Secondary Air Pollutants

Impact, Metabolism and Toxicity of Organic Xenobiotics - IMTOX*

Secondary Air Pollutants Epistomatal Wax Erosion of Scots Pine Needles 1Barbro M. Gullvfig, 2Hartmut Frank, 3yrjii Norokorpi 1 Department of Botany, AVH, University of Trondheim, N-7055 Dragvoll, Norway 2 Environmental Chemistry and Ecotoxicology, University of Bayreuth, D-95440 Bayreuth, Germany 3 Finnish Forest Research Institute, RO. Box 16, FIN-96301 Rovaniemi, Finland * See ESPR 2/96, pp 112-114

Abstract The study has been performed in a supposedly clean-air region of a Northern Finnish forest with a homogeneous stand of Scots pine. Stomatal epicuticular wax layer erosion is described using a classification system of five erosion stages. The percentage of stomatal wax within each erosion stage is calculated and the results are treated statistically, which makes the morphological study of the needle surface semi-quantitative. Severe wax degradation has already been found in the current year, increasing with the age of the needles. In this area, the wax layer erosion is correlated with secondary air pollutants, as analyses have shown high trichloroacetate (TCA) levels in needles from the same trees. The wax layer analyses are in accordance with earlier findings which have demonstrated differences in tolerance to TCA. Key words: Organic xenobiotics, impact, metabolism, toxicity; impact, organic xenobiotics; metabolism, organic xenobiotics; toxicity, organic xenobiotics; air pollutants, secondary; secondary air pollutants; epistomatal wax erosion; erosion, wax, epistomatal; pine needles; needles, spruce trees; volatile organic compounds (VOCs); photooxidation, VOCs; airborne C 2 halocarbons; haloacetic acids; trichloroacetic acid (TCA); dichloroacetic acid; monochloroacetic acid (MCA); trifluoroacetic acid; phytotoxic secondary air pollutants; ecosystems, forests; alkylbenzenes, atmospheric oxidation; nitrophenols, phytotoxic; TCA, bioindicator; bioindicator, TCA; herbicidal secondary air pollutants



Recent studies have brought attention to the importance of the formation of herbicides as secondary pollutants arising from photooxidation of various volatile organic compounds (VOCs). For example, airborne C2-halocarbons may be photooxidized to haloacetic acids (FRANK& FRANK 1989, FRANK 1991); trichloroacetic acid (TCA) and monochloroacetic acid (MCA) have been found in needles from clean-air regions where forest decline is observed. Other phytotoxic secondary air pollutants identified in forest ecosystems are derived from atmospheric oxidation of alkylbenzenes, giving rise to formation of phytotoxic nitrop h e n o l s (HINKELet al. 1989). These authors have pointed to the presence of nitrophenols in the needles of urban arESPR - Environ. Sci. & Pollut. Res. 3 (3) 159-162 (1996) 9 ecomed publishers, D-86899 Landsberg, Germany

eas, and stomatal wax often shows severe degradation in such areas (AuNE and GULLVAG1991). TCA contents in pine needles from Northern Finland are as high as, or exceed, the values found in Central Europe (FRANK et al. 1992 a, 1992 b, 1994, NOROKORPI& FRANK 1995). A dose-response relationship between the levels of TCA analyzed and needle loss has been demonstrated for a pine stand in Northern Finland (FRANK et al. 1994, NOROKORPI & FRANK1995); damage to birch trees has also been correlated to TCA levels (NOROKORPI & FRANK 1993). In these investigations, TCA is used as an indicator of secondary air pollutant deposition. In the past, TCA has found application as an herbicide, the commercial use of which was terminated in the early eighties. While the occurrence of haloacetic acids (trichloroacetic acid, dichloroacetic acid, monochloroacetic acid and trifluoroacetic acid) as secondary air pollutants is a relatively new finding, the degrading effect of TCA on epicuticular waxes has been known for more than three decades (JUNIPER 1959). Numerous studies have shown that degradation of epicuticular waxes is a sensitive indicator of plant exposure to various pollutants (e.g. GRILL 1973 a, 1973 b, TURUNEN et al. 1992). Both acid rain and photooxidants (or their combined effects) have been emphasized. Laboratory studies (e.g. PERCY & BAKER 1990) have been performed along with field studies (e.g. CAPE 1986, KARHU & HUTTUNEN 1986), many of them on Pinus species (HUTTUNEN& LAINE 1983). Scanning electron microscopical studies of needle surfaces have shown wax erosion in polluted areas but also in supposedly clean areas, so no dose-response relationship has been obtained in these previous studies. Therefore, stomatal wax as a bioindicator for air pollution damage to spruce trees was partly abandoned because of the reaction not being specific (EUTENEUER-MACHER1990). The investigations of FRANKand FRANK(1989), ERIKSSONet al. (1989) and JENSENet al. (1992), however, point at the capacity of the wax as a solvent for fat-soluble pollutants. The accumulation of such pollutants can be traced during the


Secondary Air Pollutants lifetime of a needle by means of wax analyses. The epistomatal wax may be a morphological character in the process of permanent change due to ubiquitous air pollution. An accelerated erosion process is probably quite common. In the investigated area, TCA is a pollutant present in tree foliage in concentrations up to a micromol per kilogram flesh weight. Investigation of stomatal wax erosion was therefore a logical step for the verification of our hypothesis.


Material and Methods

The site is background area in Northern Finland located about 50 km SE of Rovaniemi (66~ 26~ It is a dry sandy heath with a stand of Scots pine (Pinus sylvestris, L.) and a bottom layer of Empetrum, Vaccinium myrtillus, V. vitisidaea and Calluna vulgaris, with growth conditions being homogeneous. The trees have regenerated naturally, and are of the same age due to a forest fire 120 years ago; their mean height is 19 m. Sixteen of the outermost trees of the stand were sampled from the dominant canopy from the southwesterly quadrant 15-16 m above the ground. TCA has been determined systematically in their needles. NOROKORPI and FRANK (1995) divided the trees into two groups designated as TCA-sensitive and TCA-resistant. The sensitive ones exhibited more extensive needle losses with the same amount of TCA found in the needles. We kept this division of the material in order to see if it coincided with epistomatal wax degradation, and sampled 8 trees from each group. The twigs were stored in a deep freezer. For analysis, they were thawed overnight in a refrigerator. All needle years present on a twig were studied; they were designated C = current year, 1 = one year old, and so on. The needles were cut 2 mm from the tip and fastened to a metal holder with double adhesive tape. The material was coated with platinum in a Fine Coat Sputter JFC1 100. A JEOL JSM258 scanning electron microscope was used. For each tree, 15 to 20 stomata on the upper side of 3-5 needles of each needle age were studied. The samples were tested blindly. For each sample, 2-5 electron micrographs were taken. The fine structure was evaluated from the micrographs. 3


Each stomatal wax sample was determined to belong to one of the five different categories depicted and described in Fig. 1. The percentages found in the different categories were noted. Fig. 2 (p. 162) shows the current-year (C) samples divided into the TCA-sensitive and TCA-resistant groups (NOROKORPI and FRANK 1995). The percentage within each category is depicted for the two groups. Severe wax degradation is already apparent in the current year (year C); no stomata are found in category 1 - "fine" - and very few may be classified as category 2 - "insignificantly damaged". In the sensitive group, for the category "seriously damaged", the average percentage (44 • 26 %) is signifi-


IMTOX cantly different (P > 99 %) from the average of the TCAresistant group (23 • 15 %). In age group 1, even collapsed stomata have been found (1 and 2 %) in two trees. An overview of all needle age classes studied shows that nearly all stomata (99 %) fall into categories "damaged" and "seriously damaged" (3 and 4). The percentage of category 4 was therefore used for assessment of degradation processes in each age class, and for comparing the sensitivity between individual trees. In Fig. 3 (p. 162), all sixteen trees included in this study are ordered with increasing percentage in category 4. The tree with least needle damage (A) was in the TCA-resistant group and differed significantly from all others. The eight trees with the most seriously damaged wax layers contained five TCA-sensitive (L, M, N, O, P) and three TCA-resistant trees (F, G, H). The three most seriously damaged trees (N, P, O) were TCA-sensitive ones. 4


The population of trees is quite homogeneous. The levels of sulphuric pollutants are negligible (TuoVINEN & LAURILA 1994). TCA and ozone are the only pollutants analyzed so far in phytotoxic amounts. Differences in tolerance of individual trees, as monitored here, may be important and may be induced on different organizational levels. Other variables are not known, and there are no "controls" in this study. The category "fine" was never found during the present analysis, and the SEMG in Fig. 1-1 is from another study (AuNE & GULLV~G 1992). Previous publications (CAPE 1986, GRILL 1973 A, B, HUTTUNEN & LAINE 1983, KARHU & HUTTUNEN 1986, PERCY & BAKER 1990) on stomatal wax studies in Pinus invariably include SEMGs of noneroded wax layers. The fact that even the category "insignificantly damaged" is rarely found in year C is striking. When the epistomatal wax shows severe degradation already in year C, it is our experience from extensive studies of stomatal wax degradation from locations in Norway and Germany that pollution has reached a level with permanent damage to some trees in the area. This is in accordance with the present results. If the response of the epistomatal wax layer to pollution is graded in the semi-quantitative way, as suggested here, the wax degradation caused by acid rain is less severe than by air pollution in the so-called "clean air" region near Rovaniemi, or even in urban areas (AUNE & GULLVAG 1991) where air pollution is possibly derived from phenols and photooxidants. We conclude and emphasize that TCA may be an indicator of herbicidal secondary air pollutants causing increased wax erosion damage, increased wettability, and increased water loss. In the studies of FRANK et al. (1992 a, b), dealing with the same area and the same trees as in the present study, two groups of trees with different sensitivity to TCA have been discerned. In the present study, these groups can be distinguished as significantly different in respect to stomatal wax degradation. One tree (A) previously classed as "TCA resistant" shows a retention of epistomatal wax quality which surpasses all others. On the other hand, differences in wax degradation are found within the groups indicating ESPR- Environ. Sci. & Pollut. Res. 3 (3) 1996


Secondary Air Pollutants

Fig. 1:

Fig. 1-1: Fig. 1-2: Fig. 1-3:

Fig. 1-4: Fig. 1-5:

5 a gradient in sensitivity, whereas only the trees with a very similar degree of damage show non-significant differences with regard to wax degradation. The epicuticular wax has been designated as the first defense line of the needle (JUNIPER & Cox 1973). The expression seems very apt as it also indicates the presence of other defense lines. Physiological or biochemical effects inside the living system must add to differences in tolerance as expressed in the TCA-sensitive and TCA-resistant trees described in earlier studies.

ESPR - Environ. Sci. & Pollut. Res. 3 (3) 1996

Representative illustrations of the five categories of stomatal epicuticular wax layer erosion in Scots pine needles (white bar = 10 btm): Fine: The wax consists of fine crystals in both stomatal and interstomatal areas. Slightly damaged: The wax fine structure is somewhat obliterated on the elevated areas around stomata. Damaged: Areas with crystals as well as with complete obliteration of fine structures are found; residual crystals are coarser than usual. Seriously damaged: Fine structure almost completely lost but stomatal areas can be discerned. Collapsed: Complete destruction of stomatal wax makes identification of stomatal areas impossible


AUNE, L. & GULLV~G, B.M. (1991). The effect of different air pollution environmental exposures on the stomatal epicuticular wax layer of Picea and Pinus needles. Conference on Effects of Atmospheric Pollutants on Climate and Vegetation. Taormina, Sept. 26-29, 1991 CAPE, J. N. (1986): Effects of air pollution on the chemistry of surface waxes of Scots pine. Water, Air, Soil Pollut. 3 1 , 3 9 3 - 3 9 9 ER1KSSON, G., JENSEN, S., KYLIN, H. & STRACHAN, W. (1989): The pine needle as a monitor of atmospheric pollution. Nature 341, 4 2 4 4 EUTENEUER-MACHER,T. (1990): Morphologie und Chemie der Epicuticular-Wachse yon Picea abies (L.) Karsten unter dem Einfluf~ von Klima und Immissionen. Gebriider Borntraeger Verlagsbuchhandlung, Berlin, Stuttgart FRANK, H. (1991}: Airborne Chlorocarbons, Photooxidants, and Forest Decline. Ambio 20, 13-18


Secondary Air Pollutants

Fig. 2:

Percent distribution of stomata between wax layer erosion categories 1 to 4 (c.f. Fig. 1) in the current-year needles for each tree. The latter are divided into TCA-resistant and TCA-sensitive ones, based on a dose/response-relationship between TCA content and needle loss (NOROKORPI & FRANK 1995)

FRANK, H. & FRANK, W. (1989): Uptake of Airborne Tetrachloroethene by Spruce Needles. Environ. Sci. Technol. 23, 365-367 FRANK, H., SCHOLL, H., SUTINEN, S., ~x~ NOROKOPI, Y. (1992 a): Trichloroacetic acid, a ubiquitous herbicide in Finnish forest trees. In: Symposium on the State of the Environment and Environmental Monitoring in Northern Fernnoscandia and the Kola Peninsula. TIKKANEN,E., VARMOLA,M. & KATERMA,T. (Eds.) Oct. 6-8, 1992, Rovaniemi, Finland. Extended Abstracts, Arctic Centre Publ. 4, 259-261 FRANK, H., SCHOLL, H., SUTINEN, S., & NOROKOPI, Y. (1992 b): Trichloroacetic acid in conifer needles in Finland. Ann. Bot. Fennici 29, 263-267 FRANK, H., SCHOLL, H., RENSCHEN, D., RETHER, B., ~x~ NOROKOH, Y. (1994): Haloacetic acids, phytotoxic secondary air pollutant. J. Environ. & Sci. Pollut. Res. 1, 4-14 GRILL, D. (1973 a): Rasterelektronenmikroskopische Untersuchungen an Wachs-Strukturen der Nadeln yon Picea abies L. Karsten. Micron 4, 146-154 GRILL, D. (1973 b): Rasterelektronenmikroskopische Untersuchungen an SO2-belasteten Fichtennadeln. Phytopath. Z. 78, 75-80 HINKEL, M., RHSCHL.,A., SCHRAMM, K.-W., TRAUTNER, E, REISSINGER, M., & HUTZINGER,O. (1989): Concentration levels of nitrated phenols in conifer needles. Chemosphere 18, 2433-2439 HuTrUNEN, S. & LAINE, K. (1983): Effects of airborne pollutants on the surface wax structure of Pinus sylvestris needles. Ann. Bot. Fennici, 20, 79-86 JENSEN, S., ERIKSSON, G. &~KYLIN, H. (1992): Atmospheric pollution by persistent organic compounds: Monitoring with pine needles. Chemosphere 24, 229-245



Fig. 3:

Percentage of wax layer erosion category 4 (seriously damaged) in each needle age class by tree. Needle age classes: C = current year, 1 = C + 1 year, etc. The lettering of the trees (A to P) is the same as in Fig. 2. TCA = TCA content in ng 9g-l; NL = needle loss percentage

JUNIPER, B.E. (1959): The effect of pre-emergent treatment of peas with trichloroacetic acid on the sub-microscopic structure of the leaf surface. New Phytol. 58, 1-5 JUNIPER, B.E., & Cox, G.C. (1973): The Anatomy of the Leaf Surface: The First Line of Defence. Pestic. Sci. 4, 543-561 KARHU, M., ~x~HU'H~NEN, S. (1986): Erosion effects of air pollution on needle surfaces. Water, Air, Soil Pollut. 31,417-423 NOROKORPI, Y., & FRANK, H. (1993): Effect of stand density on damage to birch (Betula pubescens) caused by phytotoxic air pollutants. Ann. Bot. Fennici 30, 181-187 NOROKORPl, Y., & FRANK, H. (1995): Trichloroacetic acid as a phytotoxic air pollutant and the dose-response relationship for defoliation of Scots pine. Sci. Total Environ. 160/161,459-463 PERCY, K.E. & BAKER,E.A. (1990): Effects of simulated acid rain on epicuticular wax production, morphology, chemical composition and on cuticular membrane thickness in two clones of Sitka spruce (Picea sitchensis (Bong.) Carr.). New Phytol. 116, 79-87 TUOVINEN, J.-P., LAURILA,T. (1994): Variability of sulphur dioxide and ozone concentrations in Northern Finland. In: B. SIVERTSEN(Ed.): Air Pollution Problems in the Northern Region of Fennoscandia including Kola. Proceedings of the Seminar at Svanvik, Norway, June 1-3, 1993. NILU-TR 14/94:41-45 TURUNEN, M., HUTI~NEN, S., S. BACK,J., KOPONEN, J. & HUHTALA, P. (1992): Needle damage in the Scots pine of Lapland and the Kola Peninsula. In: TIKKANEN~E., VARMOLA~M., KATERMAA~T. (Eds.): Symposium on the State of the Environment and Environmental Monitoring in Northern Fennoscandia and the Kola Peninsula. Oct. 6-8, 1992, Rovaniemi, Finland. Extended Abstracts, Arctic Centre Publ. 4, 235-239

ESPR - Environ. Sci. & Pollut. Res. 3 (3) 1996

Secondary air pollutants : Epistomatal wax erosion of Scots pine needles.

The study has been performed in a supposedly clean-air region of a Northern Finnish forest with a homogeneous stand of Scots pine. Stomatal epicuticul...
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