amounts of fatty acids in early detection of effects on birch trees will be elucidated.
2. Experimental
2.1. Plant material As test material was used one year old plants of Bet&a pendula Roth., provenance Stange, Southeast Norway. The plants were raised for one year in trays with 125-ml pots in Ulvik nursery, West Norway. One month before the experiment started (April 1991) they were transferred to 2-l plastic containers with fertilized peat (Floralux). Throughout the experiment the plants were added nutrient solution equivalent to 10 g N per square meter per year (Superba blue, added NH,NO, and Ca(NO,),). The plants were watered to maintain field capacity. When the ozone exposure started in early May, the plants were about 25 cm high and the apical bud was just about to burst. By the end of the experiment in October the plants were 90-100 cm high, and most of the leaves had been dropped. Six open top chambers (OTC) were used for the ozone exposure, 3.25 m in diameter, and 3.5 m high, made of galvanized steel profiles and coated with transparent Filmtex plastic sheet (quality 201 BOl, colour 001). According to the manufacturer about 90% of the visible light penetrates the sheet. One plant was placed in each OTC. In addition, two plants were placed in field outside the chambers and given the same fertilizing and watering treatment as the plants inside the chambers. Filtered air, 300 m3/h, was added to the OTC. Natural ozone was removed first by passing the air through a filter with active carbon layer. The concentration of ozone in the filtered air was less than 10 pg 03/m3. Ozone was produced in a Fischer ozone generator 502 from
Chimica Acta 304 (1995) 209-216
oxygen of quality 4.8 (purity 99.998%), and added to the filtered air. The exposure time was 8 h daily (from 08.00 to 16.00). The ozone concentration was 300 pg 03/m3 in two chambers, 150 pg 0,/m3 in two chambers, while no ozone was added to the filtered, ozone-free air which was led to the last two chambers. In ambient air the ozone concentration generally varied between 30 pg /m3 and 70 pg/m3 at day time, but higher and lower concentrations also often occurred. Night values were always lower than day values. The ozone concentration was monitored by a Monitor Labs ozone analyser, Model 8810 UV. Twelve sampling points were connected by methanol washed PTFE tubes via an automatic switcher to the monitor. The sampling points were: 1 and 2 m above the ground in the four ozone chambers, 2 m above the ground in the two ozone-free chambers and two sampling points for ambient air. The air was sucked by a pump with 1000 m3/h capacity through the switcher into the monitor for 5 min for each channel at the time, i.e., once every hour. The results were recorded by a Campell Scientific 21X Micrologger. Two leaves were collected from each of the eight trees on July 10, August 18, and September 11, 1991. Fully developed leaves, as low as possible on the trees, but without any signs of visual damage, were carefully selected. The leaves sampled in July and August were of approximately the same age, but in September younger leaves, higher up on the trees, had to be selected from the ozone-exposed trees to avoid the damaged ones. The leaves were stored at -30°C until analysis in June 1993. 2.2. Extraction
and methanolysis
A small strip of each leaf (8-20 mg) was accurately weighed and transferred to a thickwalled glass tube with PTFE-lined screw-cap. The dry weight of the material was determined from remaining pieces of leaf. As internal standard, an accurately determined amount of heneicosanoic acid, 21:0, in chloroform solution had been added to the tube and thereafter the chloroform had been evaporated before the leaf sample was transferred to the tube. The leaf material was treated with 600 ~1 anhydrous methanol containing 2 M HCl for 15 h at 100°C in tightly capped tubes. Approximately half of the methanolic
R. Slimestad et al. /Analytica
solution was evaporated under a stream of N, gas and replaced with water. The mixture was extracted twice with 1.5 ml hexane by strong mixing on a whirl mixer. The two extracts were combined and evaporated down to 0.5 ml by passing N, gas over the solution. 2.3. Gas chromatography Gas chromatography was performed on a 30 m X 0.32 mm i.d. fused silica column, DB-23 from J&W Scientific, with a 0.25 pm thick layer of (50% cyanopropyl) methylpolysiloxane as stationary phase and helium as mobile phase with a flow rate of 2.35 ml/min. Aliquots of 1 ~1 were injected by an HP 7673 autoinjector. The column was kept at 60°C for 2 min after injection, thereafter the temperature was raised to 145°C at a rate of 30”C/min and further to 220°C at a rate of 2.8”C/min where it was kept for 3 min. The eluting components were detected on a flame ionization detector, whose output was coupled to a VG Multichrom lab-data system for storage and treatment of the chromatograms. To identify the components in the methanolysate, a mixture of 20 fatty acids methyl esters, from 14:0 to 24:l (from NuCheckPrep, Elysian, USA) was chromatographed under identical conditions as the
Chimica Acta 304 (1995) 209-216
211
samples. In this manner the 13 fatty acids presented in Table 1 and Fig. 1 were identified. To verify this, and to attempt to characterize the other components present in the methanolysate, mass spectra of the peaks were recorded from samples and a reference mixture on a HP5993C quadropole mass spectrometer connected to a gas chromatograph. The chromatograph was equipped with the same column as used earlier and run under the same conditions. 2.4. M&variate
data treatment
The amounts of the 23 most prominent components in the samples were obtained by integration of their peaks in the chromatograms (Fig. l), assuming that all had the same response factor. The amounts were expressed as percentage of the sum of the components. The average values of the four samples for each ozone concentration at each sampling time are given in Table 1. The chromatographic data, i.e., the areas of the peaks representing the 23 components, were fed into the computer for principal component analysis based on the program SIMCA [8], using the program package SIRIUS [9]. The data were first normalized to percentage of total area for each sample and then weighted by taking their logarithm, thereby giving
Fig. 1. Gas chromatogram of lipid extract from Be&a pendula Roth. In the shorthand nomenclature, A:BnC, for fatty acids, A refers to the total number of carbon atoms, B to the number of double bonds and C to the number of carbon atoms from the center of the double bond farthest removed from the carboxyl group to, and including, the therminal methyl group. When an acid contains more than one double bond, there arc always two single bonds between each double bond. By mass spectrometric analysis the 10 minor peaks, X1-X10, which could not be identified by help of fatty acid reference mixture, were tentatively characterized as: Xl: unidentified, X2: methyl 16.methylheptadecanoate [ll], X3: betuligenol derivative (121, X4: contains an aromatic moiety, X5: unidentified, X6: methyl branched eicosanoate, X7: betuligenol derivative, X8: fatty acid methyl ester, X9: unidentified and X10: fatty acid methyl ester.
10.4* 1.6 27.9+0.3
rated Unknown Total
ll.&+ 1.6 27. I + 2.9
11.6& 1.7 27.7 f 2.2
44.8k4.5
45.5 + 2.8
rated Polyunsatu-
44.7+2.0
1.0~0.2 0.2 & 0.2 27.Ok6.0 3.1kO.8 2.1kO.2 2.8kO.3 1.2+0.2 15.Ok3.3 29.8k3.1 1.4kO.3 0.2 * 0.0 2.7k 1.6 1.9kO.3 0.3 & 0.2 0.8kO.2 1.3+0.6 0.7 * 0.4 1.5 +0.4 0.2kO.l 1.2+0.7 1.7* 1.1 3.4t0.1 0.5 * 0.2 36.1+ 6.2 7.5 +0.9
l.lkO.3 0.1 kO.0 26.6 f 2.3 3.lkO.5 2.0 + 0.3 2.7 * 0.4 1.0 * 0.2 12.0+ 1.8 32.7 f 0.8 1.5 5 0.2 0.2 + 0.0 3.0* 1.7 2.0+ 0.3 0.2+0.1 0.9iO.l 1.2* 0.2 0.8 + 0.6 1.7kO.4 0.2+0.1 1.5 * 0.4 2.3+0.9 2.6* 1.0 0.4+0.1 36.2 f 2.9 7.1+0.7
0.9+0.1 0.1 f 0.0 27.1+ 1.6 3.7 + 0.2 2.5 f 0.1 2.1 kO.1 1.7+0.5 12.8k2.4 32.7 + 1.5 1.6k0.0 0.2 + 0.0 2.2+0.2 1.9*0.3 0.2LO.O 0.9*0.1 1.5 f 0.2 okJ+o.1 1.8 f 0.5 0.2kO.l 0.9 f 0.2 1.5 + 1.0 2.5 f 1.1 0.3 f 0.0 36.2k 1.6 7.8 k 0.5
14:o 14:ln5 16:0 16:ln7 18:0 18:ln9 18:ln7 18:2n6 18:3n3 20:o 20:ln9 22:o 24:0 Xl X2 X3 x4 X5 X6 x7 X8 x9 x10 Saturated Monounsatu-
11.7*1.7 21.4k7.4
40.5 + 2.8
16.9 +2.0 22.7 + 1.4
40.5 + 1.8
l.lkO.2 0.1 +o.o 26.1 k 1.6 2.6kO.2 2.8 + 0.3 2.lkO.l 1.3kO.2 8.4f0.4 32.1 k 1.8 1.6kO.l 0.2+0.1 2.6k 1.4 2.1 kO.4 1.4kO.5 0.6 + 0.0 1.3+0.2 0.9+0.3 1.7kO.5 0.5+0.1 2.OkO.4 2.8+0.6 3.6kO.8 2.1+1.5 36.3 +2.2 6.3 + 0.3
Ambient
l.OkO.2 0.1+0.1 28.2+3.7 4.0 + 1.3 2.3 f 0.3 2.8 + 0.5 l.lkO.4 16.0+ 1.4 24.5 f 2.4 1.7kO.5 0.2+0.1 3.8 + 1.7 2.7kO.9 0.4kO.l 0.5kO.l 1.5+0.6 1.1+0.6 2.1+ 1.0 0.3+0.1 1.3 + 0.6 1.5+0.4 2.6 + 0.7 0.4+0.2 39.7 + 4.2 8.2 + 1.5
August 300
150
0
Ambient
Component
July
300 pg ozone per m3 air
16.4 + 4.4 26.6 + 2.6
41.Ok3.6
1.0+0.2 0.1 f 0.0 25.3 f 1.9 2.6 + 0.7 2.3 f 0.2 2.3 f 0.2 0.9 + 0.5 8.3 f 0.5 32.7k3.1 1.3+0.1 0.2 f 0.0 2.1+ 0.3 2.1 f 0.4 0.7 f 0.3 0.9 + 0.2 1.2kO.4 l.lkO.5 1.9 f 0.6 0.4 k 0.2 2.5 f 0.4 2.7 + 0.5 3.4+0.7 1.6kO.6 34.1+ 3.1 6.1& 1.4
0
18.9k2.1 21.6 f 1.9
35.7 + 3.6
1.3 f 0.3 0.1 f 0.0 28.2 + 2.5 2.3 f 0.6 3.OkO.3 3.2 + 0.4 1.1 kO.4 11.2+0.7 24.5 f 3.5 1.7 f 0.2 0.2 + 0.0 2.2 f 0.2 2.2 f 0.2 1.0+0.2 0.3 f 0.2 1.0 + 0.2 0.3+0.1 1.4 f 0.2 0.3 + 0.0 3.3 f 0.7 3.9 f 1.2 5.2* 1.3 2.2 + 0.9 38.6 f 2.6 6.9 + 0.8
150
13.4 f 2.0 15.4 k 2.6
44.9k6.9
1.9kO.8 0.lj;O.l 23.7 + 1.4 1.9kO.4 3.4+0.5 3.3 f 0.9 0.4kO.l 16.0+2.3 28.9 + 6.5 2.3 + 0.3 0.2+0.1 3.3 f 0.4 1.2kO.l 0.9+0.2 0.3 f 0.2 1.3+0.3 0.3kO.l 1.4kO.3 0.3+0.1 2.6 + 0.5 2.2* 1.1 3.1* 1.4 1.0+0.5 35.8+ 1.8 5.9* 1.0
300
18.7L2.7 25.3*4.4
41.7k2.7
1.7kO.7 0.1 +o.o 24.9k3.1 2.OkO.4 2.4 + 0.2 2.0+0.2 0.6kO.2 9.2 + 1.0 33.5 f 2.5 1.5kO.2 0.2+0.1 2.7kO.6 1.3+0.6 1.5 +0.6 0.8-tO.2 2.OkO.6 0.5 + 0.2 1.6+0.7 0.5kO.l 2.7+ 1.4 2.2kl.l 3.0+ 1.0 3.9* 1.3 34.5 + 3.3 4.9 + 0.5
Ambient
September
18.7 + 2.0 22.4 + 1.2
21.0+2.7 16.6 + 1.6
29.7 + 1.8
1.6kO.4 0.2 f 0.1 31.1 k4.9 2.0 + 0.3 3.OkO.3 2.4 + 0.4 0.8 f 0.2 11.6+0.2 18.1+ 1.8 2.0+0.5 0.2 f 0.0 2.8 + 0.3 3.4kO.5 0.7kO.3 0.1 +o.o 2.0 k 0.6 0.3 kO.1 2.lkO.8 0.3 +0.1 2.7kO.8 3.4+ 1.8 5.9+ 1.3 3.5 +0.8 43.9 * 5.0 5.6 kO.5
1.5 +0.3 0.1 +o.o 25.7k2.1 2.4kO.3 2.7+0.3 1.9kO.3 l.OkO.2 7.8+0.6 32.0 f 1.7 1.7+0.3 0.2kO.O 2.OkO.3 2.3 + 0.6 1.9kO.5 0.6+0.1 1.3 *0.1 0.5 f 0.2 0.8 + 0.4 0.4kO.2 2.6kO.4 3.2kO.9 4.3 + 1.3 2.3kl.O 35.9 + 2.3 5.6kO.5 39.8 * 1.8
150
0
14.3 f 1.1 16.5 + 1.5
41.5 + 5.8
1.5 * 0.5 0.3 + 0.4 27.8 + 6.3 1.7+0.2 2.8 f 1.0 2.3 + 0.2 0.4 + 0.2 16.6+3.2 24.9 + 4.8 1.7+0.4 0.2+0.0 4.2+1.3 1.4 f 0.4 0.6 + 0.0 0.2 f 0.0 2.0 f 0.4 0.3kO.l 1.6+0.2 0.3+0.1 2.6 + 0.6 2.5 & 0.6 3.6 k 0.5 0.6+0.1 39.4 I) 6.8 4.9 * 0.5
300
Table 1 Relative, as percent of sum + SD., and total amounts, in rig/g dry weight, of methanolysable components, in leaves of birch exposed to ambient air, ozone-free air and 150 and
R. Slimestad et al. /Analytica
the components, or variables, present in small amounts relatively higher importance. The computer then positioned the samples in a coordinate system with one coordinate for each of the 23 variables. New coordinates in the direction of the largest and second largest variance among the samples, were computed. The larger part of the systematic variance between the samples will be described by these new coordinates, or principal components. The principal components were computed for various combinations of the samples, and the results are reported as biplots of samples and variables in the coordinate system of the first and second principal component (Fig. 2).
3. Results and discussion 3.1. Visual effects on the trees Ten days after the exposure started on May 10, the first leaves were fully developed, with normal green colour. During the summer new leaves were formed regularly from the apical bud on all trees, and leaf growth continued during the whole experimental period. The newly developed leaves looked normal in all cases. In the chambers with 300 pg 0,/m3 the lowest leaves started to turn yellow in the middle of June, while yellowing of the lowest leaves started in the first week of July on the trees exposed to 150 pg 0,/m3. Necrotic spots developed along the leaf edge and between the veins about two weeks later on the yellow leaves on the trees exposed to both concentrations. By the end of July the leaves had fallen off the lower half of the trees in the chambers with highest ozone concentrations. The same fate occurred to the trees exposed to 150 /*g 0,/m’ approximately three weeks later. The leaves on the trees in the chambers with ozone-free air and on those in the open air kept the green colour, and were attached to the plants until the time of normal leaf abscission in fall. 3.2. Chemical effects on the leaues The methanolysate of the leaves was dominated by the three fatty acids, 16:0, 18:2n6 and 18:3n3,
Chimica Acta 304 (1995) 209-216
213
which made up about 70% of the total amount of the recorded components (Table 1). Saturated fatty acids with even number of carbon atoms, from 14:0 to 24:0, make up about 36% of the total, monoenic acids make up 6% while the two polyunsaturated acids, 18:2n6 and 18:3n3, constitute about 40% of the total. The exposure to ozone caused a decrease in total amount of components in the methanolysate of the leaves (Table 1). Although the standard deviation was large, a decrease in the total amount of the leaves exposed to the highest concentration of ozone was apparent in the July samples, i.e., after 60 days of exposure. For the leaves harvested in September the decrease in total amounts was obvious for those exposed to both 150 and 300 pg 0,/m’. These variations could probably be due to the differences in stress situations. Healthy plants have a greater possibility to recover, while plants acting upon stress have got their tresholds for recovery raised [IO]. An evaluation of the relative amounts of the components indicated that there had been a change caused both by ozone exposure and by ageing of the leaves (Table 1). Of the three fatty acids present in highest amounts, 16:0 did not appear to change, while 18:2n6 was higher and 18:3n3 was lower in exposed leaves than in the unexposed and in those from trees grown in ambient air. A change with ageing of the leaves was also manifested in the unidentified and tentatively identified components (Table 1). The unidentified component Xl, the betuligenol derivative, X7, and the unidentified fatty acid, X10, increased from July to September, while there was a tendency to decrease in the 16-methyldecanoic acid, X2, and in the aromatic component, X4. To obtain a more complete picture of the changes in the fatty acid composition caused by ageing and exposure to ozone, principal component analyses (PCA) were performed on the data. Computation of all samples simultaneously did not give any clearcut pattern among the samples. The samples from each month were then treated separately. No effect was seen on the leaves sampled in July. The leaves from August showed distinct different lipid content due to the difference in ozone-fumigation. This becomes visible in Fig. 2(I) where the samples from the trees grown in untreated and ambient air formed one
R. Slimestad et al. /Analytica
214
FC2 20%
Chimica Acta 304 (1995) 209-216
X8
x2
-
e PC1 41%
PC1 36%
III
PC2 21%
Iv x4 X6
X8
J J
1 X8 16:l
\
J
Xl
18: x9
240
c PC1 62%
P PC1 56%
V
cl?J”,‘, 16:l n7 18:l n7 2O:l n9 l6:O 1833 n3 18:l nj 18:2 n6 22:o
X8
PC1 42%
R. Slimestad et al. /Analyrica Chimica Acta 304 (1995) 209-216
group in the PCA-plot, whereas those from the trees exposed to 1.50 and 300 pg 0,/m3 formed the second and third groups. This distinction was even more pronounced for the September samples, Fig. 2(II). The plots in Fig. 2(I) and Fig. 2(H) are biplots, which show the importance of the variables, fatty acids and unidentified components, for the distinction between the samples. In both plots 18:3n3, 18: ln7 and 16:ln7 lie on the left side of the origin together with the unidentified components X2, X4, X6 and X8. This indicated that these components have higher relative amounts in the unexposed leaves. On the other hand, 18:2n6, 14:ln5, 18:ln9 and the saturated acids 16:0, l&O, 20:0 and 22:0 occur in higher relative amounts in the exposed leaves. Since the total amount of components decreased in the exposed leaves, the latter group of components, i.e., 18:2n6, etc., did not increase in absolute amounts, but decreased less than the former group of components. Of the two polyunsaturated fatty acids present in high amounts in the leaves, 18:3n3 was more susceptible to attack by ozone than 18:2n6. The effects of ozone on the fatty acids of the needles of loblolly pine was found to be similar [2]. In that case the absolute amounts of 18:3n3 decreased while the 18:2n6 increased. The composition of the methanolysable components in the leaves also changed with age. When the samples from each of the four categories of exposure were computed separately, the samples from July and September were distinctly different in all cases, with the August samples falling in between, Fig. 2(111-IV). However, the age-effect on the various components appeared to be dependent on the degree of ozone exposure. In the zero ozone and ambient-air grown leaves the relative amounts of fatty acids
21s
decreased with age, while the unidentified components increased (Fig. 2(111-IV)). In the leaves exposed to increased levels of ozone, the two unidentified components X2 and X4 decreased with age (Fig. 2(V-VI)). The relative position of the fatty acids in these two plots was different from their position in the PC plots in Fig. 2(111-IV).
4. Conclusion We have here demonstrated that it is possible with our chemometric method to detect effects on Beth pendula Roth. from exposure to ozone in concentrations of 150 pg/m3 and higher for three months. No visible damage on the analyzed leaves were observed, but the leaves exposed at 300 pg 03/m7 contained a slightly, grey edge. Of the major fatty acids, the relative amounts of 18:3n3 decreases and 18:2n6. increases upon ozone exposure. The total amount of lipid components in the leaves decreases. Simultaneously an effect of ageing was observed. Therefore, when the method is applied in the search for non-visible effects of exposure to ozone, it is necessary to use reference leaves of the same age as exposed leaves.
References [l] S. Kyburz, W. Eichenberger, T. Keller, H. Rennenberg and P. SchrGder, Eur. _I. For. Path., 21 (1991) 49. [2] A. Fangmeier, L.W. Kress, P. Lepper and W.W. Heck, New Phytol., 115 (1990) 639. [3] 0. Grahl-Nielsen and T.N. Barnung, Mar. Environ. Res., 17 (1985) 218. 141 0. Grahl-Nielsen and 0. Mjaavatten, Mar. Biol., 110 (1991) 59. [5] A. Viga and 0. Grahl-Nielsen, Comp. Biochem. Physiol.. 96B (1990) 721.
Fig. 2. Principal component plots of leaves from Bet&a pendula and of the fatty acids in the leaves. The principal components, PC 1 and PC 2, are computed on the basis of the 23 fatty acids. The variance which is described by the two PCs is given as percent of the total variance among the samples. The positions of the fatty acids in the plots indicate their influence on the PCs, and thereby the influence on the position of the samples in the plots; i.e., the further away from the origin a fatty acid lies in the horizontal direction, the higher is its influence on PC I. Each letter indicate one leaf. Plots I and II show leaves collected in August and September, respectively. A are leaves from trees grown in ambient air, B leaves from trees grown in ozone-free air, C leaves from trees grown in air with 150 pg 0,/m” and D leaves from trees grown in air with 300 pg 0,/m’. Plot III shows leaves from trees grown in ambient air; J are leaves sampled in July, A leaves sampled in August and S leaves sampled in September. In a similar manner, plots IV to VI show leaves from trees grown in 0, 150 and 300 Fg 0,/m”,
respectively.
216
R. Slimestad et al./Analytica
[6] 0. Grahl-Nielsen, 0. Mjaavatten and D.O. (avstedal, Nor. J. Bot., 11 (1991) 613. [7] D.O. OvstedaI and 0. Mjaavatten, PI. Syst. EvoI., 181 (1992) 21. [8] C. AIbano et al., Proc. Symp. On Appl. Stat., Copenhagen, (1981). [9] O.M. Kvalheim and T. Karstang, Chemom. Intell. Lab. Syst., 2 (1987) 235.
Chimica Acta 304 (1995) 209-216 [lo] H. Sandermann, C. Langebartels and W. Heller, Z. Umweltchem. ijkotox., 2 (1990) 14. [ll] W.M.N. Ratnayahe, A. Timmins, T. Ohshima and R.G. Ackman, Lipids, 21 (1986) 518. [12] R. Hegnauer, Chemotaxonomie der Planzen, Vol. 3, Birkhluser Verlag, Basel, Stuttgart, 1964, p. 259.