SCOPE 53 - Methods to Assess the Effects of Chemicals On Ecosystems

9.1 INTRODUCTION

Despite growing in generally fairly clean air, forests may be subjected to a variety of pollutant: salts (Na, Ca, Mg, sulphates, chlorides, or bicarbonates), gases (SO₂, NO₂, and ozone), nitrogen compounds (HNO₃, NH₃, and NH₄⁺), heavy metals (Cd, Cu, Ni, Pb, and Zn), acids (H₂SO₄ and HNO₃). Weed control agents and other pesticides appear to have a negligible impact on forests, because, at most, they are used for only a few years in a rotation of a hundred years or more, and some countries have banned their use completely.

Based on the nature of these substances, their effects may be identified either at either of two levels: that of the trees and that of the forest ecosystem.

9.2 METHODS USED TO STUDY TREES

9.2.1 CHEMICAL STUDIES

9.2.1.1 Stress reactions at the metabolic level

Plants are exposed in their environment to numerous stress factors that may act either simultaneously or consecutively with different intensities and frequencies. Plants experience stress from several environmental influences such as (1) contact with chemicals of anthropogenic origin, (2) climate (e.g., high light intensity, drought, high temperature, and low temperature), (3) nutrient deficiency, (4) excess availability of trace elements, and (5) pathogens. At the metabolic level, many factors generate oxidative stress to plants (Polle and Rennenberg, 1993a). Therefore, metabolic effects caused by exposure of plants to oxidative chemicals cannot easily be distinguished from effects caused by other factors.

The site(s) of initial generation of oxidative stress in plants may be different for airborne chemicals and other stress factors. Atmospheric substances enter the shoot of plants predominantly via the stomata of the leaves. wherein they first contact the aqueous phase of the cell walls (apoplastic space). Reactions in this compartment are considered initiating events. Metabolic alterations in the cytoplasm may be caused by products of initial reactions in the apoplastic space or by a fraction of the parent substance. By contrast, oxidative stress from climate or nutrient deficiency may predominantly take place inside the cell with the chloroplast as the most important initial target. Compartmental analysis. therefore. may be used as a diagnostic tool to distinguish the influence of alternative stresses.

The metabolic responses of trees to air pollutants (Bytnerowicz and Grulke, 1992) and oxidative stresses (Polle and Rennenberg. 1993a. 1993b) have been reviewed recently in detail. This chapter discusses metabolic reactions in trees as diagnostic tools of damage caused by atmospheric sulphur and nitrogen compounds. photochemical oxidants, and atmospheric xenobiotics.

9.2.1.2 Metabolic effects of photochemical oxidants

During atmospheric transport of pollutants. radical reaction chains are initiated by light. producing highly reactive hydroxyl radicals and peroxides, hence the name photochemical oxidants. Ozone is believed to be the most important final product of these reaction chains. By this mechanism, emissions of volatile organic compounds contribute to the ozone troposphere. Among the many photochemical oxidants. ozone is the most studied for its toxic properties on vegetation; hence. the discussion of the metabolic effects of photochemical oxidants is focused on ozone.

Like other atmospheric pollutants ozone enters the leaves predominantly via the stomata. and first contacts the aqueous phase of the cell walls (apoplastic space). Ozone's high oxidation potential ozone will lead either to its rapid degradation to other reactive oxygen species or to its reaction with natural components in the apoplastic space. Ascorbate is known to protect against injury from ozone. is found in mM concentrations in the apoplastic space, and, therefore, plays a major part in the neutralization of ozone absorbed by leaves. When the dehydroascorbate produced in this reaction is regenerated, this reaction protects against damage from ozone (Polle and Rennenberg. 1993a). Ozone appears unlikely to pass the aqueous phase of the cell wall and enter the symplasm. Hence. ozone will damage simplistic structures and alter metabolic processes only in the apoplastic space inside the cell.

Ozone-mediated changes in apoplastic ascorbate content and peroxidase activity have been observed in fumigation experiments and field studies. However. even the direction of these changes varied between species and even for the same species in different studies (Castillo et al., 1987; Castillo and Greppin, 1988; Ogier et al., 1991; Polle et al., 1991; Polle and Rennenberg, 1992). Therefore, apoplastic markers cannot be recommended for the identification of perturbations by ozone stress.

Likewise, biochemical markers in the symplasm are unavailable. Though ozone is known to affect photosynthesis, stomatal aperture, respiration, allocation of photosynthetase, substrate levels, and enzyme activities of the antioxidant system, and ethylene biosynthesis, none of these effects are specific for ozone (Bytnerowicz and Grulke, 1992). Ozone-mediated induction of polyamine and stilbene biosynthesis and pathogen-related proteins has been observed in plants (Ernst et al., 1992; Rosemann et al., 1991; Schraudner et al., 1992); however, the specificity of these' biochemical responses to ozone remains to be elucidated.

9.2.1.3 Metabolic effects of sulphur compounds

Trees growing in regions of volcanic or geothermal activity or in heavily industrialized areas are subjected to high levels of atmospheric sulphur compounds, most predominantly sulphur dioxide (SO₂) and hydrogen sulphide (H₂S), which are both rapidly absorbed by plant shoots where it enters the plant's metabolic pathways of sulphur reduction and association (DeKok, 1990). Foliar deposition of sulphur at high concentrations can result in damage to plants, but, at low concentrations, may also be beneficial for plant growth and development.

Sulphur dioxide

Herbaceous and woody plants exposed to SO₂ have increased sulphur content in the shoots (Bytnerowicz and Grulke, 1992). The extent of sulphur accumulation varies between species, and depends on suffix nutrition to the roots, atmospheric SO₂ concentration, duration of exposure, and climatic conditions during exposure (DeKok, 1990). Shoot sulphur content cannot be used as a specific diagnostic marker of SO₂ exposure, when plants grow in high sulphur soil or are in contact with other sulphur-containing gases in high concentrations in the air (Rennenberg, 1984).

Elevated sulphur levels in shoots can predominantly be ascribed to the accumulation of sulphate in the vacuole (Rennenberg, 1984; DeKok 1990; Cram, 1990). Though a decrease in the sulphate content of shoots has been observed upon cessation of SO₂ fumigation (Maas et al., 1987a), sulphate accumulation may still be a diagnostic tool for atmospheric sulphur exposure especially in trees, because mobilization of sulphate stored in the vacuole proceeds at a low rate (Cram, 1990) and dilution of accumulated sulphate by growth may only cause injury during specific developmental stages.

Some of the SO₂ absorbed by plant shoots is reduced to the level of sulphide (DeKok, 1990), and up to 10-15 percent of absorbed SO₂ can be re-emitted into the atmosphere in the form of H₂S (Rennenberg, 1991). The sulphide is incorporated into cysteine, and used for the synthesis of other low molecular weight sulphur compounds, such as glutathione, and for protein synthesis. Since elevated levels of low molecular weight thiols are also present from various stresses besides air pollutants (Smith et al., 1990), thiol levels provide no specific diagnostic measure for SO₂ stress in plants. However, the ratio of sulphate-S/organic-S may provide a relatively specific tool to diagnose SO₂ stress in plants. In pine, this ratio varied from 0.29 at a reference location to 0.88 in an area with high SO₂ deposition (Legge et al. , 1988). No such changes have been observed from other stress factors including exposure to other atmospheric sulphur compounds or to excess sulphate in the soil.

Exposure of foliage to atmospheric SO₂ results in a transport of SO₂-derived sulphur to the roots in the form of sulphate and organic sulphur compounds like cysteine and glutathione (Garsed, 1985; Rennenberg and Polle, 1992). Sulphate and organic sulphur compounds are normal constituents of both xylem and phloem, and their long-distance transport in these pathways is important for the inter-organ regulation of sulphur nutrition (Herschbach and Rennenberg, 1991; Schupp et al., 1992). Consequently, atmospheric SO₂ interacts in plants with processes involved in the regulation of sulphur nutrition at the whole plant level, but no diagnostic tools has been developed to benefit from this observation.

SO₂ and its metabolic products affect numerous physiological processes in plant cells, including stomatal aperture and its control (Black, 1985), carbon balance and allocation of photosynthetate (Winner et al., 1985; Bytnerowicz and Grulke, 1992), stromal and thylakoid functions (Wellburn, 1985), and antioxidant defence mechanisms (Rennenberg and Polle, 1992). None are specific for SO₂ exposure, or to that of other air pollutants. Therefore, they are not suitable as diagnostic measures.

Likewise, the analysis of apoplastic processes is not helpful diagnostically. SO₂ that has entered the leaves rapidly dissolves in the aqueous phase of the cell walls and reacts with water to form bisulphite. Finally, determination of apoplastic sulphate can presently not be recommended for the diagnosis of atmospheric SO₂ exposure.

Hydrogen sulphide

Fumigation of plants with H₂S results in elevated sulphur contents of the shoot. Only a minor part of this increase can be ascribed to enhanced levels of sulphate. The bulk of the H₂S absorbed by leaves is directly incorporated into reduced sulphur compounds. Consequently, elevated levels of sulphydryl compounds, mainly glutathione and cysteine, are found in shoots fumigated with H₂S (DeKok, 1990). Several other stress factors cause elevated levels of sulfhydryl compounds, mainly glutathione (Smith et al. , 1990) Therefore, this finding is unsuitable as a diagnostic measure of exposure to H₂S. In spinach, prolonged H₂S exposure results in an accumulation of sulphate in the roots (Maas et al., 1987b), and such accumulation in the roots may be used as an indication of H₂S exposure, provided the phenomenon can be generalized.

9.2.1.4 Metabolic effects of nitrogen compounds

Anthropogenic activities have led to high emissions of gaseous nitrogen compounds into the atmosphere¾specifically nitrogen oxides released during the combustion of fossil fuels by power plants and by automobiles. Agricultural activities result in the emission of high amounts of ammonia and ammonium into the atmosphere. Consequently, critical loads for nitrogen to forests are exceeded in many regions (Hadwiger-Fangmeier et al., 1992). Although excess nitrogen input may initially be beneficial to forests growing under nitrogen limitation, prolonged exposure will result in nutritional disorders and enhanced susceptibility to other stresses. Furthermore, the species composition of forest ecosystems will change dramatically during long-term exposure to excess nitrogen (Hadwiger-Fangmeier et al., 1992).

Nitrogen oxides

The combustion of fossil fuel produces nitrogen monoxide (NO) as the primary oxide of nitrogen. In the presence of ozone, NO is rapidly oxidized to nitrogen dioxide (NO₂). Both gases can contact the stomata of the leaves, a major path of entry in plants. Deposition velocities are one order of magnitude higher for NO₂ than for NO, due to differences in solubility. Both NO₂ and NO react with water to form nitrate and nitrite; for NO, this reaction is much slower than for NO₂ (Wellburn, 1990). Having entered a leaf and been solubilized in the aqueous phase of the cell wall, each species reacts with other constituents of the apoplastic space (e.g., with ascorbate), perhaps a very significant group of interactions (Thoene, et al., 1991).

As reaction products of NO and NO₂ in the aqueous phase of the cell wall, nitrite and nitrate may provide a diagnostic tool to identify exposure of trees to nitrogen oxides. In herbaceous plants, nitrite levels rarely rise, and changes in the level of total nitrate in response to NO₂ depend on the N supply to the roots. When red spruce seedlings were fumigated with NO₂ in the winter, nitrite accumulated in the apoplastic space, whereas nitrate levels remained unchanged (Wolfenden et al., 1991). In the same study, the catabolism of nitrite was slower than its production from NO₂. The finding of nitrate in the xylem sap of control plants in this study was unexpected, and may be an indication of insufficient mycorrhization of the seedlings. Nitrate reduction in mycorrhized trees is supposed to proceed in the fungi associated with the roots. From these fungi amino acids are transferred to the roots and transported to the leaves with the transpiration stream (Martin and Botton, 1993). Therefore, the occurrence of nitrate in leaf extracts from mycorrhized trees may be an indication of exposure to nitrogen oxides. Whether the fungi withhold the nitrate because of the high concentrations in soil is unknown. Furthermore, total nitrogen content of the leaves provides no diagnostic measure, since it often declines in response to exposure of plants to nitrogen oxides, presumably due to enhanced export of nitrogen from the leaves (Wellburn, 1990).

For many trees, the assumption is incorrect that the conversion of NO and NO₂ to nitrite and nitrate in the aqueous phase of the cell wall (passed the plasmalemma) indicates a normal pathway of nitrate reduction followed by synthesis of amino acids and protein. Nitrate reduction and amino acid synthesis may take place in the mycorrhizal fungi of the roots and not in the leaves. Thus, nitrate reduction and amino acid synthesis are not considered normal processes in the leaves of mycorrhized trees, although their leaves remain capable of reducing nitrate and synthesizing amino acids. Nitrate reductase (NaR), which catalyses the reduction of nitrate to nitrite, is induced by its substrate; therefore, the level of its activity is determined by the nitrate supply. Studies of several tree species lacking significant nitrate reduction in the leaves have shown that exposure to atmospheric NO₂ concentrations results in increased NaR activity of the shoots (Thoene et al., 1991). While nitrite reductase activity has been shown to increase, the change is less at normal atmospheric NO₂ concentrations (Wellburn, 1990; Thoene et al., 1991). Therefore, the elevated NaR activity is postulated to be a reliable diagnostic tool to identify nitrogen oxide exposure to mycorrhized trees.

By eliciting oxidative stress, NO and NO₂ produce numerous physiological changes similar to those found with other oxidants. The responses include membrane damage due to inhibition of lipid biosynthesis and lipid peroxidation and reduction in net photosynthesis (Wellburn, 1990; Bytnerowicz and Grulke, 1992). Since none of these effects is specific for NO and NO₂, they are considered to be diagnostic measures.

Ammonia and ammonium

Stomatal aperture provides the only resistance for the influx of gaseous ammonia or ammonium into plant shoots, a phenomenon that may be due to the high water solubility of ammonia or ammonium that facilitates penetration through the aqueous phase of the cell wall. Dry deposition of these compounds accounts for 60 percent of the total, and takes place in close proximity to the source, because of the high deposition velocity (i.e., 65 percent within a distance of 30 km and 90 percent within 90 km from the source (Hadwiger-Fangmeier et al., 1992). Therefore, the forests adjacent to agricultural activities are a large sink for atmospheric ammonia and ammonium.

Exposure (e.g., soil fertilization) of herbaceous and woody plants to atmospheric ammonia and ammonium increases the concentrations of these compounds in foliage and shoots. Such exposures also increased the amino acid content, especially arginine, in the leaves, and glutamine synthetase activity was found to be enhanced (Hadwiger-Fangmeier et al., 1992). Physiological reactions of the exposure to atmospheric ammonia and ammonium included increased stomatal conductance connected with increased photosynthesis, enhanced transpiration, reduction of the water potential in water stressed plants, and mineral disorders such as reduced K and P content in the leaves (Hadwiger-Fangmeier et al., 1992). These physiological changes are related to differences in susceptibility to other biotic and abiotic stresses such as drought, frost resistance, attack by herbivores, and fungi. Mycorrhization was found to be reduced (Hadwiger-Fangmeier et al., 1992). Apparently, increased ammonia and ammonium contents of the shoots provides a reliable measure of atmospheric ammonia and ammonium input into plants. Elevated total nitrogen of the shoots may be the result of atmospheric as well as pedospheric input. Reduced K and P contents of the leaves may be used to diagnose atmospheric ammonia input at sufficient K and P supply in the soil.

9.2.1.5 Metabolic effects of airborne xenobiotics

Halogenated hydrocarbons and pesticides are found in the atmosphere of highly industrialized areas, in the vicinity of agricultural regions, and in remote sites, such as alpine forests (Koval et al., 1986; Elling et al., 1988; Herterich and Herrmann, 1990). Xenobiotics can be transported in the atmosphere over long distances, and may be deposited preferentially in forests of mountain slopes. Trees growing in this environment are already exposed to various natural stresses like extreme temperatures, elevated ozone concentrations, high irradiation, and drought. The deposition of xenobiotics, as evident from the accumulation of xenobiotics in conifer needles (Gaggi and Bacci, 1985; Reichl et al., 1987; Hinckel et al., 1990), provide additional stress to these trees. Numerous crops, weeds, and even the needles of conifer species are known to contain glutathione S-transferases (GSTs), a group of constitutive and inducible isoenzymes capable of detoxifying halogenated and nitrated xenobiotics (Lamoureux and Rusness, 1989; Schroder et al., 1990a, 1990b; Schroder and Rennenberg, 1992). GSTs may be induced by exposure to xenobiotics that they metabolize (Lamoureux and Rusness, 1989); thus, analysis of the GST isoenzyme pattern may be a useful means of identifying xenobiotic exposure of trees. Such an approach could be misleading, if GST activity was also induced by oxidative stresses such as ozone (Price et al., 1990), since protection from oxidative stress may be considered a natural function of this group of enzymes.

9.2.1.6 Conclusions

Numerous biochemical parameters and physiological processes have been studied in herbaceous and woody plant species to ascertain the impact of exposures to photochemical oxidants, atmospheric sulphur and nitrogen compounds, and airborne xenobiotics. Though many effects of these pollutants have been identified, only few have proven to be sufficiently specific to serve as diagnostic measures for the identification of exposure to an individual pollutant. The ratio of sulphate-S/organic-S may be used to identify sulphur dioxide exposure; apoplastic or cellular nitrate and nitrite as well as nitrate reductase activity of the leaves may be suitable to identify exposure of mycorrhized tree species to nitrogen oxides. Several biochemical parameters may be useful to identify atmospheric or pedospheric ammonia and ammonium input into trees. GST isoenzyme analysis for xenobiotics remains a research interest. A reliable diagnostic measure of exposure to photochemical oxidants is unavailable.

9.2.2 ANATOMICAL STUDIES

9.2.2.1 Microscopic diagnosis of damage to needles and leaves

The sudden occurrence of widespread "new type forest decline" in Central Europe and North America during the early 1980s caused an intensive search for diagnostic criteria to evaluate possible causal pathogenic factors. Although abiotic agents seemed to be the main toxic agents, discussions focused on whether symptoms seen in the field were caused by the direct impact of acid precipitation on the foliage or of gaseous pollutants on the leaves, or by indirect disturbances in mineral nutrition from the increased deposition of acidity and nitrogen (Roberts et al., 1989).

Several approaches have been developed to construct criteria that could identify the actual causative factors. These criteria were first deduced from comparisons of healthy control trees with those subjected to artificial acid precipitation, controlled fumigation in chambers, or controlled experiments with varying mineral nutrition. These criteria were then applied in the field, using material from healthy and declining trees. Most diagnostic approaches use quantitative biochemical analysis of homogenates of needles or leaves, looking for carbohydrates and associated metabolites, lipids, antioxidants, or enzymes. Limitations of these indicators include unspecific reactions to numerous stress factors and only occasionally the determination of the impact of specific stresses.

To improve the possibility of diagnosing damage in needles, the reactions to stress of individual cells in the complex tissue of conifer needles and angiospermous leaves is an appropriate starting point for exploring more detailed diagnostic approaches. Qualitative changes within specialized cells and tissues were chosen as a criterion instead of quantitative changes in a homogenate of all cell types. The approach, originally developed in plant hormone research (Osborne, 1978, 1984), focused on finding target cells in needles and leaves that react differentially to external influences. The needles of Norway spruce (Picea abies (L.) Karst.) have been intensively studied for this purpose. The regular structures in these needles and leaves and in those from other species can be compared to alterations induced in controlled experiments to establish causal relationships, and the results then can be applied to damage patterns observed in the field.

9.2.2.2 Results from controlled experiments

Acid precipitation

The influence of acid precipitation is first investigated at the leaf/needle surface with epidermis, cuticle, and epicuticular waxes. In controlled experiments with simulated acid rain or mist, the amorphous and fibrillar crystalline waxes (especially in the epistomatal chambers) become partially fragmented and eroded, and partially baked together to amorphous masses, which is especially pronounced on long-lived leaves such as conifer needles. On one side, formation of these waxes on young leaves is retarded by the acidic conditions, whereas on the other side acid erosion is accelerated by 2-5 times the rate without acid; the regeneration capability is reduced by acid (Haines et al., 1985; Riding and Percy, 1985; Crossley and Fowler, 1986; Grill et al., 1987; Mengel et al., 1987, 1989; Schmitt et al., 1987; Tuomisto, 1988; Rinallo et al., 1986; Turunen and Huttunen, 1989, 1991; Percy and Baker, 1990).

Acid precipitation may also lead to punctual necrosis of epidermal cells on the adaxial surface, frequently close to leaf veins or stomata, and at the bases of trichomes or glandular hairs. If cells adjacent to stomata collapse, the stomata may stay open continuously. At pH values below about 3.5, larger epidermal necroses may occur, which then may spread to adjoining cells of the palisade parenchyma and eventually spongy mesophyll. The neighbouring undamaged mesophyll cells then react with hypertrophy; the cells divide again; and, by hyperplasia, they may finally lead to circular or ring-like (with necrotic tissue in the centre) proliferations of the leaves, somehow resembling insect galls. These tissues form a delineation, resembling early stages of wound periderm formation, and may thus limit further progress of damage. In extreme cases, the damage may also proceed to the vascular bundles after most of the mesophyll has already collapsed. In general, developing young leaves are more affected by acid rain than older leaves (Evans et al., 1978; Evans and Curry, 1979; Swiecki et al., 1982; Paparozzi and Tukey, 1983; Adams et al., 1984; Evans, 1984; Crang and McQuattie, 1986; Rinallo et al., 1986). This pattern is mainly confined to angiospermous leaves. At pH-values below 2.5, similar epidermal necroses may occur in young needles (Whitney and Ip, 1991; Zobel and Nighswander, 1991).

After acid rain-treatment, non-lethal changes may be seen in chloroplast structures. Chloroplasts in outer mesophyll cells are more irregular in form with fewer thylakoids and larger starch grains. Sometimes thus treated needles show more severe changes in chloroplast structure after they have been subjected to cold treatments (Holopainen and Nygren, 1989).

Fumigation with gaseous air pollutants

Within a leaf or needle, gaseous pollutants primarily attack the mesophyll, especially that directly adjacent to the substomatal cavities; they sometimes directly attack the guard cells where the pollutants contact the stomata. The vascular bundle is mainly unaffected during initial exposure. This observation has been confirmed for many conifer needles and deciduous leaves subjected to SO₂, O₃, NO_x, HF, Cl₂, singly or in combination; in long-living conifer needles or evergreen leaves cumulative damage proceeds through additional necrosis in subsequent years (Hartig, 1896; Evans and Miller, 1972, 1975; Stewart et al., 1973; Percy and Riding, 1981; Soikkeli 1981a, 1981b, 1981c; Carlson and Gilligan, 1983; Fink, 1988, 1989; Kozlowski and Constandinidou, 1986; Ruetze et al., 1988; Hafner et al., 1989; Ebel et al., 1990). The damaged mesophyll cells show plasmolysis and shrinkage, breakdown of internal compartmentalization, and finally necrosis and discolorations; the mesophyll cells directly bordering necrotic cells may react with slight discolorations, thickenings of their cell walls, or hypertrophy; a specific reaction in conifer needles is the hypertrophy of epithelial cells of the resin sacs leading to their occlusion. Collapse of mesophyll may lead to depressions in the overlying epidermis. The vascular bundles generally remain unaffected during an initial phase, and only after extensive damage to the mesophyll the sieve elements may also show some necrosis, accompanied by a hypertrophy of adjacent cells of the phloem and transfusion parenchyma. Damage by O₃ preferentially affect the palisade parenchyma, even if this is located on the upper side of a leaf, i.e., opposite the stomata that are on the lower side in hypostomatic leaves; O₃ hardly affects the vascular tissue. HF seems to induce first injury to the spongy mesophyll, which spreads to the vascular tissue. SO₂ induces a more general damage to all mesophyll cells, spreading only later to the vascular tissues.

Epicuticular waxes can be affected when SO₂ dissolves in moisture upon the leaf surface, and then causes the same reactions as acid precipitation. Ozone seems to have no effect on the structure of the already developed waxes, but obviously reduces the biosynthesis of waxes in the young needle (Gwrthardt-Goerg and Keller, 1987; Schmitt et al., 1987; Percy et al., 1992).

Before lethality in the mesophyll, the most pronounced changes produced by gaseous pollutants are alterations in the structure of chloroplasts: granulation of stroma, curling and swelling of thylakoids, reduction of grana, accumulation of plastoglobuli, and small, round chloroplasts with occasional irregular outlines. Most changes are rather non-specific signs of premature senescence, and only few specific changes for the different pollutants may be distinguished speculatively. SO₂ seems to lead more to reduced grana, swollen thylkoids, and light plastoglobuli; O₃ and other oxidants produce generally smaller and flattened chloroplasts that may have irregular outlines with granulated stroma partially containing crystaloids or fibrils, and accumulation of electron-dense material between the two membranes of the chloroplast envelope; NO₂ produces tubular protrusions from the chloroplast envelope; and BF produces stretched chloroplast envelopes and swollen or curled thylakoids (Thomson et al., 1966, 1974; Thomson, 1975; Athanassious et al., 1978; Athanassious, 1980; Soikkeli, 1981a, 1981b, 1981c; Soikkeli and Karenlampi, 1984; Karenlampi and Houpis, 1986; Sutinen, 1987c; Ruetze et al., 1988; Ebel et al., 1990; Sutinen et al., 1990).

Induced mineral deficiencies

The widespread mineral deficiencies typical of many declining forests have structural impacts different from those caused by gaseous air pollutants. In conifer needles, deficiencies of magnesium, potassium, and calcium cause initial changes in the vascular bundle with early necrosis of the sieve cells and hypertrophy and hyperplasia of cambial cells and adjacent parenchyma cells, as well as alterations of the transfusion parenchyma. The mesophyll cells are only affected at a later stage, and may continue to live, even though their organelles may be severely modified (Fink, 1988, 1989, 1990a; Holopainen and Nygren, 1989; Holopainen et al., 1992). These changes may be explained by the importance of these cations for the stabilization of membranes and cell walls, by their role in osmotic regulations and hormone metabolism, and by their part in modulating enzyme activities (e.g., Mg-dependent ATPase responsible for loading of sugars into the phloem). For deficiencies of Mg, K, Ca, the sequence of degeneration (with regard to necrosis) is as follows: vascular bundle, then mesophyll, and finally epidermis which remains mainly unaffected through development of damage (Fink, 1988).

Deficiencies of Mg and K lead to a reduction and swelling of the thylakoids in chloroplasts, which are frequently also distorted because of the occurrence of large starch grains; reduced and irregularly organized grana compartments are typical, as are increased amounts of plastoglobuli (Whatley, 1971; Hamzah and Gomez, 1979; Fink, 1989; Holopainen and Nygren, 1989; Holopainen et al., 1992).

9.2.2.3 Patterns from damaged foliage in the field

The first visible and most pronounced damage in declining trees is seen macroscopically as alterations of colour (turns yellow), partial necrosis, or premature abscises. By applying results from controlled experiments to the field, more conclusive microscopic criteria were sought to discriminate among possible causes of such alterations. Several microscopic criteria are discussed below.

Integrity of epicuticular waxes

In current research on forest decline, several studies have reported on possible changes in wax structure in field material as an indicator of the direct action of acid rain. Heavy degradations, accompanied by contamination by particulates, were indeed found in pine needles from urban areas of Berlin (Hafner, 1986; Hafner et al., 1989). Under much less polluted conditions, fused stomatal waxes and degraded surface waxes were reported from declining trees in various areas in Central Europe (Sauter and Voss, 1986; Badot et al., 1988). A close correlation of wax structure exists with the vitality of the trees, but not necessarily with type or amount of emissions. Some conflicting data exist, e.g., for the Black Forest, since other researchers could find no significant difference in wax structure between healthy and declining trees, because of the large variation of wax structure even within a single tree. A perplexing result is that the healthiest trees in Scotland have the least intact waxes (highest wettability), while the most diseased trees in the Black Forest have the most intact ones and the slowest weathering rates (Cape et al., 1988). These findings strongly suggest that, outside "classical" heavily polluted industrial areas, the structure of surface waxes may be mainly determined by natural (climatic) influences, water stress, mineral nutrition, and indirectly by the general vitality of the trees (Guth and Frenzel, 1989; Ylimartino et al., 1992; van Gardeningen et al., 1991).

Mesophyll necrosis

The types of primary mesophyll damage caused by gaseous pollutants have been found widespread in areas with well-established ozone damage to susceptible species, mainly in the northeastern and southwestern parts of the USA (Costonis and Sinclair, 1969; Costonis, 1970; Karenlampi, 1986; Evans and Leonard, 1991). Concerning forest decline in Germany, some instances of this type of damage were found close to the borders to CSSR and GDR, where with easterly winds occasionally very high SO₂ concentrations occur (Cape et al., 1988; Forschner et al., 1989; Hasemann and Wild, 1990). Furthermore, they were encountered in heavily polluted urban regions of Berlin, where dead mesophyll cells already appear in green needles (Hafner, 1986; Hafner et al., 1989). In leaves from sensitive hardwoods (Fraxinus, Prunus), necrotic palisade parenchyma cells (pointing to ozone damage) were claimed to have been found during the summer of 1989 in the Black Forest and the Alps with that region's unusually high ozone concentrations. No mesophyll necrosis, however, is associated with the most widespread yellowing of conifer (especially spruce) needles, which may stay living and turgescent for 2-4 years without necrosis of mesophyll cells, being totally yellow. Besides limited reports on mesophyll necrosis in some areas (Sutinen, 1987b; Forschner et al., 1989) only little evidence exists that widespread direct damage by gaseous pollutants is involved with the typical "montane yellowing."

Some problems arise to avoid confounding pollution-caused necroses with necroses of other causes. This is especially a problem for several necrotic flecks that frequently occur on many leaves and needles in the field. In conifer needles (especially at higher altitudes), an increasing density of necrotic flecks is found on the upper surfaces as the needles age. No clear relationship has been observed between the occurrence of such flecks and either air quality or the vitality of the trees. A possible link between these necrotic spots on conifer needles and damage by acid rain has been hypothesized (Whitney and Ip, 1991). These necrotic spots are characterized by initial damage to mesophyll cells, frequently beneath the hypodermis, whereas the epidermis continues to live, thereby excluding the possibility of damage by acid precipitation (Fink, 1990b). Hypotheses that these spots had been caused by ozone (Lang and Holdenrieder, 1985; Sutinen, 1986) are not in accordance with this damage pattern, so that the explanation remains speculative, "winter fleck injury" remaining the most likely explanation (Daniker, 1923; Schmidt, 1936; Miller and Evans, 1974; Brennan, 1988; Fink 1990b); however, acidity and ozone should be rejected as possible causative agents.

Phloem alterations

Yellow needles from declining conifers that show symptoms of "montane yellowing" (associated mainly with Mg-deficiency) frequently exhibit symptoms characterized by drastic changes in their vascular bundles (sieve cell necrosis, hypertrophy and hyperplasia of cambium and phloem parenchyma), and less alterations in their still living mesophyll cells. These symptoms have been found in decaying yellow spruces and firs in the Black Forest (Fink, 1983, 1988, 1989; Parameswaran et al., 1985) and the Hunsruck mountains (Forschner et al., 1989; Hasemann and Wild, 1990). The observations of Schmitt et al. (1986) on early changes in the phloem, transfusion parenchyma, and endodermis (while mesophyll cells were still intact) already in green needles from decaying trees in northern Germany are in accordance with these observations of damage starting in the vascular bundle. These observations can be exclusively attributed to mineral deficiencies, which are probably caused by a combination of soil acidification, increased input of nitrogen, and climatic stresses. Further evidence for this phenomenon arises from the fact that at least moderately yellow needles may repair their chloroplasts and start to produce functional sieve cells as soon as the deficient minerals are added as fertilizers (Huttl and Fink, 1988).

Chloroplast ultrastructure

Alterations of chloroplast ultrastructure have been studied mainly in yellowing needles from declining trees in the field, and consist of reduction of thylakoids and accumulation of numerous plastoglobuli (Parameswaran et al., 1985; Sutinen, 1987b; Jung and Wild, 1988; Zellnik and Gailhofer, 1989). These changes are comparable with those induced by controlled nutrient deficiencies. The type of swelling and distortion produced experimentally by ozone or SO₂ has been described as that from heavily polluted industrial and urban areas (Soikkeli, 1981b, 1981c; Hafner, 1986). However, these changes are only partly pollutant-specific and partly signs of senescence, either the natural rate of ageing or accelerated senescence caused by air pollutants or mineral deficiencies (Dodge, 1970; Whatley, 1971; Hecht-Buchholz, 1972, 1983; Chabot and Chabot, 1975; D' Agostino and Pennazio, 1981; Soikkeli, 1981 a, 1981b, 1981c; Cunninghame et al., 1982; Sutinen, 1987a).

Swelling, thylakoid disorganization, formation of vesicles, and partial accumulation of large starch grains and lipid bodies have been found in needles from declining conifers to be caused by ozone, SO₂, water deficit, or mineral deficiencies (Thomson et al., 1966,1974; Whatley, 1971; Thomson, 1975; Pell and Weissberger, 1976; Vapaavuori et al., 1984; Fink, 1988, 1989, 1992).

Observations on separation and dilatation of the double membranes of the thylakoids, forming electron-translucent spaces and inducing a swelling of the chloroplasts to roundish bodies, have stimulated hypotheses about the possible effects of organic atmospheric pollutants; however, no evidence yet supports this contention (Meyberg et al., 1988). Similar symptoms have been reported with SO₂, O₃, NO_x, and mineral deficiencies (Wellburn et al., 1972; D' Agostino and Pennazio, 1981; Soikkeli, 1981a, 1981b; Hecht-Buchholz, 1983; Platt-Aloia et al., 1983; Soikkeli and Karenlampi, 1984). Sutinen (1986, 1987b, 1987c) and Sutinen et al. (1990) suggested that smaller and flatter chloroplasts with reduced thylakoids and more plastoglobuli may be typical for impacts of ozone (alone or together with SO₂), but the same changes are reported for different mineral deficiencies (Fink, 1988, 1989). Other types of chloroplast changes originally were thought to be typical for ozone or acid rain, but apparently may also be caused by water stress, such as crooking of the chloroplasts and the formation of thylakoid-free parts of the stroma, in which crystalline structures may occur (Thomson et al., 1966, 1974; Athanassious et al., 1978; Athanassious, 1980; Vapaavuori et al., 1984; Crang and McQuattie, 1986; Fink, 1989).

Generally, chloroplast changes seem to be non-specific, and may offer little help in determining the cause of damage in needles from declining trees (Fink, 1988). However, such studies may help to identify early damage in needles (without determining the causes), while these still may look green and healthy from the outside (Grill et al., 1989; Barsig et al., 1990). Application of qualitative and quantitative morphometric measurements at the cell and chloroplast levels may help in more detailed analysis, as currently applied to a large-scale project in Finland (Jokela et al., 1992).

Histochemical tests

The structural changes described above represent relatively late stages of damage, since metabolic changes usually start long before they finally become manifest in the form of structural consequences. The search for early indicators of damage should be investigations combining biochemical and structural (cell, tissue) measures such as histochemical tests for the localization and quantification of substances (e.g., starch) and enzyme activities.

Gaseous pollutants and mineral deficiencies can induce similar pathological starch accumulations (Hanson and Stewart, 1970; Fischer and Bussler, 1988; Ebel et al., 1990; Luethy-Krause and Landolt, 1990). However, with gaseous pollutants, the inhibition of carbohydrate translocation from the chloroplasts seems to be inhibited at the level of the single cells, whereas with mineral deficiencies translocation apparently is inhibited at the more central level of the phloem. These changes lead to patchy, heterogeneous distribution of starch grains in the first case and a homogeneous distribution in the second case (Fink, 1992). Thus, the pattern of starch distribution may give more information than a measure of the concentration, although other factors (e.g., insect damage or viruses) may lead to similar patterns in starch accumulation. Starch occurrence in hardened mesophyll cells in late autumn and winter may reflect disturbances in the hardening process due to impact of acid precipitation (Back and Huttunen, 1992).

The distribution of calcium oxalate in conifer needles may be a sensitive indicator for pathogenic influences (Fink, 1991 a, 1991b, 1991c). In the epidermis, minute crystals are normally distributed within the epidermal cell walls and especially in the cuticular layer between the cuticle proper and the cell walls. Under the impact of acid precipitation, this pattern of distribution now becomes altered, and only very small amounts of crystals occur in the cuticular layer and the outer epidermal cell walls, whereas most of them remain restricted to the inner epidermal cell wars. This effect probably reflects "leaching" of Ca²⁺ from the outer cell walls by the impact of acid precipitation, whereas the inner parts of the walls are unaffected by the acidity. Already existing crystals of calcium oxalate are unlikely to become dissolved by the acid; rather the cations are leached out of the apoplast before they are precipitated as oxalate. Physiologically, whether Ca²⁺ is leached away from the apoplast and is lost to the needle or would otherwise become precipitated as insoluble calcium oxalate and thus also disappear physiologically matters little. This observation is consistent with the microscopical detection of crystals of calcium sulphate on the surface of needles subjected to acid rain (Bosch et al., 1983; Nebe et al., 1988; Fiedler et al., 1990; Huttunen et al., 1990).

Ozone alters the localization of the calcium oxalate as well. In the epidermis, numerous small crystals suddenly occur within the lumen of the cell, whereas the cell walls remain free of them and in the mesophyll cells where the crystals usually occur outside the cell walls towards the intercellular spaces, these crystals also grow from cell wall projections into the cells. In some mesophyll cells, free crystals can be found within the vacuoles, although these seem to become dissolved again quite rapidly. Generally, the mesophyll and epidermal cells in conifer needles seem to have a strong tendency to keep excess Ca²⁺ outside in the apoplast and to prevent entrance into the symplast and the vacuole. Under the impact of ozone, however, permeability of the plasma membranes may increase, and calcium may leak into the cells. This effect could provoke defense reactions such as callous deposition, which further can develop into wall ingrowths, or can lead to a breakthrough into the vacuole. Historically, the effects of ozone on mineral nutrition have been attributed to enhanced leaching of minerals and afflux out of the needles; however, recent findings suggest that the main effect may consist in a disturbance of the internal mineral compartmentalization and, at least for calcium, an increased influx from the apoplast into the symplast.

Early accumulation of phenolic compounds in cells close to the stomata have been regarded as possible early indicators for the direct impact of acid rain on conifer needles (Zobel and Nighswander, 1991).

Enzyme activities have been measured by histochemical means in only a few cases so far. However, such approaches might offer quite sophisticated tools for detailed analysis of pathological changes in affected needles or leaves.

9.2.2.4 Conclusions

Compound-specific reactions of target cells hypothetically offer an improved differentiated diagnostic approach over the quantitative analysis of parameters in tissue homogenates. For conifer needles, the main target cells of acid precipitation are those in the epidermis; for gaseous pollutants, the mesophyll cells; and for Mg^- or K^- deficiency, the sieve cells. Histochemical techniques offer promise for the localization of early metabolic changes in affected cells. Nevertheless, additional investigations are needed on the influence of natural factors (e.g., cold shocks, insects, water deficiency) on the structure of tissues under controlled conditions. Neither of the mentioned structural approaches offer a totally secure diagnosis, but always has to be combined with other techniques (e.g., biochemical analysis or nutrient analysis).

9.3 METHODS TO IDENTIFY EFFECTS ON FOREST ECOSYSTEMS 

9.3.1 INTRODUCTION

Measurable effects of chemicals in trees are signs of on-going change, or perhaps even damage, as chemicals transported by air or water are deposited on leaves or needles or enter the soil and are absorbed by roots. They may accumulate in soils or in soil animals or micro-organisms with no immediate effects in trees, but perhaps changes in soil composition or constituents. These changes may be signs of an excessive chemical load and of a hazard to be manifest in the future. To the extent that they confirm assessments made directly on trees or animals, they may be useful in detecting or confirming the chemical load on an ecosystem.

Methods to study the effects of chemicals on forest ecosystems include those of soil solid phase, soil solutions, species composition, and chemical composition of ground vegetation, microfauna, and microflora.

9.3.2 SOIL SOLID PHASE STUDIES

9.3.2.1 Total soil chemical composition

Total element concentrations in soil are easy to determine by acid digestion. In several areas and forest types, concentrations of cadmium, copper, nickel, lead, and zinc have been measured, shown to vary widely according to region and soil layer, and are higher in industrial regions (Southern Norway: Steinnes et al., 1989; Austria, Weiner Wald, beech: Kazda and Glatzel, 1984; Germany, Black Forest: Zottl, 1985; Germany, Solling, spruce: Heinrichs and Mayer, 1980; Germany, Nordrhein Westfalien, beech: Neite, 1989; Germany Hoglwald (Bavaria), spruce: Schierl and Kreutzer, 1991; USA, Vermont, Camel Humps, Boreal forest: Friedland et. al, 1984; Canada, boreal spruce fir forest: Geballe et al., 1990; Canada, Turkey Lake, Ontario, maple and beech: Morrison and Hogan, 1986; USA, New England, Great Smoky Mountains: Turner et al., 1985; USA, North-East, Coniferous forest 1000 m above sea level: Herrick and Friedland, 1990). Such data are of limited utility, because potential effects of chemicals depend on their form and availability. Besides, even the highest concentrations (e.g., in solling area) are too low to cause toxicity. Nonetheless, the measurement of total heavy metal concentrations, particularly in the forest floor where most of these metals accumulate, may be of value. If the concentrations are not of the same order as the highest values, a low risk of toxicity exists; but periodic measurements assess the evolution of concentrations, because heavy metals (especially Pb) accumulate because the rate at which they are drained from the soil is slower than that at which they are deposited.

The total concentrations considered to be without injurious consequences in garden soils (Kloke, 1981) can be used to estimate acceptable levels in forest soils. In mg/kg, these tolerable concentrations are As, 20; B, 25; Be, 10; Br, 10; Cd, 3; Co, 50; Cr, 100; Cu, 100; F, 200; Hg, 2; Mo, 5; Ni, 50; Pb, 100; Sb, 5; Se, 10; Sn, 50; U, 5; V, 50; and Zn, 300. These values take into account risks to human health. If phytotoxicity alone were to be addressed, the following values in mg/kg would be obtained: water soluble B, 3; total Cu, 130; Cu (EDTA), 50; total Ni, 70; exchangeable Ni, 20; total Zn, 300; and exchangeable Zn, 130.

9.3.2.2 Available forms of elements

Several extraction processes allow one to obtain fractions of cadmium, copper, lead, nickel, or zinc in soil that are considered available to trees (ammonium acetate, acetic acid, NH₄CI, EDT A) (Austria, Wiener Wald-beech: Kazda and Glatzel, 1984; several European beech forests: Wittig and Neite, 1989; Germany, Nordhein, Westfalien, beech forests: Neite, 1989). Such values are more useful than total concentrations, as they can be compared to quantities experimentally added to soil samples where seedlings are grown. Toxic concentrations (mg/kg) of several heavy metals have been reported for trees (Kahle et al., 1989; Geballe et al., 1990; Seiler and Paganelli, 1987; Chappelka et al., 1991; Kristodorov et al., 1989; Smith and Brennan, 1984; Denny and Wilkins, 1987; Brown and Wilkins, 1985; Burton et al., 1984; Patterson and Olson, 1983; Nakos, 1979).

Some of these toxicity values are probably incorrect. For instance, when seedlings are grown in sand, the determined toxic value is probably lower than when grown in soil, because sand has a lower exchange capacity; thus the solution in equilibrium with sand is more concentrated than soil solution in the presence of clay and organic matter. Furthermore, only results with mycorrhizal seedlings are valid, because fungi are a barrier against toxic element uptake. Moreover, most of the published results of experiments in soil samples suppose that the whole quantity of a toxic metal added in the form of salt (mainly chloride) remains in available form, which is incorrect most of the time. Such experiments should be completed by the analysis of the actual available contents of toxic metals at the end of the experiment, as done by Burton et al. (1986); otherwise estimated toxic levels may be higher than the actual ones.

Regrettably, research efforts have focused predominantly on cadmium and lead. The toxicity levels of each vary according to species and authors. The toxic level of lead is in the area of 50-300 mg/kg; toxicity of cadmium begins at 0.3-5 mg/kg. In some areas, present levels of available lead may be toxic to beech seedlings since they are higher than 55 mg/kg, the value which was found toxic by Kahle et al. (1989). No data have been reported for nickel, copper, and zinc.

For aluminium, Devevre (1990) found that the yellowing of young Norway spruce seedlings, the death of roots, and poor mycorrhization occurred in French and German soils containing more than 80 meq/kg (720 mg/kg) Al extracted by NH₄Cl. However, different salts used in extracting exchangeable Al give different results; KCl and NH₄Cl generally extract more Al than does BaCl₂. Also, the level of exchangeable Al must be assessed according to the exchangeable Ca and Mg contents. Ca/Al and Mg/Al ratios of 0.05 and 0.02, respectively, seem to be threshold values below which adult coniferous trees such as Picea abies or Abies pectinate may suffer from aluminium toxicity (Mohamed, 1992; Bonneau et al., 1992). The corresponding values for young spruce plantations, where roots are less developed, are 0.15 and 0.04 (Bonneau et al., 1992).

The heterogeneous distribution of heavy metals in a forest stand must be ascertained according to the distance of the sampling point from the tree stems, since heavy metals are leached from tree crowns and concentrated in the vicinity of the trunks (Kazda and Glatzel, 1984).

9.3.3 SOIL SOLUTION STUDIES

Concentrations of cadmium, copper, nickel, lead, and zinc in soil solution have been measured (mg/L) and reported (Germany, SoIling: Godbold and Huttermann, 1985; USA, Turkey Lake, drainage water: Foster and Nicolson, 1986; USA, Northern Vermont, drainage water: Friedland and Johnson, 1985). However, some studies were conducted on freely drained water. Heavy metal concentrations should be determined in micropore water extracted by centrifugation or by displacement, i.e., applying a water flux at low pressure on a soil column, because tree uptake occurs mainly from this water that is representative of risks of injury .Other works have attempted to determine levels of heavy metals corresponding to toxicity thresholds (Godbold and Huttermann, 1985; Burton et al., 1986; Chappelka et al., 1991; Lozano and Morrison, 1982; Clarke and Brennan, 1980; Russo and Brennan, 1979; Mitchell and Fretz, 1977; Smith and Brennan, 1984). Results are often incomparable, because many were obtained by adding nutrient solutions with varied heavy metal concentrations to plants grown in soils or with non-mycorrhizal seedlings. Realistic toxicity thresholds can only be obtained from experiments where nutrition solutions are applied to sand cultures. Toxic element absorption by soil colloids and the reduction of their concentration in the solution can hence been avoided. It is also necessary to experiment with mycorrhizal seedlings as indicated for available elements. Threshold levels greatly vary according to tree species and to elemental interactions; for instance Pb toxicity is significantly increased by the addition of 2 ppm Cd (Kahle et al., 1989). Toxicity thresholds are of the order of 0.5-5 mg/L Cd, 10-100 mg/L Cu, 10-50 mg/L Ni, more than 200 mg/L Pb and 0.4-2 mg/L Zn. Such levels are far from being reached in soil solutions for Cd, Cu, and Pb, but might be reached for Zn.

For aluminium, toxicity levels seem to be controversial, and vary according to tree species. In species well adapted to acid soils, such as Picea abies, root growth is hampered only by high concentrations: 800-1200 micromoles/L (27 mg/L), while Ca uptake is depressed by lower concentrations (100 micromoles/L, i.e. 2.7 mg/L). Sugar maple is much more sensitive: its growth is depressed by about 3 mg/L; such concentrations are rare in forest soils. In coniferous forests in the Vosges, a concentration of 4 mg/L was rarely found in drainage water (Gras et al., 1989). Under usual conditions, the concentrations are in the order of 1-3 mg/L in drainage and in micropore water (extracted by the displacement technique). As for the exchangeable elements, the Ca:Al and Mg:Al ratios should be taken into account. The value of Ca:Al equivalent ratios corresponding to toxicity varies from 0.06-0.12 in very tolerant species (Fagus sylvatica, Pseudotsuga menziesii) to 0.6 in rather tolerant species (Picea abies) (1 in molar ratios) (Ulrich, 1984). An Mg:Al equivalent ratio of 0.7 in micropore water in the Vosges corresponds to a very healthy spruce stand, while values of 0.3-0.5 correspond to yellow, Mg- deficient stands (Mohamed, 1992; Ranger et al., 1993).

When comparing metal soil solution concentrations to toxicity thresholds, only ionic forms of metals are toxic, while metals complexed in organic molecules are not. The extracted soil solution must be percolated through exchange resins to assess toxicity (Schierl, 1989). The composition of soil water may be indicative of tree sensitivity to salts: very sensitive species do not tolerate more than 2 g/L NaCl, the resistant ones up to 9 g/L.

The following conclusions can be drawn from this information:

1. Aluminium's toxicity, which differs by tree species, can be assessed by determining exchangeable Al concentrations and Ca:Al and Mg:Al ratios of exchangeable elements, or Ca:Al and Mg:Al ratios in the micropore water; and only the ionic forms of Al must be considered.

2. To assess heavy metal toxicity, the concentrations of exchangeable metals in the soil can be measured and compared to toxicity levels determined in appropriate experiments, in which different quantities of heavy metal salts are added to soil samples containing seedlings supplied with mycorrhizae, and which are incubated several days. Exchangeable concentrations are then measured at the end of the experiment.

3. To assess heavy metal toxicity, the concentrations of ionic forms of heavy metals must be measured in soil micropore water in an adequate season (end of the rainy season) and compared to toxicity levels. These toxicity levels have to be determined by percolating a sand culture (of mycorrhizal seedlings) with nutrient solutions containing different concentrations of heavy metals.

9.3.4 ELEMENT CYCLING STUDIES

Soil composition data reflect a static measurement of potential toxicity. Estimating dynamic aspects of the processes and evolution of risks in the future is not possible. The stress effects of nitrogen mineral forms (NO₃^- and NH₄⁺) cannot be investigated by static soil studies, since they are very variable with time, and do not accumulate in the system, (particularly NO₃^-). Thus, knowledge of the cycling of several elements (mainly protons and nitrogen, but also heavy metals) is essential to obtain valid views on the ecosystem function.

9.3.4.1 Protons

Proton cycling can be established only indirectly. The first difficulty is input determination, because of the interference of the forest canopy. Acidic dust or aerosols are deposited on forest leaves or needles and leached by the rain. Simultaneously, acidity is exchanged on the canopy with base cations (Ca, Mg, K) from the inside of the foliar tissues ("recretion"). Thus the total acidity input must be measured in the throughfall and is the sum of the H⁺ and of the recreted base cations (Ca, Mg, K). The excess of the flux of the base cations in the throughfall is the sum of wet, dry , and occult deposition and of the recreted base cations (Ca, Mg, K). Thus for an exact determination of acidity, a correct estimation would be required either of dry and occult deposition or of recreted basic cations. Presently, a method to estimate canopy exchanges as a function of rainfall quantity and composition does not exist. Several methods of estimating dry and occult deposition of cations exist (Lovett and Lindberg, 1984; Mayer and Ulrich, 1974, 1978), but none is totally satisfactory. Thus, the acidity added to the ecosystem cannot be estimated exactly.

Global effects of the acidity flux in the soil may be estimated from composition of the drainage water and the fluxes of elements at the base of the soil. If only basic cations (Na, Ca, Mg, K) were in the drainage water, the soil is able to buffer acidity input by weathering or exchange processes. In these conditions, the ecosystem may be considered to be stable, but its "acid neutralizing capacity" (ANC) is consumed little by little, and risks of acidification and release of aluminium or heavy metals may arise later (Van Breemen et al., 1984). If the drainage water contains high quantities of H⁺ and Al³⁺ (only in the case of acid soils), the soil is subjected to excessive stress; the acid input is too high compared to the quantity of exchangeable cations and to quantities and weathering rates of the soil minerals; the uptake of nutrients is difficult, and the toxicity of aluminium and heavy metals may ensue.

A dynamic estimate of the acid stress may be achieved by models that calculate the critical load for the ecosystem. The "critical load" of a soil is defined as the flux of acidity acceptable without ionic aluminum being in too high a concentration in the drainage water. Several models exist that take into account quantity, nature, size, and weathering rates of minerals in the soil, the quantity of exchangeable cations and the rate of their exchangeability against protons, and the potential of hydroxides for fixing SO₄^2- anions, a process that consumes protons (Sverdrup et al., 1988; Hettelingh and de Vries, 1991; Henriksen et al., 1990; Hettelingh et al., 1991). Comparison of the total acidity to which a soil is submitted with a calculated critical load may give a fairly accurate idea of the acidity stress, but to calculate the total acidity to which a soil is submitted, the external proton sources (estimated in the throughfall as indicated above) and the internal sources (uptake of ions by roots and acidity coming from litterfall, humus, root, and micro-organism decomposition and from nitrogen cycling) must be added (Van Breemen et al., 1984).

The total acidity to which a soil is submitted may also be estimated as the sum of the total output of basic cations in excess of the input, plus lack of SO₄^2- in comparison with SO₄^2- input, (if drainage of SO₄^2- is higher than input this difference must be put in the proton sources), plus the protons and aluminum in the drainage water plus the difference (C⁺-A^-) of the cations and anions immobilized annually in wood. This sum is called "the proton sinks." Comparing all the proton sinks and all the proton sources estimated as above is called the "proton balance." This balance, theoretically equalling zero, allows verification of the estimate of the proton sources and more accurate comparisons between the estimated critical load and the total proton sources.

9.3.4.2 Nitrogen

When the nitrogen supply to forest trees is in excess, mineral nitrogen is a stress factor. Independent of the fact that NO₃^- anions are often distributed to the forest ecosystems together with a proton, an excessive N uptake creates an imbalance in nutrient composition, and induces deficiencies in P, Ca, and Mg (Roelofs and Van Dijk, 1986; Nys, 1989) that may be detected easily by leaf or needle analysis. When N is brought in the form of NH₄⁺, its nitrification (that may be very active even in acid soils) supplies protons to soil and increases the acidification rate. The proton sources linked to nitrogen cycling may be calculated from input and output fluxes of NH₄⁺ and NO₃^- using the formula (Van Breemen et al., 1984):

(NH₃^- output  - NH₃^-  input) - (NH₄⁺ output - NH₄⁺  input)

Nitrogen cycling specialists hypothesize that, in a sound forest ecosystem, nearly no nitrogen loss occurs in the drainage water (Schulze, 1987). When this latter contains fairly large quantities of nitrogen, namely in the form of nitrate, stress from an excessive nitrogen supply or from other factors that depress stand health and reduce N uptake below the normal value takes place. In addition, such a high N drainage may cause problems in ground or surface water.

Comparing N output to its permits judgement as to the origin of the excess supply of N. N input of 10 to 15 kg/ha-yr and N drainage of about 10 kg/ha-yr may be considered normal. An N drainage much lower than that input means that N brought into the ecosystem from outside is consumed by trees, plants, or micro-organisms. When N drainage is as high as, or higher than, N input, the ecosystem is called "nitrogen saturated." When N drainage is higher than its input, the quantity of N mineralised in the soil is higher than uptake by trees or other living organisms. The causes of such a situation may be an unhealthy state for the forest, leading to restricted uptake or, if the stand is healthy, mineralization in the soil of excess organic nitrogen coming from an accumulation in a previous ecosystem. Feger et al. (1992) give examples in the Black Forest, Germany, of excessive organic nitrogen mineralization in a spruce stand (Schluchsee) following a beech forest of which the root system was deeper, and of an unsaturated spruce ecosystem (Viflingen) where the total N input is consumed¾a phenomenon that tends to compensate for a previous excess of N exportation by litter grubbing. Nys (1987, 1989) gives an example of a spruce stand in the French Ardennes where an excessive N drainage is linked to high N input and where the spruce stand suffers severe Mg and Ca deficiencies and severe defoliation.

9.3.4.3 Heavy metals

The drainage and input may also be compared for heavy metals. In most current forest conditions, inputs exceed outputs, mainly for Pb which is absorbed strongly in soils. Zottl (1985) has shown the input and drainage of several heavy metals in the Black Forest in Germany. Such comparisons demonstrate the rate of accumulation of these metals in forest soils and the risk of a short-term excess. For instance, Friedland and Johnson (1985) think that Pb quantities may be doubled in 40 years in the forest soils of Vermont.

The input-output balances of acidity, nitrogen, or heavy metals can be made at soil or at watershed level. If the functioning and the health of the forest ecosystem is considered, the soil level is the only valid one. If the impact of forest condition on aquatic ecosystems is paramount, the watershed level has to be taken into account together with the soil level, because many phenomena may occur in subsoil layers: nitrate denitrification, weathering of minerals in the regolith, and the resaturation of water by basic cations together with the precipitation of aluminum and heavy metals.

9.3.5 FLORA, FAUNA, MICROFLORA SPECIES AND THEIR COMPOSITION 

9.3.5.1 Species composition

Species composition may indicate the stresses that a forest ecosystem is undergoing. For instance, an abundance of N-liking species in the ground flora of an acid forest ecosystem indicates an excess of nitrogen and often of nitrates. Presently, such a situation is often described in a European forest (Becker et al., 1992; Thimonier et al., 1992). This abundance may be a result of tree defoliation that permits more light and heat to reach the soil surface and that increases the litter mineralization rate. Abundance of acidophilous species, absence of lumbricidae, abundance of collembolas and acatridae are normal in acid forests, even in a good state of health; it is difficult to interpret this as an acidification stress. Generally, the identification of stress by species composition is very uncertain, and such methods are more relevant for comparing the evolution of ecosystems over years and decades.

9.3.5.2 Chemical composition

Chemical composition of plant or animal species might be used to detect a high availability of heavy metals in the upper layers of soils. Mapping of heavy metal concentrations in the mosses Hylocomium spendens and Pleurozium Schreberi is being carried out in Europe under the supervision of Swedish researchers. High levels of metals in these mosses can localize areas with high deposition rates, but do not indicate whether the concentration of heavy metals really is a current stress agent for those ecosystems. A similar method has been used by other researchers to analyze meso- and microfauna (Zottl and Lamparski, 1981; Roth-Holzapfel et al. , 1992), but great attention must be paid to the choice of animal groups to be analyzed, since some are far more able than others to accumulate heavy metals, and the variability within species is high (Roth-Holzapfel et al., 1992). Much advance in understanding is needed before the determination of the chemical composition of microfauna species can be used for detecting real heavy metal stress.

9.3.6 CONCLUSION

Many different ways exist to detect the effects of chemicals on forest ecosystems. At the present state of knowledge, the most efficient and simple ones seem to be analyses of exchangeable heavy metals and aluminium, soil solution analyses and cycling studies, eventually in connection with the determination of the critical loads of acid input.

9.4 MONITORING ECOSYSTEM CONDITIONS

9.4.1 INTRODUCTION

All of the investigations indicated above provide information on the state of an ecosystem at a defined time; they are difficult to interpret in terms of ecosystem evolution. Monitoring characteristics of a forest stand and properties of soil or flora and fauna composition together at defined time intervals provide opportunities to improve knowledge of toxicity thresholds and of the effects of chemicals on the long-term evolution of forest ecosystems. Many countries in the world have set up permanent assessment plots since forest decline appeared in the 1980s. Roughly three kinds of plots exist according to the intensity of monitoring.

9.4.2 THE LOWEST INTENSITY

At the lowest intensity, systematic assessment networks like the 16 x 16 km ECC network exist. In the corresponding plots, there are only annual assessments of stand health, simple analyses of soil upper horizons about every tenth year: texture, pH (water and KCl or CaCl₂), cation exchange capacity, exchangeable base cations, exchangeable Al and Mn at soil pH, total C and N. Determination of the concentration of the major elements in needles or leaves is foreseen every fifth year by sampling at least five trees and analysing a composite sample.

The aim of such networks is to monitor the evolution of stand health and to detect possible relationships with the state and changes in the main soil properties and in mineral nutrition. This type of network has many major drawbacks. The position of the plots is systematic, and thus they are not very representative of the regional forest types; in addition, the stands are of any age and health state. Furthermore, the analysis of only a composite soil sample does not allow any statistical comparison of properties to be made between dates.

9.4.3 INTERMEDIATE-INTENSITY OBSERVATION PLOTS

Intermediate-intensity observation plots, generally less numerous than low-intensity plots, are chosen in order to be highly representative of the regional forest types. In addition, they have to be of adult age to be observed over several decades without being young or senile, i.e., without risk of confusing age change and stress effects. Each observation plot (about 0.5 ha) must be very homogeneous concerning soil and stand characteristics.

Soil must be sampled at a minimum of twenty or twenty-five locations, and at each point with more detail than in the low-intensity network. For instance, the organic horizons have to be taken up from a defined area (about 0.1 m² and weighed. Weight increase over the observation period may be a sign of a decrease in the mineralization rate of the litterfall. Below the organic horizons, A1 horizons and two or three mineral horizons must be sampled. Sampling must be carried out at defined depths to compensate for operator differences. The soil horizons must be collected at a minimum of 25 points, and sampling points must be defined according to a precise geometric configuration. A defined extrapolation must be anticipated from the initial points to other locations to avoid taking a second sample in a previously perturbed location. Each sample may be analysed individually, or a composite of five samples from five locations may be assembled. These precautions permit valid statistical comparisons of soil properties among sampling dates. To avoid site waste and because soil properties change very slowly, the time interval between two successive sampling times should be at least ten years. The flora beneath the stand has to be inventoried at the beginning of the work and every tenth year; this causes disturbances, and must be made only in a separate plot.

In these intermediate intensity plots, climatic data (temperature and precipitation) must be collected daily to distinguish climatic stresses from chemical ones.

In addition to observations of defoliation and colour changes, several other stand characteristics must be noted every few years (e.g., circumference) or yearly (e.g., date of bud burst and date of leaf fall). The determination of litterfall quantity is of great interest in deciduous stands as an index of primary production. In evergreen stands, interpreting litterfall is difficult, because it is a function of yearly foliage production together with duration of persistence.

Ideally, needle or leaf analysis should be performed yearly to permit detection of long-term trends. Foliage composition is submitted to annual fluctuations with the result that analyses at five-year intervals might mask long-term trends. Since yearly foliage sampling and analysis are somewhat expensive, the frequency of these operations requires professional judgement. If foliage is sampled yearly, damage to the tree crown must be prevented by previewing two sets of ten trees to be sampled alternatively every second year, or by sampling in an additional plot around the observation plot. The same precaution must be taken to set up water collection devices in high-intensity plots.

9.4.4 HIGH-INTENSITY OBSERVATION PLOTS

In these high intensity plots, the following are determinations to be made:

1. Collection of precipitation in an open field, as near as possible to the observation plot, and measurement of pH, NH₄^-, NO₃^-, SO₄^2-, Cl^-, Na⁺, and CO₃H^-.

2. Collection of throughfall inside (or around, if it is a large homogeneous site) the plot and analysis of the same elements as in precipitation with the possible addition of Al and heavy metals. Throughfall must be collected in a manner representative of the conditions throughout the whole plot (i.e., below-crown and between-crown areas);

3. Collection of drainage water at the base of the soil at three points inside or around the plot and analysis of the same elements as in the throughfall with the possible addition of SO₂, O₃, and NO_x in air in or around these plots.

In France a national systematic observation network (16 km x 1 km) and the ECC systematic network (16 km x 16 km) are already functioning. A network of a hundred intermediate and high intensity observation plots, of which 25 are high intensity plots (only 15 of which have drainage water collection), is being set up. Such networks of permanent monitoring plots are very expensive; therefore, their installation must proceed with particular care.

9.5 CHEMICAL STRESSES IN FORESTS TODAY AND IN THE FUTURE

Some chemical stresses are tightly localized. An illustration is salt stress: NaCl near the highways in temperate or northern countries. After many studies of forest decline in temperate areas, direct effects of SO₂ in its gaseous form have been rather rare and localised in regions where many factories are burning coal with a high S-content without a smoke scrubbing system. Some damage by NH₃ or HF are known in areas around large chemical industries. In developed countries such chemical effects are less common after the installation of filtering systems.

The most widespread chemical stresses are acidification in temperate and tropical forests, in the former as a consequence of pollution, and in the latter as a result of the strong weathering of soil materials, with the consequent elimination of most basic cations.

Acidification of forest soils is widespread because of HNO₃ and H₂SO₄ input. While H₂SO₄ pollution is decreasing in developed countries as a result of such countervailing measures as burning low S-content fuels, adding scrubbing systems, or changing to nuclear power, HNO₃ increases with expanding automobile traffic. The total acid pollution remains reasonably constant. In several forest areas with poor parent rocks and soils, damage to trees is clear and severe (defoliation, Mg and Ca deficiencies). Other effects are still more severe in water downstream or in lakes where aquatic life is disappearing due to high aluminium concentrations. Damage due to acidification is fortunately easy to stop by spreading limestone or dolomitic limestone. Many experiments have demonstrated that such fertilization improves forest health and drainage water quality, but soil pH is increased only in the upper soil horizons, and risks are present of increasing the nitrate concentration in drainage water in temperate forests where the N organic content is generally high. However, fertilization is very expensive, because, in many areas, the helicopter is the only way of spreading fertilizers and, therefore, is very costly to apply in developing countries.

Effects of ozone are still rather low except in particular regions (California), where a combination of high quantities of emitted NO_x from auto traffic and a sunny climate favours ozone synthesis. In temperate areas, this effect is equivocal. Claustres (1992) in an open top chamber experiment in the South-West of France found a 25 percent depression in Picea abies by a summer ozone content of air of 90-100 µg/m³. Many Mediterranean regions, with climate conditions like in California, have not been well investigated for this effect; in these regions, increased auto traffic eventually may increase ozone stress.

Except for narrow areas near industrial regions, heavy-metals are not yet a problem for tree health. However, as many heavy metals are retained in the soil and acidification favours the liberation of certain of these metals (Cd, Zn) in soil solution, this risk is increasing gradually (Kahle and Breckle, 1992). Liming forest soils may diminish the free ion concentration for several metals, but increased organic complexation may increase the concentration of others (Cu, Pb) in drainage water, and contribute to the load in the waters downstream. In addition to the health of forest trees, heavy metals, particularly Cd, Pb, and Hg in wild fruits and mushrooms, raise concerns for human health. These metals may become increasingly important in some forests with increasing acidification and spreading of sewage sludge.

With the progressive reduction of SO₂ emissions in developed countries and the possibility of liming the most acidified forests, the major problems for these countries eventually will become those effects produced by heavy metals and ozone (the latter, mainly in Mediterranean regions), while in tropical areas of developing countries, soil acidification with aluminum toxicity and with difficulty in Ca, Mg and K nutrition may eventually alter forest production along with the many other threats that exist in the forests of these regions.

9.6 REFERENCES

Adams, C.M., Dengler, N.G., and Hutchinson, T.C. (1984) Acid rain effects on foliar histology of Artendsia tilesii. Can. J. Bot. 62, 463-474.

Athanassious, R., Klyne, M.A., and Phan, C.T. (1978) Ozone effects on radish (Raphanus sativus L. cv. Cherry Belle): morphological and cellular damage. Z. Pflanzenphysiol. 90, 183-187.

Athanassious, R. (1980) Ozone effects on radish (Raphanus sativus L. cv. Cherry Belle): gradient of ultrastructural changes. Z Pflanzenphysiol. 97, 227-232.

Back, J., and Huttunen, S. (1992) Structural responses of needles of conifer seedlings to acid rain treatment. New Phytol. 120, 77-88.

Badot, P.M., Garrec, J.P., Millet, B., Badot, M.J., and Mercier, J. (1988) Deperissement et etat hydrique des aiguifles chez le Picea abies. Can. J. Bot. 66, 1693-1701.

Barsig, M., Endler, W., Weese, G., and Hafner, L. (1990) Cytomorphologische Untersuchungen an Nadeln von Kieferm (Pinus silvestris L.) eines Ballungsgebietes. II. Das Spektrum feinstruktureller Schadphanomene. Angew. Bot. 64,303-315.

Becker, M., Bonneau, M., and Le Tacon, F. (1992) Long-term vegetation changes in an Abies alba forest: natural development compared with the response to fertilization. J. Veg. Sci. 3, 467-474.

Black, V.J. (1985) SO₂ effects on stomatal behavious. In: Winner, W.E., Mooney, H.A., and Goldstein, R.A. (Eds.) Sulfur Dioxide and Vegetation: Physiology, Ecology, and Policy Issues, pp. 96-117. Stanford University Press, Palo Alto, California.

Bonneau, M., Landmann, G., and Adrian, M. (1992) La fertilisation comme remede au deperissement des forets en sol acide: essais dans les Vosges. Rev. For. Fr. 44(3), 207-223.

Bosch, C., Pfannkuch, E., Baum, U., and Rehfuess, K.E. (1983) Uber die Erkrankung der Fichte (Picea abies Karst.) in den Hochlagen des Bayerischen Waldes. Forstw. Centralbl. 102, 167-181.

Brennan, E. (1988) Winter spot: a common symptom on coniferous evergreens in New Jersey. Shade Tree 61, 44-45.

Brown, M.T., and Wilkins, D.A. (1985) Zinc tolerance of mycorrhizal Betula. New Phytol. 99, 101-106.

Bucher, J.B., and Landolt, W. (1985) Zur Diagnose von Ozonsymptomen auf Waldbaumen. Schweiz. Z Forstwes. 136, 863-865.

Burton, K.W., Morgan, E., and Roig, A. (1984) The influence of heavy metals upon the growth of Sitka spruce in South Wales forests. Plant and Soil 78, 271-282.

Burton, K.W., Morgan, E., and Roig, A. (1986) Interactive effects of cadmium, copper and nickel on the growth of Sitka spruce and studies of metal uptake from nutrient solutions. New Phytol. 103, 537-549.

Bytnerowicz, A., and Grulke, N.E. (1992) Physiological effects of air pollutants on western trees. In: Olson, R.K., Binkley, D., and Bohm, M. (Eds.) The Response of Western Forests to Air Pollution, pp. 183-232. Springer-Verlag, New York.

Cape, J.N., Paterson, I.S., Wellburn, A.R., Wolfenden, J., Mehlhorn, H., Freer-Smith, P.H., and Fink, S. (1988) Early Diagnosis of Forest Decline. Institute of Terr. Ecology, Edinburgh, 68 pp.

Carlson, C.E., and Gilligan, C.J. (1983) Histological Differentiation among Abiotic Causes of Conifer Needle Necrosis. USFS Research Paper No. INT -298. US Department of Agriculture, Forest Service, Washington, D.C., 17 pp.

Castillo, F., and Greppin, H. (1988) Extracellular ascorbic acid and enzyme activities related to ascorbic acid metabolism in Sedum album L. leaves after ozone exposure. Environ. Exp. Bot. 28, 231-238.

Castillo, F., Miller, P., and Greppin, H. (1987) Waldsterben: Extracellular biochemical markers of photochemical oxidant air pollution damage to Norway spruce. Experiential 43, 111-115.

Chabot, J.F., and Chabot, B.F. (1975) Developmental and seasonal patterns of mesophyll ultrastructure in Abies balsamea. Can J. Bot. 53, 295-304.

Chakrabarti, K., Rennenberg, H., and Polle, A. (1991) Interaction of plants and atmospheric hydrogen peroxide. In: Borren, P., Borrell, P.M., and Seiler, W. (Eds.) Proceedings of EUROTRAC Symposium, 1990, pp. 119-121. SPB Acad. Publ., The Hague.

Chappelka, A.H., Kush, J.S., Runion, G.B., Meier, S., and Kelley, W.D. (1991) Effects of soil-applied lead on seedling growth and ectomycorrhizal colonisation of Loblolly Pine. Environ. Pollut. 72, 307-316.

Clarke, B.B., and Brennan, E. (1980) Evidence for a cadmium and ozone interaction on Populus tremuloides. J. Arboric. 6, 130-134.

Claustres, J.P. (1992) Influence de la Pollution Atmospherique sur les Echanges Gazeux des Epiceas. Resultats d'une Nouvelle Methodologie. These de Doctorat, Universite de Nancy I, Specialite "Biologie Vegetale et Forestiere," 96 pp.

Costonis, A.C. (1970) Acute foliar injury of Eastern White Pine induced by sulfur dioxide and ozone. Phytopathology 60, 994-999.

Costonis, A.C., and Sinclair, W.A. (1969) Relationship of atmospheric ozone to needle blight of Eastern White Pine. Phytopathology 59, 1566-1574.

Cram, W.J. (1990) Uptake and transport of sulfate. In: Rennenberg, H., Brunold, C., DeKok, L.J., and Stulen, I. (Eds.) Sulfur Nutrition and Sulfur Assimilation in Higher Plants. Fundamental, Environmental and Agricultural Aspects, pp. 3-11. SPB Acad. Publ., The Hague.

Crang, R.E., and McQuattie, C.J. (1986) Qualitative and quantitative effects of acid misting and two air pollutants on fohar structures of Liriodendron tulipifera. Can. J. Bot. 64, 1237-1243.

Crossley, A., and Fowler, D. (1986) The weathering of Scots pine epicuticular wax in polluted and clean air. New Phytol. 103, 207-218.

Cunninghame, M.E., Hillman, J.R., and Bowes, B.G. (1982) Ultrastructural changes in mesophyl cells of Larix decidua x kaempferi during leaf maturing and senescence. Flora 172, 161-172.

D' Agostino, P.S. (1981) An ultrastructural study of the senescence induced in Gonmphrena globosa leaves by mineral deficiency. J. Submicrosc. Cytol. 13, 373-384.

Daniker, A. (1923) Biologische Studien uber Baum- und Waldgrenze. Vierteljahrschr. Naturforsch. Ges. Zurich 68, 1-102.

DeKok, L.J. (1990) Sulfur metabolism in plants exposed to atmospheric sulfur. In: Rennenberg, H., Brunold, Ch., DeKok, L.J., and Stulen, I. (Eds.) Sulfur Nutrition and Sulfur Assimilation in Higher Plants. Fundamental, Environmental and Agricultural Aspects, pp. 111-130. SPB Acad. Publ., The Hague.

Denny, H.J., and Wilkins, D.A. (1987) Zinc tolerance in Betula spp. I: effect of external concentrations of zinc on growth and uptake. New Phytol. 106, 517-524.

Devevre, O. (1990) Mise en Evidence Experimentale d'une Microflore Rhizosphe-Rique Deletere Associee au Depdrissement de I'Epicda en France et en Allemagne. DEA de Biologic Forestiere. Universite de Nancy, I/INRA, Dijon, France, 65 pp.

Dodge, J.D. (1970) Changes in chloroplast fine structure during the autumnal senescence of Betula leaves. Ann. Bot. N.S. 34, 817-824.

Ebel, B., Rosenkranz, J., Schiff gens, A., and Liitz, C. (1990) Cytological observations on spruce needles after prolonged treatment with ozone and acid mist. Environ. Poll. 64, 323-335.

Elling, W., Huber, S.J., Bankstahl, B., and Hock, B. (1988) Atmospheric transport of atrazine: a simple device for its detection. Environ. Pollut. 48, 77-82.

Ernst, D., Schraudner, M., Langebartels, C., Sanderrnann, H., Jr (1992) Ozone-induced changes of mRNA levels of b-1,3-glucanase, chitinase and "pathogen-related" protein lb in tobacco plants. Plant Molec. Biol. 20, 673-682.

Evans, L.S. (1984) Botanical aspects of acidic precipitation. Bot. Rev. 50, 449-490.

Evans, L.S., and Curry, T.M. (1979) Differential responses of plant foliage to simulated acid rain. Am. J. Bot. 66, 953-962.

Evans, L.S., and Leonard, M.R. (1991) Histological determination of ozone injury symptoms of primary needles of giant sequoia (Sequoiadendron giganteum Bucch.). New Phytol. 117, 557-564.

Evans, L.S., and Miller, P.R. (1972) ozone damage to ponderosa pine: a histological and histochemical appraisal. Am. J. Bot. 59, 297-304.

Evans, L.S., and Miller, P.R. (1975) Histological comparison of single and additive O₃ and SO₃ injuries to elongating pine needles. Am. J. Bot. 62, 416-421.

Evans, L.S., Gmur, N.F., and DaCosta, F. (1978) Foliar response of six clones of hybrid poplar. Phytopathology 68, 847-856.

Feger, K.H., Brahmer, G., and Zottl, H.W. (1992) Projekt ARINUS. VI. Stickstoffumsatz und Auswirkungen der experimentellen Ammonsulfatgabe, pp. 199-211. 8 Status Cofloquium des PEF. Kernforschungszentrum Karlsruhe.

Fiedler, H.J., Baronius, G., and Ehrig F. (1990) Rasterelektronenmikroskopische Untersuchungen gruner und chlorotischer Nadeln eines immissionsgeschaldigten Kiefernbestandes. Flora 184, 91-101.

Fink, S. (1983) Histologische und histochemische Untersuchungen an Nadeln erkrankter Tannen und Fichten im Sudschwarzwald. Allg. Forstz. 38, 660-663.

Fink, S. (1988) Histological and cytological changes caused by air pollutants and other abiotic factors. In: SchulteHostede, S., Darrall, N.M., Blank, L.W., and Wellburn, A.R. (Eds.) Air Pollution and Plant Metabolism, pp. 36-54. Elsevier, London, New York.

Fink, S. (1989) Pathological anatomy of conifer needles subjected to gaseous pollutants or mineral deficiencies. Aquilo Ser. Bot. 27, 1-6.

Fink, S. (1990a) Structural changes in conifer needles due to Mg and K deficiency. Fertil. Res. 27, 23-27.

Fink, S. (1990b) Todliche Saureflecken auf Fichtennadeln? Allg. Forstz. 45, 989-992. 

Fink, S. (1991a) The micromorphological distribution of bound calcium in the needles of Norway spruce, Picea abies (L.) Karst. New Phytol. 119, 33-40.

Fink, S. (199lb) Unusual patterns in the distribution of calcium oxalate in spruce needles and their possible relationships to the impact of pollutants. New Phytol. 119, 41-51.

Fink, S. (199lc) Comparative microscopical studies on the patterns of calcium oxalate distribution in the needles of various conifer species. Bot. Acta 104, 306-315.

Fischer, E.S., and Bussler, W. (1988) Effects of magnesium deficiency on carbohydrates in Phaseolus vulgaris. Z. Pflanzenernahr. Bodenk. 151, 295-298.

Forschner, W., Schmitt, V., and Wild, A. (1989) Investigations on the starch content and ultrastructure of spruce needles relative to the occurrence of novel forest decline. Bot. Acta 102, 208-221.

Foster, N.W., and Nicolson, J.A. (1986) Trace elements in the hydrologic cycle of a tolerant hardwood forest. Water Air Soil Pollut. 31, 501-508.

Friedland, A.J., and Johnson, A.H. (1985) Lead distribution and fluxes in a high elevation forest in northern Vermont. J. Environ. Qual. 14, 332-336.

Friedland, A.J., Johnson, A.H., Siccama, T.G., and Mader, D.L. (1984) Trace metals profiles in the forest floor of New England. Soil Sci. Soc. Am. J. 48, 422-425.

Gaggi, C., and Bacci, E. (1985) Accumulation of chlorinated hydrocarbon vapour in pine needles. Chemosphere 14, 451-456.

Garsed, S.G. (1985) SO₂ uptake and transport. In: Winner, W.E., Mooney, H.A., and Goldstein, R.A. (Eds.) Sulfur Dioxide and Vegetation. Physiology, Ecology and Policy Issues, pp. 75-95. Stanford University Press, Palo Alto, California.

Geballe, G.T., Smith, W.H., and Wargo, P.H. (1990) Red spruce seedling health: an assessment of acid fog deposition and heavy metal soil contamination as interactive stress factors. Can. J. For. Res. 20, 1680-1683.

Godbold, D.L., and Huttermann, A. (1985) Effect of zinc, cadmium and mercury on root elongation of Picea abies (Karst.) seedlings, and the significance of these metals to forest dieback. Environ. Pollut. 38, 375-381.

Gras, F., Boudot, J.P., Becquer, T., Merlet, D., and Rouiller, T. (1989) Acidification et liberation de L' aluminium dans un sol forestier vosgien, role de la nitrification et des apports atmospheriques de soufre. Journées de Travail DEFORPA (INRA, Nancy) 4, 841-848.

Grill, D., Pfeifhofer, H., Halbwachs, G., and Waltinger, H. (1987) Investigations on epicuticular waxes of differently damaged spruce needles. Eur. J. For. Pathol. 17, 246-255.

Grill, D., Guttenberger, H., Zellnik, G., and Bermadinger, E. (1989) Reactions of plant cells on air pollution: Phyton 29, 277-290.

Guth, S., and Frenzel, B. (1989) Epicuticularwachs der Tanne (Abies alba Mill.) und Walderkrankung. I. Die Wachsstrukur. Angew. Botanik 63, 241-258.

Gwrthardt-Goerg, M.S., and Keller, T. (1987) Some effects of long-term ozone fumigation on Norway spruce. II. Epicuticular wax and stomata. Trees 1, 145-150.

Hadwiger-Fangmeier, A., Fangmeier, A., and Jager, H.-J. (1992) Ammoniak in der bodennahen Atmosphar-Emission, Immission und Auswirkungen auf terrestrische Okosysteme. Forschungsberichte zum Forschungsprogramm des Landes Nordrhein- Westfalen "Luftverunreinigungen und Waldschalden" Nr. 28. Ministerium fur Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Dusseldorf.

Hafner, L. (1986) Zur Feinstruktur der geschadigten Kiefernnadel. Allg. Forstz. 41, 1119-1121.

Hafner, L., Endler, W., Wendering, R., and Weese, G. (1989) Ultrastructural studies on the needles of damaged Scotch pine trees from forest dieback areas in West Berlin. Aquilo Ser. Bot. 27, 7-14.

Haines, B.L., Jernstedt, J.A., and Neufeld, H.S. (1985) Direct foliar effects of simulated acid rain. II. Leaf surface characteristics. New Phytol. 99, 407-416.

Hamzah, S.B., and Gomez, J.C. (1979) Ultrastructure of mineral deficient leaves of Hevea. I. Effects of macronutrient deficiencies. J. Rubber Res. Inst. Malaysia 27, 132-142.

Hanson, G.P., and Stewart, W.S. (1970) Photochemical oxidants: effects on starch hydrolysis in leaves. Science 168, 1223-1224.

Hartig, R. (1896) Uber die Einwirkung des Hutten- und Steinkohlenrauches auf die Gesundheit der Nadelwaldblume. Forstl.-naturwiss. Z. 5, 245-290.

Hasemann, G., and Wild, A. (1990) The loss of structural integrity in damaged spruce needles from locations exposed to air pollution. I. Mesophyll and central cylinder. J. Phytopathol. 128, 15-32.

Hecht-Buchholz, C. (1972) Wirkung der Mineralstoffernshrung auf die Feinstruktur der Pflanzenzelle. Z Pflanzenern. Bodenkd. 132, 45-69.

Hecht-Buchholz, C. (1983) Light and electron microscopic investigations of the reactions of various genotypes to nutritional disorders. Plant Soil 72, 151-165.

Heinrichs, H., and Mayer, R. (1980) The role of forest vegetation in the biogeochemical cycle of heavy metals. J. Environ. Qual. 9, 111-118.

Henriksen, A.J., Lien, L., and Traaen, T.S. (1990) Critical Loads for Surface Waters. Chemical Criteria for Inputs of Strong Acids. Report No. D 89210 NIV A. Norwegian Institute for Water Research, Oslo.

Herrick, G.T., and Friedland, A.J. (1990) Patterns of trace metal concentrations and acidity in montane forest soils of the northeastern United States. Water Air Soil Pollut. 53, 151-157.

Herschbach, C., and Rennenberg, H. (1991) Influence of glutathione on sulphate influx, xylem loading and exudation in excised tobacco roots. J. Exp. Bot. 42, 1021-1029.

Herterich, R., and Herrmann, R. (1990) Comparing the distribution of nitrated phenols in the atmosphere of two German hill sites. Environ. Technol. Lett. 11, 961.

Hettelingh, J.P., and de Vries, W. (1991) Mapping Vademecum. National Institute of Public Health and Environmental Protection, Coordination Centre for Effects, Bilthoven, The Netherlands.

Hettelingh, J.P., Downing, R.J., and Smedt, P.A.M. (1991) Mapping Critical Loads for Europe. Technical Report No. 1. National Institute of Public Health and Environmental Protection, Coordination Centre for Effects, Bilthoven, The Netherlands, 183 pp.

Hibben, C.R. (1969) The distinction between injury to tree leaves by ozone and mesophyll- feeding leafhoppers. For. Sci. 15, 154-157.

Hinckel, M., Reichl, A., Schramm, K.-W., Trautner, F., Reissinger, M., and Hutzinger, O. ( 1990) Concentration levels of nitrated phenols in conifer needles. Chemosphere 18, 2433-2439.

Holopainen, T., and Nygren, P. (1989) Effects of potassium deficiency and simulated acid rain, alone and in combination, on the ultrastructure of Scots pine needles. Can. J. For. Res. 19, 1402-1411.

Holopainen, T., Anttonen, S., Wulff, A., Palomaki, V., and Karenlampi, L. (1992) Comparative evaluation of the effects of gaseous pollutants, acidic deposition and mineral deficiencies: structural changes in the cells of forest plants. Agric. Ecosyst. Environ. 42, 365-398.

Huttl, R.F., and Fink, S. (1988) Diagnostische Dungungsversuche zur Revitalisierung geschadigter Fichtenbestande (Picea abies Karst.) in Sudwestdeutschland. Forstw. Centralbl. 107, 173-183.

Huttunen, S., Turunen, M., and Reinikainen, J. (1990) Scattered CaSO₄-crystallites on needle surfaces after simulated acid rain as an indicator of nutrient leaching. Water Air Soil Pollut. 54, 169-173.

Jokela, A., Back, J., and Huttunen, S. (1992) Applicability of microscopic methods to forest damage research (Abstr.). In: Proceedings of the 15th International Meeting on Air Pollution and Interactions between Organisms in Forest Ecosystems, 9-11 September. 1992, p. 25. Tharandt, Dresden.

Jung, G., and Wild, A. (1988) Electron microscopic studies of spruce needles in connection with the occurrence of novel forest decline. I. Investigations of the mesophyll. J. Phytopathol. 122, 1-12.

Kahle, H., and Breckle, S.W. (1992) Blei und Cadmium. Zeithombe in unseren Waldboden. Biol. unserer Zeit. 22,21-27.

Kahle, H., Bertels, H., Noack, G., Roder, U., Ruther, P., and Breckle, S.W. (1989) Wirkungen von Blei und Cadmium auf Wachstum und Mineralstoffhaushalt von Baehenjungwuchs. Allg. Forstz. 29/30, 783-788.

Karenlampi, L. (1986) Relationships between macroscopic symptoms of injury and cell structural changes in needles of ponderosa pine exposed to air pollution in California. Ann. BOL Fenn. 23, 255-264.

Karenlampi, L., and Houpis, I.L.J. (1986). Structural conditions of mesophyll cells of Pinus ponderosa var. scopulorum after SO₂ fumigation. Can. J. For. Res. 16, 1381-1385.

Kazda, M., and Glatzel, G. (1984) Schwermetalleinreicherung und Schwermetallverfug- barkeit im Einsicherungsbereich von Stammablaufwasser in Buchenwalders (Fagus sylvatica) der Wienerwald. Z. Pflanzenernahr. Bodenk. 147, 743-752.

Kelly, J.M., Parker, G.R., and McFee, W.W. (1979) Heavy metal accumulation and growth of seedlings of five forest species as influenced by soil cadmium level. J. Environ. Qual. 8, 361-364.

Khristodorov, V., Kamenova-Yukhimenko, S., and Merakchiiska-Nikolova, M. (1989) Effects of pollution with cadmium (Cd²⁺) on the survival and growth of one-year-old seedlings of Scots pine (Pinus silvestris). Nauka-za-Gorata 2, 18-26.

Kloke, A. (1981) Sollen Richtwerte fur tolerierbare Schwermetallgehalte in landwirtschaf- dich/gartnerish genutzten Boden auch fur Forstboden gelten? Mitteil. Forstl. Bundesversuchsanstalt Wien 137, 241-248.

Koval, M., Giehl,H., Matuska, P., Scharffe, D., Scheel, H.E., Schlitt, K., Seiler, W., and Werhahn, J. (1986) Bestimmung der horizontalen und vertikalen Verteilung verschiedener Spurengase im Rahmen des TULLA-Projekts. CEC Report. Ispra.

Kozlowski, T., and Constantinidou, H. (1986). Responses of woody plants to environmental pollution. For. Abst. 47, 5-51.

Lamoureux, G.L., and Rusness, D.G. (1989) The role of glutathione and glutathione S- transferases in pesticide metabolism, selectivity, and mode of action in plants and insects. In: Dolphin, D., Poulson, R., and Avranovic, O. (Eds.) Glutathione: Chemical, Biochemical and Medical Aspects, Vol. III. Series: Enzymes and Cofactors, pp. 153-196. John Wiley & Sons, New York.

Lang, K.J., and Holdenrieder, O. (1985) Nekrotische Flecken an Nadeln von Ficea abies-ein Symptom des Pichtensterbens? Eur. J. For. Pathol. 15, 52-58.

Langebartels, Ch., Kemer, K., Leonardi, S., Schraudner, M., Trost, M., Heller, W., and Sandermann, H. (1990) Biochemical plant responses to ozone. I. Differential induction of polyamine and ethylene biosynthesis in tobacco. Plant Physiol. 95, 882-889.

Legge, A.H., Bogner, J.C., and Krupa, S.V. (1988) Foliar sulphur species in pine: a new indicator of a forest ecosystem under air pollution stress. Environ. Pollut. 55, 15-27.

Lovett, G.M., and Lindberg, S.E. (1984) Dry deposition and canopy exchange in a mixed oak forest as determined by analysis of throughfall. J. Appl. Ecol. 21, 1013-1027.

Lozano, F.C., and Morrison, I.K. (1982) Growth and nutrition of white pine and white spruce seedlings in solutions of various nickel and copper concentrations. J. Environ. Qual. 11, 437-441.

Luethy-Krause, B., and Landolt, W. (1990) Effects of ozone on starch accumulation in Norway spruce (Picea abies). Trees 4, 107-110.

Maas, F.M., DeKok, L.J., Strik-Trimmer, W., and Kuiper, P.J.C. (1987a) Plant responses to H₂S and SO₂ fumigation. II. Differences in metabolism of H₂S and SO₂ in spinach. Physiol. Plant. 70, 722-728.

Maas, F.M., DeKok, L.J., Hoffmann, I., and Kuiper, P.J.C. (1987b) Plant responses to H2S and S02 fumigation. I. Effects on growth, transpiration and sulfur content of spinach. Physiol. Plant. 70, 713-721.

Martin, F., and Botton, B. (1993) Nitrogen metabolism of ectomycorrhizal fungi and ectomycorrhizas. Adv. Plant Pathol. 9.

Mayer, R., and Ulrich, B. (1974). Conclusions on the filtering action of forests from ecosystem analysis. Oecol. Plant. 9, 157-168.

Mayer, R., and Ulrich, B. (1978) Input of atmospheric sulphur by dry and wet deposition into two central European forest ecosystems. Atmos. Environ. 12, 375-377.

Mengel, K., Lutz, H.-J., and Breininger, M.-T. (1987) Auswaschung von Nahrstoffen aus jungen intakten Pichten (Picea abies). Z. Pflanzenern. Bodenk. 150, 61-68.

Mengel, K., Hogrebe, A.M.R., and Esch, A. (1989) Effect of acidic fog on needle surface and water relations of Picea abies. Physiol. Plant. 75, 201-207.

Meyberg, M., Lockhausen, J., and Kristen, U. (1988) Ultrastructural changes in mesophyll cells of spruce needles from a declining forest in northern Germany. Eur. J. For. Pathol. 18, 169-175.

Miller, H.G., Cooper, J.M., and Miller, J.D. (1976) Effect of nitrogen supply in litter fall and crown leaching in a stand of Corsican pine. J. Appl. Ecol. 13, 233-248.

Miller, P.R., and Evans, L.S. (1974) Histopathology of oxidant injury and winter fleck injury on needles of western pines. Phytopathology 64, 801-806.

Mitchell, C.D., and Fretz, T.A. (1977) Cadmium and zinc toxicity in white pine, red maple, and Norway spruce. J. Am. Soc. Hortic. Sci. 102, 81-84.

Mohamed, A.D. (1992) Role du Facteur Edaphique dans le Fonctionnement Biogeochimique et l'Etat de Santé de Deux Pessieres Vosgiennes. Effet d'un Amendement Calci- Magnesien. These de Doctorat, Université de Nancy, I/INRA Nancy, France, 136 pp.

Morrison, T.K., and Hogan, H.D. (1986) Trace element distribution within the tree phytomass and forest floor of a tolerant hardwood stand, Algoma, Ontario. Acid precipitation, Part 2. Water Air Soil Pollut. 51, 493-500.

Nakos, G. (1979) Lead pollution. Fate of lead in the soil and its effect on Pinus halepensis. Plant Soil 53, 427-443.

Nebe, W., Schierhorm, E., and lIgen, G. (1988) Rasterelektronenmikroskopische und chemische Untersuchungen von immissionsgeschadigten Fichtennadeln (Picea abies [L.] Karst.). Flora 181, 409-414.

Neite, H. (1989) Gehalte und Losligkeit von Blei, Zink, und Kupfer in den Boden ausgewahlter Buchenwalder Nordrhein-Westfalien. Allg. Forstz. 29/30, 778-780.

Nys, C. (1987) Fonctionnement du Sol d'un Ecosysteme Forestier: Etude des Modifications Dues a la Substitution d'une Plantation d'Epicea Commun (Picea abies) a une Foret Feuillue Melangee des Ardennes. These de Doctorat, Université de Nancy INRA, Nancy, France, 207 pp.

Nys, C. (1989) Fertilisation, deperissement et production de l'epicea commun (Picea abies) dans les Ardennes. Rev. For. Fr. 41, 336-347.

Ogier, G., Greppin, H., and Castillo, F. (1991) Ascorbate and guaiacol peroxidase capacities from apoplastic and cell material extracts of Norway spruce needles after long-term ozone exposure. In: Lobarzewski, J., Greppin, H., Penel, C., and Gaspar, T. (Eds.), Molecular, Biochemical and Physiological Aspects of Plant Peroxidases, pp. 391-400. University Press, Geneva.

Osborne, D.J. (1978) Target cells¾new concepts for plant regulation in horticulture. Sci. Hortic. 31, 31-43.

Osborne, D.J. (1984) Concepts of target cells in plant differentiation. Cell Diff: 14, 161-169.

Paparozzi, E.T., and Tukey, H.B., Jr (1983) Developmental and anatomical changes in leaves of yellow birch and Red Kidney bean exposed to simulated acid precipitation. J. Am. Soc. Hort. Sci. 108, 890-898.

Parameswaran, N., Fink, S., and Liese, W. (1985) Feinstrukturelle Untersuchungen an Nadeln geschadigter Tannen und Fichten aus Waldschadensgebieten im Schwarzwald. Eur. J. For. Pathol. 15, 168-182.

Patterson, W.A., and Olson, J.J. (1983) Effects of heavy metals on radicle growth of selected woody species germinated on filter paper, mineral and organic substrates. Can. J. For. Res. 13, 233-238.

Pell, E.J., and Weissberger, W.C. (1976) Histopathological characterization of ozone injury to soybean foliage. Phytopathology 66, 856-861.

Percy, K.E., and 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.

Percy, K.E., and Riding, R.T. (1981) Histology and histochemistry of elongating needles of Pinus strobus subjected to a long-duration, low-concentration exposure to sulfur dioxide. Can. J. Bot. 59, 2558-2567.

Percy, K.E., Jensen, K.F., and McQuattie, C.J. (1992) Effects of ozone and acidic fog on red spruce needle epicuticular wax production, chemical composition, cuticular membrane ultrastructure and needle wettability. New Phytol. 122, 71-80.

Platt-Aloia, K.A., Thomson, W.W., and Terry, N. (1983) Changes in plastid ultrastructure during iron nutrition-mediated chloroplast development. Protoplasma 114, 85-92.

Polle, A., and Rennenberg, H. (1992) Field studies on Norway spruce at high altitudes: II. Defense systems against oxidative stress in needles. New Phytol. 121, 635-642.

Polle, A., and Rennenberg, H. (1993a) The significance of antioxidants in plant adaptation to environmental stress. In: Mansfield, T., Fowden, L., and Stoddard, F. (Eds.) Plant Adaptation to Environmental Stress. James & James, London.

Polle, A., and Rennenberg, H. (1993b) Photooxidative stress in trees. In: Foyer, C., and Mullineaux, P. (Eds.) Photooxidative Stresses on Plants: Causes and Amelioration. CRC Press, Boca Raton, Florida.

Polle, A., Chakrabarti, K., and Rennenberg, H. (1991) Extracellular and intracellular peroxidase activities in needles of Norway spruce (Picea abies L.) under high elevation stress. In: Lobarzewski, J., Greppin, H., Penel, C., and Gaspar, T. (Eds.) Molecular, Biochemical and Physiological Aspects of Plant Peroxidases, pp. 447-453. University Press, Geneva.

Price, A., Lucas, P.W., and Lea, P.J. (1990) Age dependent damage and glutathione metabolism in ozone fumigated barley: a leaf section approach. J. Exp. Bot. 41, 1309-1317.

Ranger, J., Discours, D., Mohamed, A.D., Moares, C., Dambrine, E., Merlet, D., and Rouiller, J. (1993) Comparaison des eaux liees et des eaux libres etudides dans les sols de trois pessieres vosgiennes. Application a l'etude du fonctionnement actuel de ces sols et consequences pour l'etat sanitaire des peuplements. Ann. Sci. For. 50.

Reichl, A., Reissinger, M., and Hutzinger, O. (1987) Accumulation of organic air constituents by plant surfaces. III. Occurrence and distribution of atmospheric organic micropollutants in conifer needles. Chemosphere 16, 2647-2652.

Rennenberg, H. (1984) The fate of excess sulfur in higher plants. Annu. Rev. Plant Physiol. 35, 121-153.

Rennenberg, H. (1991) The significance of higher plants in the emission of sulfur compounds from terrestrial ecosystems. In: Sharkey, T.D., Holland, E.A., and Mooney, H.A. (Eds.) Trace Gas Emissions by Plants, pp. 217-265. Academic Press, San Diego, California.

Rennenberg, H., and Polle, A. (1992) Metabolic consequences of atmospheric sulphur influx into plants. In: Wellburn, A., and Alscher, R. (Eds.) Proceedings of the Third International Symposium on Gaseous Pollutants and Plant Metabolism. Elsevier Science, Barking, England.

Riding, R.T., and Percy, K.E. (1985) Effects of SO₂ and other air pollutants on the morphology of epicuticular waxes on needles of Pinus strobus and P. banksiana. New Phytol. 99, 555-563.

Rinallo, C., Raddi, P., Gellini, R., and di Lonardo, V. (1986) Effects of simulated acid deposition on the surface structure of Norway spruce and silver fir needles. Eur. J. For. Pathol. 16, 440-446.

Roberts, T.M., Skeffington, R.A., and Blank, L.W. (1989) Causes of type I spruce decline in Europe. Forestry 62, 179-222.

Roelofs, J.G.M., and Van Dijk, H.F.G. (1986) The effect of airborne ammonium deposition on canopy ion-exchange in coniferous trees. In: Madiy, P. (Ed.) Direct Effects of Dry and Wet Deposition on Forest Ecosystems, in Particular Canopy Interaction. Air Pollution Research Report No.4 (EEC Workshop, Lokeberg, Sweden, 1986/10/19-23), pp. 34-39.

Rosemann, D., Heller, W., and Sandermann, H. (1991) Biochemical plant responses to ozone. II. Induction of stilbene biosynthesis in Scots pine (Pinus sylvestris L.) seedlings. Plant Physiol. 97 , 1280-1286.

Roth-Holzapfel, M., Funke, W., and Rittner, P. (1992) 'Multielementanalysen an Invertebraten im Okosystem "Fichtenforst." 7 Statuskolloguium des PEF, Karlsruhe, Band 1, pp. 171-183.

Ruetze, M., Schmitt, U., Liese, W., and Kuppers, K. (1988) Histologische Untersuchungen an Fichtennadeln (Picea abies L. Karst.) nach Begasung mit SO₂, O₃ und NO₂. Allg. Forstjagdztg. 159, 195-203.

Russo, F., and Brennan, E. (1980) Evidence for a cadmium and ozone interaction on Populus tremuloides. J. Arboric. 6, 103-134.

Sauter, J.J., and Voss, J.-U. (1986) SEM¾observations on the structural degradation of epistomatal waxes in Picea abies (L.) Karst.¾and its possible role in the "Fichtensterben." Eur. J. For. Pathol. 16, 408-423.

Schierl, R. (1989) Bestimmung des Komplexierungsgrades von Al, Fe, und Mn in Bodenlosungen durch Kationenaustausch. Vom Wasser 73, 161-16).

Schierl, R., and Kreutzer, K. (1991) Einflusse von saurer Beregnung und Kalkung auf die Schwermetalldynamik im Hoglwaldexperiment. In: Kreutzer, K., and Gottlein, A. (Eds.) Okosystemforschung Hoglwald. Beitrage zur Auswirkung von saurer Beregnung und jakkung in einem Fichtenalthbstand, pp. 204-211. P. Parey, Hamburg.

Schmidt, E. (1936) Baumgrenzenstudien am Feldberg irn Schwarzwald. Thar. Forstl. Jahrb. 87, 1-43.

Schmitt, U., Liese, W., and Ruetze, M. (1986) Ultrastruktyrekke Veranderungen in gruen Nadeln geschadigter Fichten. Angew. Bot. 60, 441-450.

Schmitt, U., Ruetze, M., and Liese, W. (1987) Rasterelektronenmikroskopische Unter-suchungen an Stomata von Fichten-und Tannennadeln nach Begasung und saurer Beregnung. Eur. J. For. Pathol. 17, 118-124.

Schraudner, M., Ernst, D., Langebartels, Ch., and Sandermann, H. (1992) Biochemical plant responses to ozone. III. Activation of the defense-related proteins b-l,3-glucanase and chitinase in tobacco leaves. Plant Physiol. 99, 1321-1328.

Schroder, P., and Rennenberg, H. (1992) Characterization of glutathione S-transferase from dwarf pine needles (Pinus mugo Turra). Tree Physiol. 11, 151-160.

Schroder, P., Lamoureux, G.L., Rusness, D.G., and Rennenberg, H. (1990a) Glutathione S- transferase activity in spruce needles. Pest. Biochem. Physiol. 37, 211-218.

Schroder, P., Rusness, D.G., and Lamoureux, G.L. (1990b) Detoxification of xenobiotics in spruce trees is mediated by glutathione S-transferases. In Rennenberg, H., Brunold, Ch., De Kok, L.J., and Stulen, I. (Eds.) Sulfur Nutrition and Sulfur Assimilation in Higher Plants. Fundamental, Environmental and Agricultural Aspects, pp. 245-248. SPB Acad. Publ., The Hague.

Schulze, E.D. (1987) Tree responses to acid depositions into the soil. A summary of the COST workshop at Julich 1985. In: Mathy. P. (Ed.) Air Pollution and Ecosystems. Inter- national EEC Symposium, 18-22 May, 1987, Grenoble, France, pp. 225-241. Reidel, Dordrecht.

Schupp, R., Schatten, T., Willenbrink, J., and Rennenberg, H. (1992) Long-distance transport of reduced sulphur in spruce (Picea abies L.). J. Exp. Bot. 43, 1243-1250.

Seiler, J.R., and Paganelli, D.J. (1987) Photosynthesis and growth response of red spruce and loblolly pine to soil applied lead and simulated acid rain. For. Sci. 33, 668-675.

Smith, G.C., and Brennan, E. (1984) Response of silver maple seedlings to an acute dose of root-applied cadmium. For. Sci. 30, 582-586.

Smith, I.K., Polle, A., and Rennenberg, H. (1990) Glutathione. In: Alscher, R.G., and Cumming, J.R. (Eds.) Stress Responses in Plants: Adaptation and Acclimation Mechanisms, pp. 201-215. Wiley & Sons, New York.

Soikkeli, S. (1981a) A review of the structural effects of air pollution on mesophyll tissue of plants at light and transmission electron microscope level. Savonia 4, 11-34.

Soikkeli, S. (1981b) The types of ultrastructural injuries in conifer needles of northern industrial environments. Silva Fennica 15, 399-404.

Soikkeli, S. (1981c) Comparison of cytological injuries in conifer needles from several polluted industrial environments in Finland. Ann. Bot. Fenn. 18, 47-61.

Soikkeli, S., and Karenlampi, L. (1984) Cellular and ultrastrural effects. In: Treshow, M. (Ed.) Air Pollution and Plant Life, pp. 159-174. Wiley & Sons, Chichester, New York.

Steinnes, E., Solberg, W., Petersen, H.M., and Wrer, C.D. (1989) Heavy metal pollution by long range atmospheric transport in natural soils of southern Norway. Water Air Soil Pollut. 45, 207-218.

Stewart, D., Treshow, M., and Harner, F.M. (1973) Pathological anatomy of conifer needle necrosis. Can. J. Bot. 51, 983-988.

Sutinen, S. (1987a) Cytology of Norway spruce needles. I. Changes during ageing. Eur. J. For. Pathol. 17, 65-73.

Sutinen, S. (1987b) Cytology of Norway spruce needles. II. Changes in yellowing spruces from the Taunus Mountains, West Germany. Eur. J. For. Pathol. 17, 74-85.

Sutinen, S. (1987c) Ultrastructure of mesophyll cells of spruce needles exposed to O₃ alone or together with SO₂. Eur. J. For. Pathol. 17, 362-368.

Sutinen, S., Skarby, L., Wallin, G., and Seliden, G. (1990) Long-term exposure of Norway spruce, Picea abies (L.) Karst., to ozone in open-top chambers. II. Effects on the ultrastructure of needles. New Phytol. 115, 345-355.

Sutinen, S. (1986) Ultrastructure of mesophyll cells in and near necrotic spots on otherwise green needles of Norway spruce. Eur. J. For. Pathol. 16, 379-384.

Sverdrup, H.U., and Warfvinge, P. (1988) Weathering of primary silicate minerals in the natural soil environment in relation to a chemical weathering model. Water Air Soil Pollut. 38, 387-408.

Swiecki, T.J., Endress, A.G., and Taylor, O.C. (1982) The role of surface wax unsusceptibility of plants to air pollutant injury. Can. J. Bot. 60, 316-319.

Takahama, U., Veljovic-Iovanovic, S., and Heber, U. (1992) Effects of the air pollutant SO₂ on leaves. Inhibition of sulfite oxidase in the apoplast by ascorbate and of apoplastic peroxidase by sulfite. Plant Physiol. 100, 261-266.

Thimonier, A., Dupouey, J.L., and Timbal, J. (1992) Floristic changes in the herb-layer vegetation of a deciduous forest in the Lorraine Plain under the influence of atmospheric deposition. For. Ecol. Manage. 55, 149-167.

Thoene, B., Schreder, P., Papen, H., Egger, A., and Rennenberg, H. (1991) Absorption of atmospheric NO₂ by spruce (Picea abies L. Karst.) trees. I. NO₂ influx and its correlation with nitrate reduction. New Phytol. 117, 575-585.

Thomson, W .W .(1975) Effects of air pollutants on plant ultrastructure. In: Mudd, J.B., and Kozlowski, T.T. (Eds.) Responses of Plants to Air Pollution, pp. 179-194.

Thomson, W.W., Dugger, W.M., Jr, and Palmer, R.L. (1966) Effects of ozone on the fine structure of the palisade parenchyma cells of bean leaves. Can. J. Bot. 44, 1677-1682 + 6 plates.

Thomson, W.W., Nagashi, J., and Platt, K. (1974) Further observation on the effects of ozone on the ultrastructure of leaf tissue. In: Dugger, M. (Ed.) Air Pollution Effects on Plant Growth, pp. 83-93. ACS Symposium Series 3. American Chemical Society, Washington, D.C.

Tuomisto, H. (1988) Use of Picea abies needles as indicators of-air pollution: Epicuticular wax morphology. Ann. Bot. Fenn. 25, 351-364.

Turner, R.R., BogIe, M.A., and Baes, C.F., III. (1985) Survey of Lead in Vegetation, Forest Floor and Soils of the Great Smoky Mountains National Park. Report No. ORNL/TM 9416, Publication No.2425. Oak Ridge National Laboratory, Environmental Sciences Division, Oak Ridge, Tennessee.

Turunen, M., and Huttunen, S. (1989) A review of the response of epicuticular wax of conifer needles to air pollution. J. Environ. Qual. 19, 35-45.

Turunen, M., and Huttunen, S. (1991) Effect of simulated acid rain on the epicuticular wax of Scots pine needles under northerly conditions. Can. J. Bot. 69, 412-419.

Ulrich, B. (1984) Interaction of indirect and direct effects of our pollutants in forests. In: Troyanowsky, C. (Ed.) Air Pollution and Plants. Second European Conference on Chemistry and the Environment, 21-24 May, 1984, Lindau, FRG, pp. 148-181. Federation of European Chemical Societies/VCH Verlagsgesellschaft.

Van Breemen, N., Driscoll, C.T., and Mulder, J. (1984) Acidic deposition and internal proton sources in acidification of soils and waters. Nature 307, 599-604.

Van Gardeningen, P.R., Grace, J., and Jeffree, C.E. (1991) Abrasive damage by wind to the needle surfaces of Picea sitchensis (Bong.) Carr. and Pinus sylvestris L. Plant Cell Environ. 14, 185-193.

Vapaavuori, E.M, Korpilathi, E., and Nurrni, A.H. (1984) Photosynthetic rate in willow leaves during water stress and changes in the chloroplast ultrastructure, with special reference to crystal inclusions. J. Exp. Biol. 35, 306-321.

Wellburn, A.R. (1985) SO₂ effects on stromal and thylakoid function. In: Winner, W.E., Mooney, H.A., and Goldstein, R.A. (Eds.) Sulfur Dioxide and Vegetation. Physiology, Ecology and Policy Issues, pp. 133-147. Stanford University Press, Palo Alto, California.

Wellburn, A.R. (1990) Why are atmospheric oxides of nitrogen usually phytotoxic and not alternative fertilizers? New Phytol. 115, 395-429.

Wellburn, A.R., Majernik, O., and Wellburn, F.A.M. (1972) Effects of SO₂ and NO₂ polluted air upon the ultrastructure of chloroplasts. Environ. Pollut. 3, 37-49.

Whatley, J.M. (1971) Ultrastructural changes in chloroplasts of Phaseolus vulgaris during development under conditions of nutrient deficiency. New Phytol. 70, 725-742.

Whitney, R.D., and Ip, D.W. (1991) Necrotic spots induced by simulated acid rain on needles of Abies balsamea saplings. Eur. J. For. Pathol. 21, 36-48.

Winner, W.E., Mooney, H.A., Williams, K., and von Caemmerer, S. (1985) Measuring and assessing SO₂ effects on photosynthesis and plant growth. In: Winner, W.E., Mooney, H.A., Goldstein, R.A. (Eds.) Sulfur Dioxide and Vegetation. Physiology, Ecology and Policy Issues, pp. 118-132. Stanford University Press, Palo Alto, California.

Wittig, R., and Neite, H. (1989) Distribution of lead in the soils of Fagus sylvatica forests in Europe. In: Ozturk, M.A. (Ed.) Plant and Pollutants in Developed and Developing Countries, pp. 199-206. Izmir, Turkey.

Wolfenden, J., Pearson, M., and Francis, J. (1991) Effects of over-winter fumigation with sulphur and nitrogen oxides on biochemical parameters and spring growth in red spruce (Pices rubens Sarg.). Plant Cell Environ. 14, 35-45.

Ylimartino, A., Paakonen, E., and Holopainen, T. (1992) Foliar nutrient concentrations and epicuticular wax formation of Scots pine needles. (Abstr.) In: Proceedings of the 15th International Meeting on Air Pollution and Interactions between Organisms in Forest Ecosystems, 9-11 September 1992, p. 81. Tharandt, Dresden.

Zellnik, G., and Gailhofer, M. (1989) Feinstruktur der Chloroplasten von Picea abies verschiedener Standorte im Hohenprofil "Zillertal." Phyton 29, 147-161.

Zobel, A., and Nighswander, J.E. (1991) Accumulation of phenolic compounds in the necrotic areas of Austrian and red pine needles after spraying with sulphuric acid: a possible bioindicator of air pollution. New Phytol. 117, 565-574.

Zottl, H.N. (1985) Heavy metal levels and cycling in forest ecosystems. Experientia 41, 1104-1113.

Zottl, H.W., and Lamparski, F. (1981) Schwermetalle (Pb, Cd) in der Bodenfauna des Sudschwarzwaldes. Mitt. dt. Bodenkdl. Ges. 32, 509-518.