### EPICUTICULAR WAX OF NORWAY SPRUCE NEEDLES AS

INDICATOR OF TRAFFIC POLLUTANT DEPOSITION

EEVA-LIISA VISKARI University of Kuopio, Department of Ecology and Environmental Science, P.O.Box 1627, FIN-70211 Kuopio, Finland (e-mail: Eeva-Liisa.Viskari@tpu.fi; present aadress: Tampere Polytechnic, P.O. Box 21, 33521 Tampere, Finland)

(Received 20 February 1999; accepted 21 September 1999)

Abstract. Two-year-old Norway spruce ( Picea abies (L.) Karst) seedlings were exposed to traffic emissions along roadsides with three different traffic densities and speed limits; highway, street and a quiet local road. Deposition of the polyaromatic hydrocarbons (PAH) was determined at all sites using moss bags as an indicator of organic traffic pollutant deposition. Short term concentrations of atmospheric nitrogen oxides (NO x ) and aerosol black carbon (BC) were also recorded from the sites. The responses of the needle epicuticular wax structure of the exposed spruce seedlings were analysed under the scanning electron microscopy (SEM). The epistomatal wax structure of needles was distinctively fused and aggregated along the highway and street with relatively high traffic densities. Along the local road, where the traffic density was low, no significant degradation of needle epicuticular wax was observed. The NO x and BC data, along with PAHs accumulated in moss bags indicate significant deposition of gaseous pollutants and particulate organic compounds along the highway and street and these are suggested as cause of the epicuticular wax degradation. Epistomatal wax structure is thus suggested to be an early indicator of traffic exposure effects.

Keywords: epistomatal wax, exhaust gas, Norway spruce, polyaromatic hydrocarbons, road traffic



### 1. Introduction

Norway spruce ( Picea abies (L.) Karst.) needles are covered with thick, rod-like crystals of wax, which fills the epistomatal area with a dense meshwork (Huttunen,1994; Bermadinger-Stabentheiner, 1994). Cuticular waxes are composed of lipophilic organic compounds, which form the first barrier between the plant and its environment (Schreiber and Schönherr, 1992). Ageing of needles causes changes in the epicuticular wax. The wax structure is degraded and converted into flattened and fused formations (Huttunen, 1994). Similar changes have been observed with exposure to air pollutants and other environmental stresses, such as wind and rain (Turunen and Huttunen, 1990; Tuomisto and Neuvonen, 1993; Günthardt-Goerg, 1994; Günthardt-Goerg et al ., 1994). These changes in epistomatal wax have been concluded to be due to accelerated ageing (Sauter and Voß, 1986; Günthardt-Goerg

et al ., 1994; Huttunen, 1994). Previous results indicate an accelerated structural degradation of epistomatal wax due to motor vehicle exhaust (Sauter and Voß,

Water, Air, and Soil Pollution 121: 327–337, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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### 1986; Sauter et al ., 1987) and the degradation is most likely related to the lipophilic hydrocarbon fraction of the exhaust gas as reported by Sauter and Pambor (1989). There are also indications of the damaging role of atmospheric particulate deposition on epicuticular wax structure (Simmleit et al ., 1986; Bermadinger et al

., 1988; Cape et al ., 1989; Kulsreshtha et al ., 1994), since organic air pollutants often are adsorbed onto particles (Finlayson-Pitts and Finlayson, 1986). In this paper we present results for potted Norway spruce ( Picea abies (L.)Karst) seedlings exposed to traffic emissions along roadsides for a growing season and the response of the epicuticular wax structure to roadside exposure. The results are discussed in relation to air pollutant concentrations: oxides of nitrogen (NO
x 

### ), aerosol black carbon (BC) (see Viskari et al ., 1999b) and polyaromatic hydrocarbons (PAH).

2. Material and Methods

2.1. S TUDY SITES



### Two-year old Norway spruce seedlings ( Picea abies (L.) Karst.) were placed on roadsides with three different traffic densities and speed restrictions and exposed there for eleven weeks, from May 26 to August 10 1995. The sites were located in the city of Kuopio (62 ◦ 

### 50 0 

### N, 27 ◦ 

### 30 0 

### E) in Eastern Finland: a two-lane highway, an urban street junction and a quiet local road with daily traffic densities and speed

limits of 27 500 and 120 km hr

− 1 

### , 16 000 and 60 km hr

− 1 

### , less than 1000 vehicles day

− 1 

### and 60 km hr

− 1 

### , respectively (Viskari et al ., 1999). For each exposure site a

reference site was also selected. The reference site was situated at least 50 m from the roadside and represented similar microclimatic conditions to the exposure site. The spruce seedlings were potted in plastic pots with fertilised peat and sand (1:1). The seedlings were transferred to the sites and remained in situ for eleven weeks, from May 26 to August 10 1995. The seedlings were fertilised once, four weeks after beginning of the exposure; on June 26, with 0.01% Superex-6 solution and irrigated when necessary. The detailed description of the sites and growing conditions of the Norway spruce seedlings are presented in Viskari et al . (1999c).Weather conditions during the exposure period May-August 1995 and corresponding long term average weather data from the years 1961–1990 presented in Table I.

2.2. A IR POLLUTANT MONITORING



### At the same exposure and reference sites, moss-bags were placed for passive monitoring to define polyaromatic hydrocarbon (PAH) pollution from the atmosphere. Polyaromatic hydrocarbons were selected as indicators of organic pollutant load from road traffic, since earlier studies have shown that motor vehicle emissions explicitly contribute to atmospheric PAH (Nielsen, 1996; Westerholm et al ., 1996). Red-stemmed feather moss ( Pleurozium schreberi ) was collected in a remote rural

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### 329

TABLE I
The monthly temperature and precipitation from the exposure period in summer 1995 and long term average values from the years 1961–1990

Temperature ( ◦ C) Precipitation (mm)

Month 1961–1990 1995 1961–1990 1995

May 8.8 8.5 38 64

June 14.6 16.8 56 61
July 16.8 15.2 66 45
August 14.5 15.1 79 94



### area about one kilometre from the nearest quiet local road. In the laboratory, green parts of the moss were removed and used for the moss-bag preparation. 15 g of green moss was used in each bag. Six samples were taken from the native moss fordetermining the background concentrations of PAHs. Six moss bags were transferred to each study site on June 15 and exposed to the ambient air for eight weeks until August 10. After the exposure the moss samples were stored in aluminium

foil at –20 ◦ 

### C until analysis. PAH concentrations were analysed from the moss samples using GC-MSD as described previously (Viskari et al ., 1997). Procedural blank samples were prepared in every set of Soxhlet extraction and sixteen PAHs were identified from the samples. Background concentrations of PAHs were also analysed from unexposed moss. Aerosol black carbon (BC) and NO
x 

### concentrations as indicators of traffic emissions were also monitored from the roadsides for one week during the exposure period (Viskari et al ., 1999c). The BC concentration was measured by Aethalometer (Magee Scientific, AE-9) and the NO
x 

### -concentrations with Environnement AC 30 m (France) NO
x 

### analyser.

2.3. E PICUTICULAR WAX



### For scanning electron microsopical (SEM) studies two current- and one-year old needles from ten seedlings per exposure site were sampled and a total of 40 stomata were analysed per needle. The sampling was performed at the end of the growing season on August 8 1995, 9 weeks after the bud break, from the middle part of the leading shoot. The needle samples were air-dried at room temperature and the middle part of the needle was mounted on aluminium stubs with double-sided tape.Samples were sputtered with 60 nm layer of gold-palladium under vacuum (Polaron Equipment Limited, SEM Coating Unit E5100) and needle surface structures were studied with scanning electron microscope, SEM (JEOL JSM-35, 20 kV). The structural degradation of the epistomatal wax was classified into four stages as

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### described earlier by Sauter et al . (1987); stage I: healthy, epistomatal wax crystalloids without visible degradation, stage II: slightly damaged, beginning structural degradation and aggregation of the wax rodlets, stage III: moderately damaged, advanced structural degradation and aggregation of wax rodlets on more than 30 up to 80% of the surface of the epistomatal chamber and beginning formation of a flat layer of wax, and stage IV: severely damaged, most severe aggregation and degradation of wax and formation of continuous layer on 90% or more of the epistomatal chamber leading to their structural occlusion.

2.4. S TATISTICAL METHODS



### The statistical analyses were carried out using the SPSS/PC+ package. The T-test for independent samples was used to test the difference between roadside and corresponding reference sites. The differences between roadside sites was tested with oneway variance analysis (ANOVA) and Tukey’s multiple range test. The qualitative classification of epistomatal wax injuries was changed into numerical variables by calculating the proportion of epistomatal wax in each injury class. The statistical analyses were then performed using data from tree-specific average values. The normality of the data was examined by the Kolmogorov-Smirnov test and logarithmic transformation was performed on the data, when needed. Pearson’s correlation coefficient was calculated for the proportion of injured wax and PAH

concentration data.

3. Results

3.1. A IR POLLUTANT MONITORING



### The NO
x 

### concentrations showed great diurnal variation even during the one week of measurement. At the street site, the concentrations varied between 5 and 102 ppb (hourly means), compared with 4 and 120 ppb at the highway site. Often the highest

NO
x 

### concentration episodes occurred at night. At the local road reference site, NO
x 

### concentrations were close to zero throughout the measurement period. The BC data showed that the average concentrations were about an order of magnitude higher along the highway (995 ng m

− 3 

### ) and street (779 ng m

− 3 

### ) than at the background area (76 ng m

− 3 

### ) (Viskari et al ., 1999c). The concentrations of several individual PAH compounds as well as total PAH concentration, as analysed from the moss samples, were significantly greater along the highway than at the corresponding reference site (Table II). At the street site, the concentrations of acenaphthylene, anthracene and chrysene were significantly greater along the street than at the corresponding reference site. At the local road, the concentrations of anthracene, benzo(b+k)fluoranthene and indeno(1,2,3)pyrene were significantly greater along the road than at the corresponding reference site.
The results also show that the concentrations of PAH at the reference sites were

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### 331TABLE IIConcentrations of polyaromatic hydrocarbons, PAH (ng g−1FW) (SE) on moss-bag samples exposed along roadside and corresponding control sites(N = 35)SiteHighway Street Local road BackgroundRoadside Control Roadside Control Roadside ControlNaphthalene 37.2 (3.9) 36.5 (3.3) 34.7 (1.9) 35.9 (3.6) 44.3 (7.6) 50.5 (3.3) 14.4 (1.5)Acenaphthylene 2.2 (0.2) 2.4 (0.2) 3.2 (0.2)(10)1.7 (0.2) 2.5 (0.5) 1.7 (0.3) 0.1 (0.1)Acenaphthene 0.8 (0.0)(1)a0.2 (0.1) 0.8 (0.4) 1.2 (0.3) 0.5 (0.2)(13)0 0Fluorene 0.5 (0.5) 0 1.9 (1.1) 0 1.7 (1.2) 0 0Phenanthrene 49.4 (4.5)(2)10.0 (4.3) 50.7 (9.8) 29.5 (5.3) 12.3 (7.0) 1.6 (1.3) 0.8 (0.8)Anthracene 8.8 (0.8)(3)2.0 (0.5) 5.6 (0.3)(11)4.1 (0.3) 2.0 (0.5)(14)0.5 (0.2) 1.5 (0.1)Fluoranthene 104.2 (12.0)(4)29.6 (3.9) 73.1 (10.1) 60.6 (4.7) 21.7 (4.1) 13.5 (5.8) 19.7 (4.2)Pyrene 90.9 (11.9)(5)22.3 (2.4) 50.3 (4.2) 47.8 (2.7) 18.9 (3.0) 11.8 (3.6) 24.8 (2.2)Benzo(a)anthracene 10.6 (1.7)(6)2.9 (0.1) 3.6 (0.5) 2.7 (0.5) 3.2 (0.2) 2.2 (0.2) 3.6 (0.1)Chrysene 23.0 (2.9)(7)10.0 (0.4) 20.0 (1.9)(12)14.5 (0.6) 11.4 (0.6) 8.9 (0.5) 10.7 (0.6)Benzo(b+k)fluoranthene 40.2 (4.8)(8)26.1 (0.9) 26.6 (2.4) 30.0 (1.7) 21.3 (1.6)(15)26.4 (1.3) 18.3 (2.5)Benzo(a)pyrene 13.9 (0.6) 14.2 (0.6) 12.4 (0.5) 13.1 (1.7) 11.4 (0.7) 13.3 (1.3) 9.6 (0.5)Indeno(1,2,3-cd)pyrene 7.5 (0.9) 5.5 (0.3) 7.2 (0.7) 8.5 (2.9) 4.7 (0.6)(16)6.5 (0.3) 4.0 (0.6)Dibenzo(ah)anthracene 0 0 0 0 0 0 0Benzo(ghi)perylene 14.9 (1.4) 4.2 (0.7) 9.6 (0.5) 7.1 (1.8) 4.9 (0.9) 6.1 (0.3) 7.8 (0.5)Total PAH 403.9 (40.0)(9)165.8 (8.3) 299.6 (27.1) 256.7 (11.8) 160.9 (18.1) 143.1 (12.8) 115.2 (8.2)(1)p= 0.001(2)p= 0.000(3)p= 0.000(4)p= 0.000(5)p= 0.000(6)p= 0.006(7)p= 0.007(8)p= 0.033(9)p= 0.002(10)p= 0.001(11)p= 0.007(12)p= 0.034(13)p= 0.060(14)p= 0.018(15)p= 0.033(16)p= 0.026.aIndicates statistically significant difference from the corresponding control site (t-test for independent samples).

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### close to the background concentrations which were measured from the non-exposed moss samples. The exception was the street reference site, where the concentrations of acenaphthene, phenanthrene, fluoranthene and pyrene were exceptionally high when compared to other reference sites.

3.2. E PICUTICULAR WAX



### No visible injuries were found in needles of exposed seedlings. However, SEM revealed deleterious effects of exposure on the epistomatal wax of needles. Theepicuticular wax around the stomata in current-year needles was injured to a significantly greater degree along the highway site when compared to the corresponding reference site. Only 44% of the needles had healthy epistomatal areas (stage I), while 56% of the needles exhibited different stages of injury in their epistomatal areas (stages II–IV). At the corresponding reference site over 70% of the needles showed healthy epistomatal area and only 30% injured. Similar trends could be observed along the street (Table III). No differences between the roadside and corresponding reference were observed along the local road site, where 70 to 80%of the needles showed healthy epistomatal area. In the one-year old needles the differences between exposure and reference sites were slight and the only statistically significant differences were found at the local road (Table III). Pearson’s correlation coefficient between the proportion of injured epistomatal wax (stages II–IV) and total PAH concentrations was r = 0.977 ( p = 0.001 ∗∗∗ 

### ) for current-year needles and r = 0.631 ( p = 0.179) for one-year old needles suggesting deleterious effect of organic pollutant load on the epistomatal wax for current-year

needles. SEM micrographs from the typical in juries of needle epistomatal waxes are presented in Figures 1a–d. SEM revealed fungal infection on exposed needles (Figure 1c) and indications of particulate deposition around stomatal area can be found (Figure 1d).

4. Discussion

All of the measured parameters: NO
x 

### , PAH in moss bags and BC, indicated gaseous and particle deposition along the street and highway (Viskari et al ., 1999c). Along the local road, no significant pollutant load was observed which can be explained by the low traffic density. The ratio of NO/NO 2 

### was around 1:1 along the roadsides, which indicates quick cooling and oxidation of NO to NO 2 

### in the atmosphere (Nielsen et al ., 1996; Palmgren et al ., 1996). The NO
x 

### data along the street and highway indicate that at the close range area (within tens of metres) from the emission source the gaseous pollutants such as NO
x 

### are diluted rapidly in the atmosphere (Viskari et al ., 1999c). The great variation in NO
x 

### and BC concentrations along the street and highway can be explained with changes in weather conditions,

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### 333TABLE IIIPercentages of epistomatal areas (SE) of current- (C) and one-year old (C+1) needles in different stages of injury alongroadside and corresponding control site. N = 58 in each needle-year class. For injury classes see Sauteret al. (1987)SiteHighway Street Local roadRoadside Control Roadside Control Roadside ControlCSTAGE I 43.8 (6.5) 70.8 (5.9)(1)a52.4 (6.9) 63.0 (4.9) 73.5 (5.9) 80.8 (4.6)STAGE II 27.0 (4.2) 17.6 (2.0)(2)20.0 (2.4) 14.0 (2.4)(4)10.9 (2.3) 11.3 (2.3)STAGE III 13.8 (2.4) 7.9 (3.0) 16.4 (3.0) 12.0 (2.0) 9.0 (2.3) 4.0 (1.7)STAGE IV 15.5 (5.3) 3.8 (1.7)(3)11.0 (3.8) 10.0 (3.1) 6.7 (2.9) 3.9 (2.7)C+1STAGE I 0 7.0 (4.1) 7.5 (2.2) 10.0 (3.8) 8.6 (3.5) 34.3 (8.5)(5)STAGE II 12.5 (7.2) 25.6 (4.2) 27.5 (5.2) 22.5 (4.7) 31.5 (5.3) 25.0 (4.5)STAGE III 37.5 (6.7) 37.4 (4.6) 40.0 (4.8) 34.0 (5.7) 37.0 (4.2) 29.0 (6.4)STAGE IV 50.0 (10) 30.0 (6.7) 25.0 (7.8) 33.5 (9.3) 22.9 (8.4) 11.8 (5.7)(1)p= 0.007(2)p= 0.051(3)p= 0.041(4)p= 0.088(5)p= 0.012.a= The difference between roadside and corresponding control site is statistically significant (T-test for independent samples).

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### 334 E.-L. VISKARI

Figure 1. Scanning electron microscopic (SEM) micrographs of the typical injuries of needle epistomatal waxes at different roadsides and corresponding control sites. (a) Stage I. Healthy lookingepistomatal wax at street cross control site, current-year needle. (b). Stage II. Slightly injured epistomatal wax at the street control site, current-year needle. (c) Stage III. Moderately injured epistomatal wax at the highway site, with fungal hyphae, one year old needle. (d) Stage IV. Severely injured epistomatal wax at the highway site, current-year needle. For stages of injury see Materials and

Methods and Sauter et al . (1987).



### e.g. precipitation (Table I) and traffic density (Nielsen et al ., 1996; Palmgren et al

., 1996).At the reference sites the PAH concentrations were close to background concentrations with the exception of the street reference site. The street reference site wasin an open field about 50 m from the street. According to earlier studies the concentrations of the deposited pollutants from road traffic have been close to background level at 60 m from the road (Viskari et al ., 1997). Thus the street reference site,

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### 335

located about 50 m from the road, may have received some of the traffic related deposition, which increased the PAH concentrations in the moss bags.The exposure to traffic emissions was associated with significant fusion and degradation of the epicuticular wax, especially along the highway, confirming earlier studies where exhaust gas exposure of spruce seedlings has been observed to cause significant degradation of the waxes (Sauter and Voß, 1986; Sauter et al ., 1987; Sauter and Pambor, 1989; Viskari et al ., 1999b). At the local road site, where the traffic density was low (about 1000 vehicles day

− 1 

### ), there were no significant injuries compared to the reference sites. This indicates that along roadsides with low traffic densities the vegetation effects of traffic pollutants are insignificant. At all sites the wax degradation in the one year old needles was more severethan in current-year needles, which indicates the effect of ageing. Normal ageing causes fusing and flattening of the wax crystals. In polluted air, however, the

erosion rate of the wax structures becomes enhanced. This has been observed in several microscopic observations of epicuticular wax structures with conifers (Sauter and Voß, 1986; Turunen and Huttunen, 1990; Tuomisto and Neuvonen, 1993; Günthardt-Goerg et al ., 1994; Huttunen, 1994) and our present and earlier results in the laboratory support this view (Viskari et al . 1999b). There were no differences between the street site and corresponding reference site in epistomatal wax injuries. These sites were the warmest and sunniest in this study (Viskari et al ., 1999c). It is possible that other environmental factors (Turunen and Huttunen, 1990; Günthardt-Goerg, 1994; Günthardt-Goerg et al ., 1994) such as temperature accelerated the epistomatal wax degradation at these sites and thus partially masked the pollutant effect. The possible fraction of exhaust gas emissions deposited to the street reference site (Viskari et al ., 1997) also may have increased the epistomatalwax degradation in those seedlings. The positive correlation between the currentyear needle injuries in epistomatal waxes and total PAH concentrations indicate the deleterious effect of organic pollutant deposition from the atmosphere (Sauter and Pambor, 1989). Our present and previous results (Viskari et al ., 1999b) suggest that the condition of epicuticular wax can be used as an early indicator of traffic pollutant effects which is also suggested by earlier studies with other air pollutants (Cape et al ., 1989)Terpenes emitted from the needles can cause injuries to epistomatal waxes (Tollsten and Müller, 1996). However, our results showed no differences in monoterpene concentrations in needles as a consequence of traffic exposure (Viskari et al

., 1999c). The most likely cause of the wax fusion is the hydrocarbon deposition from the traffic (Sauter and Pambor, 1989, Viskari et al ., 1999b). Previous studies have shown that the epicuticular wax acts as significant sorbent of atmospheric organic pollutants. This is shown to enable uptake of these pollutants which in turn may lead to phytotoxic consequences (Schreiber and Schönherr, 1992). The ageing of needles increases the permeances of cuticular membranes (Schreiber, 1994), which in turn may enhance the uptake of pollutants into needles possibly leading to more severe injury.

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### 336 E.-L. VISKARI



### There were indications of particulate deposition in and around the epistomatal area of exposed spruce needles. According to previous studies (Burkhardt et al

., 1995) atmospheric particles preferentially deposit in the stomatal regions of the needles, which is in agreement with our observations with SEM. Since organic hydrocarbons, such as PAH, usually are adsorbed onto particles (Finlayson-Pitts and Finlayson, 1989), the particulates containing reactive substances on needle surfaces may accelerate the degradation of epistomatal wax (Grill and Golob, 1983; Bermadinger et al ., 1988) and there is evidence that this may disturb the stomatal function of the exposed plant (Kammerbauer et al ., 1987; Viskari et al ., 1999b).

5. Conclusions

The results show the contribution of road traffic to atmospheric NO
x 

### , particulate and PAH pollution. Scanning electron microscopy revealed deposition of particles and significant degradation of epicuticular waxes of needles as a result of traffic emission exposure in field conditions. The possible cause for these injuries were the organic and particulate pollutant fractions of vehicle exhaust. Epistomatal wax structure can be used as an early indicator of traffic exposure effects.

Acknowledgements

The author wish to thank Ms Katalin Urban for her assistance with laboratory analyses. This study was financially supported by the Academy of Finland.

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