Interactions between calcium and heavy metals in Norway spruce Accumulation and binding of metals in wood and bark

Ann Helén Österås

Department of Botany, Stockholm University, 2004 Doctoral dissertation Ann Helén Österås Stockholm University April 16, 2004

Front cover: The distribution of Ca (red), Mn (blue) and Cu (green) in a 400µm thick stem section from a 2-year-old Norway spruce analysed with microbeam X-ray fluorescence. The area scanned was 450 x 3510 µm, and the step-size was 15µm.

Back cover: The distribution of Ca (red) and Cu (blue) in the same stem section as on the front cover.

 Ann Helén Österås 2004 ISBN 91-7265-821-5, pp. 1 – 52. Printed at PrintCenter, Stockholm University Stockholm, Sweden, 2004 ABSTRACT

Waste products from the forest industry are to be spread in forests in Sweden to counteract nutrient depletion due to whole tree harvesting. This may increase the bioavailability of calcium (Ca) and heavy metals, such as cadmium (Cd), copper (Cu) and zinc (Zn) in forest soils. Heavy metals, like Cd, have already been enriched in forest soils in Sweden, due to deposition of air pollutions, and acidification of forest soils has increased the bioavailability of toxic metals for plant uptake. Changes in the bioavailability of metals may be reflected in altered accumulation of Ca and heavy metals in forest trees, changes in tree growth, including wood formation, and altered tree species composition. This thesis aims at examining: A) if inter- or intra- specific differences in sensitivity to Cd occur in the most common tree species of Sweden, and if so, to study if these can be explained by the uptake and distribution of Cd within the plant: B) how elevated levels of Ca, Cd, Cu and Zn affect the accumulation and attachment of metals in bark and wood, and growth of young Norway spruce (Picea abies): C) how waste products from the forest industry, such as wood ash, influence the contents of Ca, Cd, Cu and Zn in wood and bark of young Norway spruce. Sensitivity to Cd, and its uptake and distribution, in seedlings of Picea abies, Pinus sylvestris and Betula pendula from three regions (southern, central and northern parts) of Sweden, treated with varying concentrations of Cd, were compared. Differences in root sensitivity to Cd both among and within woody species were found and the differences could to some extent be explained by differences in uptake and translocation of Cd. The root sensitivity assays revealed that birch was the least, and spruce the most, sensitive species, both to the external and to tissue levels of Cd. The central ecotype of the species tested tended to be most Cd resistant. The radial distribution, accumulation and attachment of, and interactions between Ca and heavy metals in stems of two-year-old Norway spruce trees treated with elevated levels of Cd, Cu, Zn and/or Ca, were investigated. Further, the influence of these metals on growth, and on root metal content, was examined. Accumulation of the metals was enhanced in wood, bark and/or roots at elevated levels of the metal in question. Even at low levels of the metals, similar to after application of wood ash, an enhanced accumulation was apparent in wood and/or bark, except for Cd. The increased accumulation of Zn and Cu in the stem did not affect the growth. However, Cu decreased the accumulation of Ca in wood. Higher levels of Cu and Cd reduced the stem diameter and the toxic effect was associated with a reduced Ca content in wood. Copper and Cd also decreased the accumulation of Zn in the stem. On the other hand, elevated levels of Ca increased the stem diameter and reduced the accumulation of Cd, Cu, Zn and manganese (Mn) in wood and/or bark. When metals interacted with each

3 other the firmly bound fraction of the metal reduced was in almost all cases not affected. As an exception, Cd decreased the firmly bound fraction of Zn in the stem. The influence of pellets of wood ash (ash) or a mixture of wood ash and green liquor dregs (ash+GLD), in the amount of 3000 kg ha-1, on the contents of Ca, Cd, Cu and Zn in wood and bark of young Norway spruce in the field was examined. The effect of the treatments on the metal content of bark and wood was larger after 3 years than after 6 years. Treatment with ash+GLD had less effect on the heavy metal content of bark and wood than treatment with ash alone. The ash treatment increased the Cu and Zn content in bark and wood, respectively, after 3 years, and decreased the Ca content of the wood after 6 years. The ash+GLD treatment increased the Ca content of the bark and decreased the Zn content of bark and wood after 3 years. Both treatments reduced, or tended to decrease, the Cd content in wood and bark at both times. To conclude, small changes in the bioavailability of Ca, Cu, Cd and Zn in forest soils, such as after spreading pellets of wood ash or a mixture of wood ash and green liquor dregs from the forest industry, will be reflected in an altered accumulation of metals in wood and bark of Norway spruce. It will not only be reflected in changed accumulation of those metals in which bioavailability in the soil has been enhanced, but also of other metals, probably partly due to interactions between metals. When metals interact the exchangeable bound fraction of the metal reduced is suggested to be the main fraction affected. The small alterations in accumulation of metals should not affect the growth of Norway spruce, especially since the changes in accumulation of metals are low, and further since these decrease over time. However, as an exception, one positive and maybe persistent effect of the waste products is that these may decrease the accumulation of Cd in Norway spruce, which partly may be explained by competition with Ca for uptake, translocation and binding. A decreased accumulation of Cd in Norway spruce will probably affect the trees positively, since Norway spruce is one of the most sensitive species to Cd of the forest trees in Sweden. Thus, spreading of waste products from the forest industry may be a solution to decrease the accumulation of Cd in Norway spruce. In a longer perspective, this will decrease the risk of Cd altering the tree species composition of the forest ecosystem. An elevated bioavailability of Ca in forest soils will, in addition to Cd, probably also decrease the accumulation of other less competitive heavy metals, like Zn and Mn, in the stem.

Ann Helén Österås © Ann Helén Österås 2004 Department of Botany ISBN 91-7265-821-5, pp. 1-52 Stockholm University, Sweden [email protected]

4 LIST OF PAPERS

This thesis is based on two published papers and three manuscripts, which will be referred to by their Roman numerals.

I Österås A.H., Ekvall L. and Greger M. 2000. Sensitivity to, and accumulation of, cadmium in Betula pendula, Picea abies, and Pinus sylvestris seedlings from different regions in Sweden. Can. J. Bot. 78: 1440-1449.

II Österås A.H. and Greger M. 2003. Accumulation of, and interactions between, calcium and heavy metals in wood and bark of Picea abies. J. Plant Nutr. Soil Sci. 166: 246-253.

III Österås A. H. and Greger M. Interactions between Ca and Cu or Cd in Norway spruce. Submitted manuscript.

IV Österås A. H., Rindby A. and Greger M. Radial distribution, attachment of and interactions between Ca and heavy metals in stems of young Norway spruce. Submitted manuscript.

V Österås A. H., Sunnerdahl I. and Greger M. Calcium and heavy metal contents in wood and bark of young Norway spruce after application of waste products from the forest industry. Submitted manuscript.

Papers I and II are reproduced with the permission of the publishers concerned.

My contributions to the papers were as follows: I was responsible for the writing of all papers, with help from the co-writers, especially my supervisor Assoc. Prof. Maria Greger. I planned the experiments in papers II – IV and performed the laboratory work in papers II and III, and part of the laboratory work in paper IV. Further, I took part in the planning of the experiments and laboratory work in paper I and field collection and analyses in paper V.

5 TABLE OF CONTENTS

ABSTRACT...... 3 LIST OF PAPERS ...... 5 TABLE OF CONTENTS ...... 6 ABBREVIATIONS...... 7 INTRODUCTION ...... 8 Background ...... 8 Uptake, transport and translocation of metal ions...... 11 The morphology of wood and bark in softwoods ...... 12 Wood chemistry...... 14 Objectives...... 16 COMMENTS ON MATERIAL AND METHODS ...... 18 Laboratory and Field studies...... 18 Plant material...... 18 Nutrient and metal additions ...... 18 Treatments...... 19 Metal analyses ...... 20 RESULTS AND DISCUSSION ...... 21 Calcium, essential role and influence on wood formation ...... 21 Heavy metals (Zn, Cu, Cd), function and toxicity ...... 23 Differences in sensitivity to heavy metals ...... 25 Metal binding in bark and wood...... 28 Radial distribution and accumulation of metals in the stem...... 29 Interactions between metals ...... 34 Influence of waste products from the forest industry on the Ca and heavy metal content in wood and bark ...... 38 CONCLUSIONS...... 42 ACKNOWLEDGEMENTS ...... 44 REFERENCES...... 45

6 ABBREVIATIONS

AAS Atomic absorption spectrophotometry Ash Wood ash Bark The whole bark part, including the cambial region CEC Cation exchange capacity EDTA Ethylenediaminetetraacetic acid-solution ext-TT95b Toxicity threshold value for the external concentration GLD Green liquor dregs int-TT95b Toxicity threshold value for the internal concentration of the plant New wood Wood formed during the treatment time Old wood Wood formed before the treatment time Outer bark The bark part outside the inner phloem µ-XRF Microbeam X-ray fluorescence

7 INTRODUCTION

Background In Sweden about 65% of the land is covered by forest and the forest is of great importance both for the national economy and for recreation (SNA, 1990). Harvest of whole trees and acidification of forest soils is depleting the base cation pool of the forest ecosystem, decreasing the availability of essential nutrients, such as calcium (Ca), and increasing the availability of toxic metals to plants (Falkengren-Grerup et al., 1987; Hallbäcken, 1992; Mannings et al., 1996; Olsson et al. 1996). According to Olsson et al. (2000), harvest of whole trees may negatively affect the nutrient balance in forest trees. To counteract nutrient depletion, waste products from the forest industry, especially wood ash, but also wood ash mixed with green liquor dregs, have been suggested to be recycled as liming and fertilising agents in forest soils of Sweden (Eriksson HM, 1998; Greger et al., 1998; Arvidsson and Lundkvist, 2003). Wood ash is produced from burning logging residues for energy production and green liquor dregs are produced during the recovery boiling process in sulphate pulp production (Greger et al., 1998). In order to compensate for base cation losses at whole-tree harvesting, the National Forestry Board in Sweden has suggested an application of 3-tonnes of wood ash per hectare (Anon., 2001). Wood ash contains most mineral nutrients, except nitrogen, which is lost during combustion. The most abundant elements in wood ash are the base cations Ca, K and Mg. Wood ash also contains heavy metals, such as cadmium (Cd), copper (Cu) and zinc (Zn) (Table 1). Calcium is generally the dominant element both in wood ash (Table 1) and in the leaches from the wood ash (Steenari et al., 1999). The leakage rate of heavy metals, like Cd, Cu and Zn, from wood ash, is generally low. However, leakage studies with wood ash have shown that there may be a temporary increase of soluble heavy metals in the soil after application (Eriksson, 1998). This increase of heavy metals was suggested not to originate from the wood ash itself; instead it may be due to the salt effect of the wood ash releasing exchangeable heavy metal ions of the mor layer. The leakage rate of metals from loose wood ash can be decreased by stabilising the ash into granulated, crushed or pelletised form (Greger et al., 1998). Field studies have shown that wood ash application can increase the concentration of Ca as well as heavy metals, such as Cd, Cu and Zn, in the forest soil solution (Bramryd and Fransman, 1995; Rumpf et al., 2001; Arvidsson and Lundkvist, 2003). Heavy metals are, and have already been, enriched in forest soils by other human activities of different kinds (Hüttermann et al., 1999). Changed bioavailability of metals in forest soils will probably be reflected in a changed accumulation of metals in wood and bark of forest trees (Figure 1).

8 Table 1. The contents of Ca, Cd, Cu and Zn in pellets of wood ash from the pulp and paper industry and the amount of metals added to one hectare of forest soil with an addition of 3000 kg ha. Data from Greger et al. (1998) Metal Content in ash pellets (mg/g) Load of metals (g/ha) Ca 215 645 000 Cd 8.5 x 10-3 26 Cu 88.7 x 10-3 266 Zn 3880 x 10-3 11640

In addition to an expected temporary increase in the bioavailability of Cd in forest soils by spreading of wood ash from the forest industry (Nohrstedt, 2001), the bioavailability of Cd in the mor layer has increased in Sweden, especially in the southern parts, due to acid deposition and deposition of air pollution (Andersson et al., 1991; SNV, 1993). Cadmium can be accumulated in plants and have a toxic effect on plant growth at low concentrations. Further, heavy metal sensitivity, uptake and translocation are known to differ both between and within tree species (Jastrow and Koeppe, 1980; Patterson and Olson, 1983; Geburek and Scholz, 1989; Arduini et al., 1996; Greger, 1999). Thus, if there are differences in the sensitivity to Cd between and within forest tree species it may cause different effects on different species and populations, due to differences in uptake and translocation of Cd, which in a longer perspective may alter the tree species composition of the forest ecosystems (Figure 1). Metals with similar physiochemical properties, such as ion-size and charge, compete with each other for binding sites in plants (Marschner, 1995), thereby affecting the uptake, translocation and accumulation of each other. Thus, if the bioavailability of metals is changed in forest soils, due to whole tree harvesting, acidification, deposition of heavy metal air pollutions, spreading of liming material such as wood ash etc., this can affect the accumulation of metals in wood and bark of forest trees, due to competition between metals (Figure 1). Calcium is known to have a positive influence on the growth of plants and also to ameliorate the toxicity of heavy metals (Marschner, 1995; Hagemeyer, 1999). Further, in nutrient solutions, Ca has been found to decrease the Cd, Cu, Mn and Zn contents in roots and/or shoots of plants (Chaudhry and Loneragan, 1972; Jarvis et al., 1976; Wallace et al. 1980; Tyler and McBride, 1982; Kawasaki and Moritsugu, 1987; Saleh et al., 1999). Similarly, heavy metals, like Cu and Cd, have been found to reduce the Ca, Mn and Zn contents in roots, shoots and/or leaves of trees (Burton et al., 1986; Gussarsson, 1994; Arduini et al., 1998). Calcium is a very important plant nutrient, being strongly involved in wood- formation and in crosslinking within wood structure (McLaughlin and Wimmer, 1999). Thus, if the Ca nutrient pool in the forest soil is decreased and the availability of heavy metals is increased this may result in a deficiency of Ca in

9 the wood of trees caused by competition for uptake, translocation and binding with heavy metals, which may negatively affect tree growth and the properties of the wood formed in trees (Figure 1). If, instead, the bioavailability of Ca is increased in the forest soil it may decrease the uptake and accumulation of toxic metals in the trees, thereby ameliorating the toxicity of heavy metals.

Acid deposition Heavy metal Altered tree species air pollution composition of the forest ecosystem

Differences in sensitivity to Cd between and within Whole tree forest tree species? harvesting

Enhanced accumulation of metals in bark and wood? Application of fertilising and Changed proportions of liming agents, metals in wood and bark e.g. wood ash due to interactions Changed concentrations and between metals? proportions of plant available Ca and heavy metals, such as Cd, Cu and Zn. Altered tree growth and wood formation?

Figure 1. Whole tree harvesting, deposition of air pollution and application of liming and fertilising agents, such as wood ash, in forest ecosystems changes the concentrations and proportions of plant-available metals in the forest soils. This may be reflected in changes in metal accumulation in wood and bark of forest trees, changes in tree growth, including wood formation, and altered tree species composition.

The influence of metal interactions on the accumulation and attachment of metals in wood and bark of forest trees is an area that is scarcely investigated. Examining the content of metals in wood and bark is of interest since these plant parts finally will end up either in the paper pulp or in waste products, such as wood ash. Even though an enhanced accumulation of heavy metals in the wood may not be toxic to the trees, it may cause problems for the production of paper pulp. Accumulation of metal ions in the bleach plant can result in scaling

10 problems and filtration failures and some of the metals, such as Cu and Mn, can also catalyse the decomposition of the bleaching chemicals, thereby reducing the bleaching efficiency, and increasing the consumption of chemicals and production cost (Colodette et al., 1988; Brooks et al., 1994; Ulmgren, 1997). The main purpose of this thesis was to study the influence of elevated levels of Ca and heavy metals on the growth, and metal accumulation in bark and wood, of Norway spruce.

Uptake, transport and translocation of metal ions Plant species and genotypes differ in their ability to take up metals and translocate metals from roots to shoots (Greger, 1999). Metals may enter trees via roots, leaves or bark, however, roots are the major pathway of entrance for most metals (Lepp, 1975). The uptake of metal cations by roots from the soil solution is affected by their bioavailability (Marschner, 1995). The bioavailability is influenced by several soil factors such as pH, redox potential, organic matter content, competing ions and cation binding capacity. Bioavailability is also influenced by plant factors, such as the release of organic acids and hydrogen ions from the roots, which may increase the solubility and uptake of metal ions (Marschner, 1995). Another important factor that may affect the uptake of, and sensitivity to, metals of trees under natural conditions is the association between fine roots of temperate trees and mycorrhizal fungi (Godbold et al., 1998; Hütterman et al., 1999; Jentschke and Godbold, 2000). In some cases, ectomycorrhiza may ameliorate the toxicity of metals to forest trees by restricting the uptake of the toxic metal into the host plant or by improving the mineral nutritional status of the host plant. Generally, as the concentration of the metal ions in the external solution increases, the uptake increases (Greger, 1999). First, the metal ions reach the apoplast of the roots, via diffusion or mass flow (Marschner, 1995). Before metal cations are taken up at the plasmalemma into the root cells they have to pass the root apoplast. The root apoplast is thought to have a great importance for the uptake of nutrients because of its negative charges that can bind and accumulate cations (Thornton and Macklon, 1989; Sattelmacher, 2001). Most of the metals are retained in the apoplast bound to the cell wall or are transported further in the apoplast (Greger, 1999). Part of the metals is also transported through the plasmalemma into the cytoplasm of root cells, where they are bound to various macromolecules (Greger, 1999). Membrane transport proteins probably generally mediate the influx of metal ions from the apoplast into the cytoplasm (Piñeros and Tester, 1997; Lindberg et al., 2004). The transport of heavy metals and Ca in plant tissues is thought to be mainly via the apoplastic

11 pathway (Greger, 1999; White, 2001). To reach the xylem via the apoplast the metal ions have to be taken up in root tips, since the Casparian strips are not yet fully developed there, or via lateral roots of basal root zones, where the endodermis has been ruptured (Marschner, 1995). When the metal ions reach the xylem they are translocated up the tree bole by interactions with nondiffusible anions of the cell wall of the xylem vessels, which leads to a separation of cation transport from water flow (Wolterbeek, 1987). The xylem is thought to act as an ion exchange column that has the potential to impede movement up the bole for months or years (Bell and Biddulph, 1963; Momoshima and Bondietti, 1990). The translocation of metal cations can be enhanced if they form complexes with organic compounds, thus reducing their affinity for the fixed negative charges in the cell walls (White et al., 1981; Cataldo et al., 1988). That roots contain much higher levels of metals than shoots is a common finding for higher plants (Greger, 1999). Even though accumulation of heavy metals in plants is generally restricted to root tissue, due to binding to the cell walls, they can also be translocated to the shoot (Greger et al., 1991; Gussarsson et al., 1995) and be accumulated in wood and bark of trees.

The morphology of wood and bark in softwoods In Sweden, the forest trees are dominated by two gymnosperms, Norway spruce (Picea abies (L.) Karst) and Scots pine (Pinus sylvestris L.) (SNA, 1990). Of the trees belonging to angiosperms, birch (Betula spp.) is the most abundant species in Sweden. Trees belonging to gymnosperms are classified as softwoods or coniferous woods, while trees belonging to angiosperms are classified as hardwoods (Sjöström, 1993). As a result of a rhythmic activity of the cambium in the stem of trees in temperate regions, annual growth rings in the wood are produced (Figure 2). During spring and early summer the activity of the cambium is high and thin-walled cells with large cavities are produced, giving a light-coloured and porous wood, so-called early wood, with the main function to transport liquids (Sjöström, 1993). Later, as the activity of the cambium ceases, cells with thicker cell walls are produced, giving a dark and dense wood, so- called late wood, with the main function to provide mechanical support. In the centre of the stem, the pith is located, which is the wood formed during the first year of growth (Figure 2). In older trees, the outer lighter part that conducts water is called sapwood, whereas the inner dark-coloured dead part is called heartwood (Sjöström, 1993).

12 Outer bark Inner bark Cambium Late wood Early wood Figure 2. Transverse view of Pith a coniferous stem showing the growth rings, consisting of early and late wood (Modified from Sjöström, 1993).

Growth ring

The wood or the xylem of softwoods consists mainly of tracheids (90-95%), which are dead cells that conduct water and nutrients, and give the stem mechanical strength (Figure 3; Sjöström 1993). Other cells present in the xylem of softwoods are ray cells (5-10%), including ray parenchyma cells and ray tracheids. The ray parenchyma cells are living cells that, together with the ray tracheids, extend in a radial direction from the cambium toward the pith and can provide liquid transport in the radial direction. Epithelial cells are also present and line the cavities of resin canals, which are intercellular spaces building up a uniform channel network in the trees. resin canal tracheids Lumen P CZ DX MX

Cell wall

periclinal

Ray radial Figure 3. Transverse section of xylem and phloem of 5.5-months-old Norway spruce (Picea abies). CZ, cambial zone; P, phloem; DX, differentiating xylem; MX, mature xylem with ray cells (ray). The periclinal and radial directions of the wood are marked with arrows. Epithelial cells line the resin canal.

13 The bark of the stem is located outside the cambium, and can be divided into living inner bark and dead outer bark (Sjöström, 1993; Figure 2). The inner bark or phloem mainly consists of sieve elements, parenchyma cells, and sclerenchymatous cells. The main function of sieve elements is to transport liquid and nutrients, of parenchyma cells to store nutrients, and of sclerenchymatous cells to support the tissue. The outer bark functions as a protective layer to the wood against mechanical damage and environmental stress, and mainly consist of periderm or cork layers. The cell wall of tracheids can be divided into several layers depending on differences in structure and chemical composition (Figure 4; Esau, 1977; Sjöström, 1993). The middle lamella is located between the cells and, in early stages of the growth, consists mainly of pectins, later becoming highly lignified. It serves the function of binding the cells together. Next to the middle lamella there is a thin and lignin-rich layer, the primary wall. Three layers of secondary wall follow the primary wall and they consist mainly of cellulose, lignin and hemicelluloses. The middle layer of the secondary wall forms the main portion of the cell wall and its thickness varies in early and late wood. On the inner surface of the cell wall there is a thin amorphous membrane of unknown composition, the warty layer (Sjöström, 1993).

W

S3 Figure 4. The cell wall layers of a tracheid, showing the middle lamella (ML), the primary wall

(P), the outer (S1), middle (S2), S2 and inner (S3 ) layers of the secondary wall, and the warty

S1 layer (W) (Modified from Côté, P 1967). M

Wood chemistry The main components of wood are cellulose, lignin and hemicellulose (Esau, 1977; Sjöström, 1993). Lignins are a heterogeneous three-dimensional group of polymers that contribute to the rigidity of the cell wall. Cellulose is a linear polysaccharide of β-D-glucose and hemicelluloses are a heterogeneous group of

14 polysaccharides dominated by glucomannan and glucuronoxylan. Cellulose forms the framework of the cell wall that is embedded and supported by a matrix of hemicellulose and pectins. Pectins, the major component of which is polygalacturonic acids, exist in minor quantities of softwood. Hemicellulose and pectic acids, and to a lesser degree lignin, contain negative charges originating from carboxyl groups, thereby the cell walls of tracheids function as cation exchangers (Sjöström, 1989). However, only a certain fraction of the carboxyl groups in the wood, about 20-50% of the original amount, participates in ion exchange, since some of the carboxyl groups are located in inaccessible regions of the cell walls. Another minor component of the wood that also contains carboxyl groups and can bind cations is extractives, including free fatty acids and resin acids (Sjöström, 1989). These are mainly concentrated in the resin canals and in the ray parenchyma cells. The chemical composition of the bark is more complex than the wood and varies between species, and between the cell types of the bark (Sjöström, 1993). The bark is rich in soluble constituents such as pectin and phenolic compounds. The principal cell wall constituents of bark are polysaccharides (dominated by cellulose and hemicellulose), lignin and suberins. In addition to these organic constituents, the wood and bark also contain inorganic constituents (metals) in minor quantities (Sjöström, 1993). The inorganic content of wood is less than 1% of the dry wood weight, whereas of bark it is higher, 2-5% of the dry bark weight. The main inorganic elements found in wood are Ca, Mg and K, which make up 70-80% of the total inorganic content of the wood (Fengel and Wegener, 1989). Similarly, in the bark the main metals found are Ca and K, and most of the Ca occurs as Ca-oxalate crystals (Sjöström, 1993). Other metals, like Mn, Fe and Cu, are also present in wood and bark but in smaller amounts. Most metals are present as metal salts, such as carbonates, silicates, oxalates, and phosphates, and in the wood the main part of the metals are thought to be bound to carboxylic groups of hemicellulose and pectins (Sjöström, 1993; Bailey and Reeve, 1996; Sundén et al., 2000). Some metals (Fe, Cu and Zn) have been shown to have a more heterogeneous distribution than others in the wood of Norway spruce (Ca and Mn) (Berglund et al., 1999).

15 Objectives The main purpose of this thesis was to study the influence of elevated levels of Ca and heavy metals on the growth, and metal accumulation in bark and wood, of Norway spruce. The knowledge in this area is of importance since waste products from the forest industry are to be spread in the forest in Sweden and these will change the bioavailability of Ca and heavy metals in forest soils and thereby probably also the metal content in the trees. Accumulation of certain metals in the wood can negatively affect the production of paper pulp. Further, heavy metals can be toxic, reducing the growth of trees, and may alter the tree species composition of the forest ecosystem, if the sensitivity to heavy metals varies between species.

The objectives and hypotheses of the thesis were as follows:

Firstly, two questions were asked: Do inter- or intra-specific differences in sensitivity to Cd occur in woody species? If so, can these be explained by the uptake and distribution of Cd in plants? The hypothesis was that inter- and intra-specific differences in sensitivity to and accumulation of Cd exist in woody species.

Secondly, the distribution and accumulation of, and interaction between, Ca and heavy metals (Cd, Cu, Zn) in wood and bark of young Norway spruce were examined. Further, the effect of changed accumulation of, and interaction between, Ca and heavy metals on the wood formation of Norway spruce was studied. The hypothesis was that elevated metal additions, e.g. comparable to wood ash application, would enhance the accumulation of metals in wood and bark. In addition, it was also hypothesised that the content and proportions of these metals would change in the wood and bark due to interactions between Ca and heavy metals, which in turn may affect wood formation.

Thirdly, the influence of an elevated Ca level on the accumulation of Cu or Cd in roots, wood and bark, and vice versa, of young Norway spruce was examined. It was hypothesised that elevated Ca levels would reduce the accumulation of Cu or Cd in roots, wood and bark, and vice versa, due to interactions.

Fourthly, microbeam X-ray fluorescence was used to examine the effect of elevated Ca, Cu and Cd addition on the radial distribution and accumulation of Ca, Cu, Mn and Zn in stems of young Norway spruce. Further, the

16 attachment of these metals within the stem and the influence of Ca, Cu and Cd on the binding of other metals were examined. Elevated levels of Ca, Cu or Cd were expected to interact with the uptake, translocation and binding of other similar metals and thereby decrease their concentration. It was hypothesised that enhanced accumulation of one metal ion, e.g. Ca, would decrease the exchangeable bound fraction of other similar metal ions, e.g. Mn, since the main part of metals in the wood is thought to be bound to these sites, and the other part, which is firmly attached, should not easily be exchanged.

Fifthly, the influence of pellets of wood ash or a mixture of wood ash and green liquor dregs on the contents of Ca, Cd, Cu and Zn in wood and bark of young Norway spruce three and six years after application was examined. The hypotheses were that changes in metal content in wood and bark would be found, since the bioavailability of metals in the soil ought to change after application of the waste products. The effect on the heavy metal content of wood and bark was expected to be larger after 3 years, than after 6 years, since the bioavailability of heavy metals in the soil was only expected to increase initially after spreading of the waste products.

17 COMMENTS ON MATERIAL AND METHODS

Laboratory and Field studies This thesis is based on results from both laboratory and field experiments (I - V). Three experiments were performed with nutrient solutions to gather information on the influences of one or two metals on growth of, and metal accumulation in, plants. The field experiment (V) was performed as a complement to the lab experiments, to elucidate what really happens under normal conditions where several factors influence the results.

Plant material Three types of plant material were used in this thesis, seedlings of different tree species, 2-year-old Norway spruce plug-plants and young Norway spruce trees grown in the field. In paper I, seedlings grown from seeds of Norway spruce (Picea abies (L.) Karst.), Scots pine (Pinus sylvestris L.) and European white birch (Betula pendula Roth) from three different regions in Sweden (southern, central and northern) were used. The seedlings were grown in continuous light since the northern provenance of spruce otherwise would have failed to grow. In papers II to IV, two-year-old Norway spruce plug-plants were used. In paper II the spruce trees originated from Grythyttan, Sweden (59˚36’ - 59˚40’), and in papers III and IV the spruce trees originated from Byelorussia (54˚38’). Norway spruce was used in these papers, since it was shown to be the most sensitive species to Cd in paper I and also because it is one of the most commonly used species in the Swedish forest industry. In the last study (V), young Norway spruce plants, grown for 4 or 7 years, at a felling site in central Sweden were used.

Nutrient and metal additions Different methods of nutrient and metal additions were used in papers I and II, compared with III and IV. An exponential nutrient addition method by Ingestad (1971, 1979) was used in papers I and II. In paper I, the main purpose of using this method was to control the relative growth rate of the plants so the results of the different ecotypes of the same species would be comparable with each other. In paper II, the main purpose was to imitate the availability of nutrients in a real soil solution. In both papers I and II the exponential nutrient addition method was also used to keep the element concentration and the risk of complex formation low. The addition of the metals in papers I and II was made

18 exponentially in proportion to the nutrients. This method of addition was used since it has been shown that it is the ratio between the toxic metal and nutrients rather than the concentration of the metal that determines the toxic effects (Greger et al., 1991). The spruce plants used in papers III and IV were grown according to a traditional method, with a single fixed addition of nutrients and metal treatment once per week, since it was not necessary to imitate the availability of nutrients in a soil solution. The nutrient solution used in these studies (III, IV) was based on the same nutrient ratios (Ingestad, 1979) and the same nutrient composition used for spruce in papers I and II.

Treatments In paper I, several Cd additions, chosen from prestudies, were used to obtain dose-response curves for each species and ecotype. Dose-response curves were needed to evaluate the sensitivity of the species and ecotypes to the Cd concentration in the medium (external) and internal concentration of Cd in the root with the modified Weibull function (Taylor et al., 1991). The modified Weibull function was chosen since it has been shown to be an excellent tool for comparison of dose-response curves and since important parameters can be estimated with it. One of these parameters is the empirical toxicity threshold

(TT95b), which is the concentration of metal that results in a yield reduction of 5% (Taylor et al., 1991). When comparing two values, a higher TT95b for a plant means that it is less sensitive to the metal than the plants with a lower TT95b. The Cd treatments lasted for 2 weeks. In paper II, the spruce plants were treated with either a low or a high addition of Ca, Cd, Cu or Zn for 3 months (Table 1). The low metal treatments in paper II were chosen to resemble the elevations of metals observed in field and leakage studies with ashes (Bramryd and Fransman, 1995; Eriksson, 1998). The high metal treatments were chosen for comparison with the low treatments. The high Cd treatment (Cd/K = 0.009) used in paper II was also used in paper I. In papers III and IV the spruce plants were treated with either an elevated addition of Ca, Cd or Cu, or as combinations of Ca with Cd or Cu for 3 months. Concentrations of the different metal treatments were chosen to affect the accumulation of metals in, but not to negatively affect the growth of, the plants. In the field experiment, in paper V, 3000 kg ha-1 pellets of wood ash (ash) or a mixture of wood ash and green liquor dregs (ash+GLD) was spread on plots in a felling site of central Sweden in 1996. To examine the effect of the treatments over time, young spruce trees that had been planted on the felling site in 1995,

19 were harvested, three (1999) and six (2002) years after the waste products were spread.

Metal analyses Two different methods for metal analyses were used, atomic absorption spectrophotometry (AAS) (I – III, V), and microbeam X-ray fluorescence (µ- XRF) (IV). Microbeam X-ray fluorescence was used as a complement to the measurements with AAS to be able to more accurately pinpoint where in the stem the changes in metal content occurred. However, this method was not able to detect Cd in the analysed samples. In paper I, the Cd contents of roots and shoots were analysed, and in papers II and III the Ca, and Cd, Cu or Zn, contents in bark (the whole bark part, including inner phloem and cambium), wood formed during the treatment time (new wood), and wood formed before the treatment time (old wood) were analysed (Figure 5). The plants in papers II and III were brought in from the tree nursery during the winter season when they were dormant and therefore the wood formed during the treatment time (new wood) could be cut from the rest of the wood (old wood) at the late wood border of the previous growth ring. This border had been studied under the light microscope in stems kept both from before the treatment and after. In paper III the Ca, and Cu or Cd, content of roots was also analysed. In paper V the metal content in bark and wood was analysed. In paper IV the relative concentrations of Ca, Cu, Mn and Zn in a stem section were analysed with µ-XRF, and then the measured relative metal concentrations were calculated for different regions of the stem: outer bark, inner phloem + cambium, new wood, wood (the whole wood part, except the pith) and pith (Figure 5).

Figure 5. To the left, a photo of part of a transverse stem section Outer Bark bark from a 2-year-old Norway spruce. To the right, an image of the BarkBark IP + C density of the stem section, within GapGap Gap the rectangle of the photo, New analysed with µ-XRF. The lighter Wood wood areas represent higher intensities, i.e. denser wood. Marked in the Wood photo and image are the division of Old the stem in different analysed and wood calculated areas. IP + C, inner phloem + cambium. Modified from Pith Figure 1 in paper IV. Density

20 RESULTS AND DISCUSSION

Calcium, essential role and influence on wood formation Calcium is an essential macronutrient with diverse functions in trees (McLaughlin and Wimmer, 1999). Calcium plays an important regulatory role in cell division, cell extension, cell wall and membrane synthesis and function (Jones and Lunt, 1967; Demarty et al., 1984; Hanson, 1984; McLaughlin and Wimmer, 1999). Calcium also functions as a second messenger in the signal transduction between environmental factors and plant responses (Marschner, 1995). The various roles of Ca in plant systems depend on its unique chemical properties that allow it to exist in a wide variety of binding states (Hepler and Wayne, 1985). The concentration of Ca in the cytosol is very low, since Ca is cytotoxic in higher concentrations (Marschner, 1995). Instead, the main part of Ca in plant tissues is located in the apoplast, bound to the cell wall, the outer surface of the plasma membrane and other structures (Demarty et al., 1984; Marshner, 1995). In the cell wall Ca is mainly bound to exchangeable sites in the middle lamella. By binding to carboxylic groups of the polygalacturonic acids (pectins) and crosslinking the pectic chains of the middle lamella, Ca strengthens the cell wall and controls its rigidity. The cell wall has to be weakened for the cells to be able to extend by the turgor pressure. Divalent cations as Ca can prevent wall weakening and subsequent cell extension and cell enlargment (Tagawa and Bonner, 1957; Cleland and Rayle, 1977; Demarty et al., 1984). In 2-years-old Norway spruce plants an increased content of Ca in bark and wood was associated with an increased stem diameter growth (III). The increased Ca addition also increased the total fresh weight growth and root dry weight of the spruce plants. A similar increase was found in 5.5-month-old spruce plants (Table 2; Österås and Greger, 2000). Furthermore, trees fertilised with Ca, K and Mg in the field showed a significant increase in the periclinal cell division compared with control trees (Dünisch and Bauch, 1994). Thus, if the availability of Ca is limited, an increased Ca addition may lead to increased growth and wood formation. How Ca influenced the wood formation was also studied in 5.5-month-old spruce plants treated with elevated levels of Ca. The size and the cell wall thickness of the tracheids tended to change at elevated Ca additions (Table 2; Österås and Greger, 2000). The radial diameter of the lumen seemed to increase at increasing Ca additions and the periclinal diameter of the lumen seemed to decrease at the highest Ca addition. Further, the cell wall thickness of the tracheids tended to increase when the Ca addition was 4 times higher than the control. A low elevation of the Ca content of stems may thereby increase the cell

21 size and cell wall thickness, whereas a high elevation may produce cells with different cell dimensions, i.e. larger radial and smaller periclinal diameter. Calcium has been found to be especially important for the secondary wall synthesis and lignification (Dünisch et al., 1998). According to Eklund and Eliasson (1990), the deposition of cell wall material and the synthesis of lignin are inhibited by Ca deficiency. Furthermore, at increasing Ca levels the cell wall deposition and synthesis of lignin are increased. Thus, elevated Ca levels can change the properties of the wood formed in trees.

Table 2. Ca content of stem, stem diameter, periclinal (LP) and radial (LR) lumen diameter and cell wall thickness (CWT) of the tracheids in 5.5-month-old Norway spruce trees treated with elevated levels of Ca during 3.5 months. The additions of Ca are given times the control. The values of the Ca content in the stem and stem diameter are the means of 7 - 8 replicates ± SE. The values for lumen diameter and cell wall thickness are the means of approximately 500 cells in 4 replicates (no significant differences were found). Data from Österås and Greger (2000). Significant differences (P ≤ 0.05) between the different treatments are marked with different letters

Castem Stem diam. LR LP CWT Treatment (mg/g dw) (mm) µm µm µm Control 1.09 ± 0.04a 1.90 ± 0.03a 10.4 8.8 2.0 Ca x 2 1.26 ± 0.05b 2.02 ± 0.04b 10.5 8.6 1.9 Ca x 4 1.47 ± 0.05c 2.16 ± 0.03c 10.6 8.8 2.3 Ca x 17 3.46 ± 0.48d 2.55 ± 0.05d 10.9 8.2 2.0

Four times higher Ca addition did not change the proportions of cellulose, hemicellulose and lignin in the wood of 2-year-old spruce plants (Österås and Greger, 2000). However, the relative content of galactan, one of the carbohydrates of hemicellulose, tended to increase in the newly formed wood. Elevated Ca additions in spruce plants have been shown to increase the deposition of cell wall material, and this material did not seem to be cellulose, but appeared to be made up of other polysaccharides (Eklund and Eliasson, 1990). Calcium can alter the composition of the cell wall by influencing the activity of structural synthesising enzymes (Brummel and MacLachlan, 1989). To summarize, an increased Ca content of stems may affect the synthesis of cell wall material and the composition of the cell wall, and thereby the wood quality.

22 Heavy metals (Zn, Cu, Cd), function and toxicity Zinc and copper are essential micronutrients with several functions in plants, whereas cadmium is, up to now, considered to be a toxic element with no function (Marschner, 1995; Hagemeyer, 1999). Normal contents in plants are 0.1-1.0 mg/kg of Cd, 1-10 mg/kg of Cu, and 10-100 mg/kg of Zn (Pais and Jones, 1997). Copper is a component of several enzymes involved in physiological redox processes, such as photosynthetic electron transport, respiration, detoxification of superoxide radicals, and lignification (Welch, 1995). Zinc is also a component of several enzymes and is required for several functions in plants such as membrane integrity, enzyme activation, gene expression and regulation, carbohydrate metabolism, protein synthesis and detoxification of superoxide radicals. Heavy metals, like Cd, Cu and Zn, exist naturally in low concentrations in soils, originating from weathering of the parent rock (Kabata-Pendias et al., 1992). However, by human activity of different kinds, such as burning fossil fuel, mining activity and spreading of lime, waste products and fertilisers, heavy metals become accumulated in forest soils, since they are not naturally removed or degraded (Hüttermann et al., 1999). Accumulation of heavy metals in forest soils has been suggested to be partly responsible for forest declines (Gawel et al., 1996). Cadmium and Cu are well-known to be very toxic to plant growth in low concentrations, whereas Zn is supposed to be the least toxic of the heavy metals (Balsberg-Påhlsson, 1989; I, II). Copper toxicity in plants generally occurs when the tissue level exceeds 20 – 30 mg Cu/kg (Pais and Jones, 1997). Even though Cd is not an essential element it can be taken up by plants, including forest trees (Jastrow and Koeppe, 1980; Gussarsson et al., 1995; I; II; III), and generally growth reduction in plants occurs at a tissue level of 3 mg Cd/kg (Pais and Jones, 1997). Accumulation of heavy metals in plants may affect many metabolic and physiological processes. The toxic effects of heavy metals are caused either by direct interaction with structural components or indirectly by changes in signal transduction and/or metabolism (Barcelo and Poschenrieder, 1999). Heavy metals can damage the function of the plasma membrane by stimulating the formation of free radicals and reactive oxygen species, which may lead to lipid peroxidation (Dietz et al., 1999). Heavy metals in high concentrations or exposed in low concentrations for long periods may lead to growth inhibition (Greger et al., 1991). Effects on photosynthesis, growth processes and uptake of water, are some of the causes giving this effect (Barcelo and Poschenrider, 1990; Greger et al., 1991; Greger and Ögren, 1991; Greger and Bertell, 1992).

23 Cadmium was shown to be very toxic to the spruce trees once a certain level (toxicity threshold) was reached in the external solution and in the tissue (I and II). This seems also to be the case for Cu (paper II). The Cd treatment Cd/K = 0.009 was shown to decrease the root length and root dry weight of spruce seedlings after 2-weeks treatment time (I). Similar results were found for root dry weight of 5.5-month-old spruce trees treated with a Cd addition of Cd/K = 0.006 for 3.5 months (Österås and Greger, 2000; I). The root dry weight of 2- year-old spruce trees treated with the same Cd addition or with a high Cu addition tended to decrease and the roots were clearly stunted (II). Generally, root length is considered to be the most sensitive growth parameter to measure the toxicity of a heavy metal in higher terrestrial plants, since this is the main entrance site for metal ions (Breckle, 1991). The inhibition of root extension growth may be the result of interference with cell division or cell elongation. The high Cd and Cu treatment in paper II gave a smaller stem diameter of the 2-year-old spruce trees, compared with the control, implying that the wood formation was affected. Similar results were found for 5.5-month-old spruce plants treated with Cd (Österås and Greger, 2000). One explanation of the reduced stem diameter may be that Cd in toxic concentrations can decrease the diameter of xylem vessels in plants and thereby reduce the annual growth rings in Norway spruce (Barcelo and Poschenrider, 1990; Hagemeyer et al., 1994). The toxic effect of Cd and Cu on wood formation is suggested to be related to a deficiency of Ca in wood and bark, since a high accumulation of Cu and Cd in the stems, together with a decreased stem diameter, was associated with a large decrease of the Ca content in wood and bark (II). Low additions of Cd, Cu, and Zn, comparable with spreading of wood ash, as well as the high Zn addition, did not affect any of the measured growth parameters in spruce (II). Similar results were found for young spruce trees (Österås and Greger, 2000). Thus, at a low elevation of heavy metals in forest soil, such as after spreading of wood ash, the tree growth and wood formation should not be affected in a short-term perspective. Higher Cd concentrations in forest soils can probably be expected than those used in the low Cd treatment in paper II, since the Cd content of bark and wood of spruce in the low Cd treatment was 2 to 5 times lower than those found in bark and wood of young Norway spruce in the field (V). Compared with the toxicity threshold value of the internal Cd concentration of the root tissue (Table 2; I), the Cd content of bark in 5-year-old spruce trees on control plots in the field was about 16 times lower (V). Compared with the high Cd treatment in paper II that gave an effect on the stem diameter, the Cd content of bark in 5- year-old spruce trees from control plots in the field was about 8 times lower (V). Thus, the Cd content of wood and bark of spruce trees found in the field should

24 not influence the growth. However, if the Cd content of bark is increased 8 times, toxic effects may occur.

Differences in sensitivity to heavy metals Differences in the response of tree species to heavy metal toxicity have been reported. In accordance with these findings, and as suggested by the hypothesis, differences in sensitivity to Cd among forest tree species were found (I). Deciduous species seem to be more sensitive than conifers (Greszta et al., 1979; Heale and Ormrod, 1982; Patterson and Olson, 1983; Arduini et al., 1996). However, in contrast to these findings, the conifers, pine (Pinus sylvestris L.) and spruce (Picea abies (L.) Karst.), were shown to be more sensitive than the deciduous species, birch (Betula pendula Roth.) (I). The root sensitivity among species increased in the following order: birch < pine < spruce, according to the toxicity threshold (TT95b) values (Table 3). That pine was more resistant than spruce is in agreement with earlier findings (Heale and Ormrod, 1982; Hutchinson et al., 1986; Arduini et al., 1993). Tree species sensitive to heavy metals will be less successful than tolerant species and therefore the pattern of natural regeneration may be affected if heavy metals are accumulated in forest soils (Hüttermann et al., 1999). Thus, an increased accumulation and bioavailability of Cd in forest soils may lead to an altered tree species composition of the forest ecosystem. Even though most tree species are more sensitive to heavy metals than herbaceous plants, some fast-growing species such as Betula spp. (Denny and Wilkins, 1987), Salix spp. (Kahle, 1993) and Acer pseudoplantanus (Turner and Dickinson, 1993) have evolved tolerance to metalliferous soils. Borgegård and Rydin (1989) showed that birch trees not specifically selected for metal tolerance survive when planted in toxic mine spoils, implying that birch has an innate tolerance to heavy metals. However, the shoot dry weight of birch seedlings was decreased at elevated Cd addition but not in spruce and pine in general (I). Shoot growth in birch was inhibited by a lower external Cd concentration than root growth when tested with the modified Weibull function (I). That shoot growth is more sensitive to Cd than root growth in birch has also been shown by Gussarsson et al. (1996). Although the Cd levels tested did not affect shoots of spruce, the effect of Cd on its roots still suggests that spruce is the most sensitive species. However, when comparing shoot and root sensitivity, the differences in sensitivity between the species became smaller than when comparing only root sensitivity. Based on these results (I), it is not enough to evaluate the sensitivity to Cd on a root basis only, but instead the whole plant has to be considered.

25 Table 3. Assessing the response of root dry weight of spruce, pine and birch seedlings from three different ecotypes to the external or internal Cd concentration using the modified Weibull function. Data from Tables 5 and 6 in paper I External Internal

Cd conc. TT95b Cd conc. TT95b Ecotype µg Cd/g FW µg Cd/g FW µg Cd/g DW µg Cd/g DW Spruce southern 0 - 675 3.4 2 - 615 26 central 0 - 675 4.9 4 - 544 27 northern 0 - 675 0.7 5 - 711 25 Pine southern 0 - 1833 6.9 6 - 3413 76 central 0 - 1833 31.0 5 - 4346 162 northern 0 - 1833 9.4 16 - 2441 143 Birch southern 0 - 11123 299 8 - 36393 273 central 0 - 11123 496 2 - 17617 -* northern 0 - 11123 276 7 - 26599 500 Note: The external Cd conc. range is given as total amount of added Cd during 14 d per FW of whole plants in each pot at the start of the treatment. The internal Cd conc. range is the Cd conc. of roots in each treatment. * The central birch ecotype could not be analysed for the internal Cd concentration with this method.

Sensitivity to metals not only varies between tree species but also among clones, varieties and populations within tree species, despite not being subjected to toxic levels of metals (Jastrow and Koeppe, 1980; Geburek and Scholz, 1989; Schaedle et al., 1989; Landberg and Greger, 1994). In accordance with these findings, and as suggested by the hypothesis, differences in sensitivity to Cd within forest tree species were found (I). The roots of the central ecotype were more resistant than roots of the southern and northern ecotypes to Cd in the medium used. This was apparent, since the root dry weight and root length were inhibited at a higher toxicity threshold (ext TT95) in the central ecotype in all three species (Table 3; I). However, differences in sensitivity to Cd in the medium used were significant between the ecotypes of pine and birch, but not of

26 spruce. That the central ecotype of pine was less sensitive than the other ecotypes was also found for the tolerance of roots to internal Cd, i.e. Cd concentration of root tissue (Table 3). Thus, if the Cd content of forest soils is increased it may not only affect different species differently, but also the same species originating from different regions. Populations of the same tree species from different regions have also been shown to vary in parameters such as growth (Ingestad and Kähr, 1985), sensitivity to air pollution (Oleksyn and Bialobok, 1986) and tolerance to cold and drought (Sholz and Stephan, 1982; Mckay, 1994). To tolerate or avoid different kinds of stress, plants have developed different strategies (Baker, 1987; Patra et al., 1994). Some of the strategies of tolerance and avoidance are common for several types of stress whereas other strategies are very specific for the stress type (Ernst et al., 1992). There is little evidence that trees can develop a heritable tolerance to heavy metals (Kahle, 1993). Phenotypic plasticity may be an alternative mechanism to resist metals for long-lived woody plants (Thompson, 1991). Differences in sensitivity to heavy metals between species and ecotypes may depend on several factors such as differences in uptake, accumulation and regulation mechanisms (I). Birch and pine differed in root sensitivity (Table 3) but had similar concentrations of Cd in the root (I). Thus, the more tolerant birch roots must have an internal property to decrease the toxicity of the Cd ion. The higher internal resistance (int TT95) of the root in the central pine ecotype compared with the other ecotypes (Table 3) implies that it also uses properties to take care of the metal in the tissue to avoid Cd toxicity. Increased retention of metals in the roots might be of adaptive significance if shoots are more sensitive than roots (Ernst et al., 1992). Lower uptake of Cu in resistant than in sensitive plants of Silene cucubalus has been found (Lolkema et al., 1986), and sensitive Minuartia hirsuta exhibit proportionately higher shoot translocation of Cu than resistant ones (Ouzounidou et al., 1994). Other evidence is also available for differences in uptake and sensitivity to heavy metals among various plant species, clones, varieties and ecotypes of non-woody plants, but only a few studies have been performed on forest tree species. In birch and spruce, the central ecotype seemed to have the lowest Cd concentration in roots compared with the other ecotypes (I). This implies that in birch and spruce, the central ecotype is more resistant due to low Cd concentration in the roots. These results may be attributed to low net uptake of Cd, the most common property in metal-resistant clones of Salix viminalis (Landberg and Greger, 1996). In addition, the central ecotype of both conifers had a lower Cd translocation to the shoot than the other two ecotypes (I). It is possible that restricted Cd translocation to the shoot confers plant resistance because photosynthesis remains unaffected. Such suggestions were

27 made earlier for different populations of Holcus lanatus and Salix spp (Coughtrey and Martin, 1978; Landberg and Greger, 1996) and negative correlations between resistance and translocation have been found for Salix spp growing on contaminated sites (Landberg and Greger, 1996).

Metal binding in bark and wood The plant cell wall acts as a cation exchanger due to its negative charges, originating from carboxylic groups of different wall substances. In the bark, some of the metals are bound to the negative charges of carboxylic groups of the bark substances (Sjöström, 1993). In the wood the majority of the metals are believed to be bound in a more or less readily exchangeable form to the electrostatic attraction of the negatively charged groups (carboxylic acids) on hemicellulose and pectins (polygalacturonic acids) (Sjöström, 1993; Bailey and Reeve, 1996; Sundén et al., 2000). Other wood compounds that also contain carboxylic groups and can bind metals are lignin and extractives, including free fatty acids and resin acids (Sjöström, 1989). Not all metals are easily exchangeable or soluble, some are present as less soluble salts and some are firmly bound in structures (Sjöström, 1993; Marschner, 1995). The attachment of metal ions was examined in stem sections of Norway spruce by analysing the metal concentration before and after treatment with EDTA (IV). EDTA was expected to remove all metals that were loosely bound to the exchangeable binding sites in the stem, only leaving the fraction firmly bound to the cell walls or other molecules, or in a non-soluble form, e.g. precipitated as salts. As was hypothesised in paper IV, the results suggest that the main part of Ca, Mn and Zn are loosely bound, whereas Cu is firmly bound, to both the wood and the bark matrix. This was apparent, since treatment with EDTA effectively removed most of Mn, Ca and Zn in the stem sections, but did not affect Cu (Table 4; IV). Manganese is suggested to be the metal most loosely bound in the stem of the metals analysed, since it was the metal most easily removed from the outer bark and inner phloem + cambium (Table 4; IV). Similar results have earlier been found and suggested for the metal attachment in the wood of Norway spruce (Sundén et al., 2000). Manganese has earlier been reported to be loosely attached to the wood material (Brelid et al., 1998). Within the stem there were differences in the attachment of Ca, but not of Mn and Zn, since after treatment of the stem sections from the control treated plants with EDTA there still were, or tended to exist, concentration differences of Ca, but not of Mn and Zn, between, e.g., outer bark and wood, and wood and pith (Table 4; IV). This result suggests that the outer bark and pith contain more Ca that is firmly bound or in a non-soluble form, e.g., Ca-oxalate, compared with

28 the wood. However, the Ca peak at the inner phloem + cambium had disappeared, indicating that this Ca-fraction was loosely attached. It was also indicated that especially the outermost part of the outer bark contains a lot of the non-soluble or irreversibly bound fraction of Ca, since the remaining Ca concentration of the outer bark decreased towards the inner phloem + cambium (IV). Enrichment of Ca-oxalate crystals in the secondary phloem of conifers has been suggested to function as a constitutive defence (Hudgins et al., 2003).

Table 4. Remaining concentrations (%) of Ca, Mn, Zn and Cu after treatment with EDTA in different parts of a stem section, of two-year-old Picea abies. The values represent the mean ± SE (n = 2). Significant differences (P ≤ 0.05) between the metals are marked with different letters Inner phloem + Outer bark Wood Pith cambium Metal % % % % Ca 33 ± 6a 15 ± 0a 14 ± 0a 23 ± 5a Mn 5 ± 5b 3 ± 3b 9 ± 9a 14 ± 14a Zn 35 ± 4a 23 ± 5a 30 ± 30a 23 ± 23a Cu 95 ± 5c 104 ± 2c 110 ± 1b 138 ± 2b

Radial distribution and accumulation of metals in the stem Heavy metals can be accumulated in the wood of trees and this can sometimes be used to monitor historical changes in trace metal deposition (Watmough, 1999). The radial distribution and accumulation of metal ions in the wood are dependent on several factors, such as the cation exchange capacity of the tissue, the radial translocation of elements in the stem, the ionic charge density of the ions, the charge density of the nondiffusible anions, the pH of the xylem sap and the activity of competing cations (Nabais et al., 2001). The µ-XRF analysis performed on stem sections from young Norway spruce showed that there are radial distribution patterns of metals in the stem (IV). Calcium, Mn and Zn had a similar radial distribution in the stem, whereas Cu differed from the others (Figure 6, IV). All analysed metals had a higher concentration in outer bark than in wood, even though it is not apparent for Cu in Figure 6. Similarly, the contents of Ca, Cd, Cu and Zn were found to be higher in bark than in wood of young Norway spruce when analysed with AAS (II, III). At the border between bark and wood, of the bark part, where the inner phloem and cambium is located, there was a concentration peak of Ca, Mn and Zn, but not of Cu. These concentrations were the highest found in the stem.

29 Wood

Outer bark IP+C NW Pith

0.30 Ca

0.20

0.10 Figure 6. The radial distributions of Ca, Mn, Zn and Cu (black line), and radial density (grey line), 0.00 in a stem section from a Mn control-treated 2-year-old Norway spruce, analysed 0.20 with µ-XRF. In the upper part of the figure the division of the stem in 0.10 different structural regions are shown. IP + C, inner phloem + cambium; NW, new wood. Left part of 0.30 figure 2 in paper IV. Zn

0.20 Relative and density concentration metal Relative

0.10

0.00 Cu

0.20

0.10

0.00 05001000 1500 2000 2500 Distance (µm)

30 At the pith there were also peaks of Ca, Mn and Zn, but not of Cu, with higher concentrations compared with the rest of the wood (Figure 6). Higher metal contents of bark than in wood of trees are commonly found (Sjöström, 1993), and have earlier been found in Picea abies and also in other tree species (Lövestam et al., 1990; Hagemeyer and Lohrie, 1995). Further, a higher Zn and/or Cd concentration of pith than in the rest of the wood has earlier been found in Picea abies and Fagus sylvatica (Hagemeyer and Lohrie, 1995; Hagemeyer and Schäfer, 1995). As was expected, the content of the metals (Ca, Cu, Cd or Zn) in bark and wood of young Norway spruce increased with elevated addition of the metal in question (Table 5; II; III). Further, as was hypothesised, even low elevated levels of Ca, Cd, Cu or Zn in nutrient solutions, comparable to wood ash application, were found to increase the accumulation of the metal in question in wood and/or bark, except for Cd (Table 5; II). The accumulation of the metals was always higher in bark than in wood. At low addition of the metal in question, similar to wood ash application, the accumulation was enhanced in bark and new wood for Ca, in new wood and old wood for Cu, and in bark, new wood and old wood for Zn, whereas no enhanced accumulation was found for Cd (Table 5, II). At higher addition of Ca, Cd, Cu or Zn all the metals were accumulated in both bark and wood, and the highest increase in accumulation of the metals was in outer bark for Ca, in outer bark and new wood for Cu, in old wood for Cd and in whole wood for Zn (Table 5; II - IV). There were no indications of an elevated accumulation of Ca or Cu in inner phloem + cambium or pith at elevated addition of the metal in question (IV). At toxic additions of Cu and Cd the increase in accumulation was highest in bark for Cu and in whole wood for Cd (Table 5; II). Thus, the accumulation pattern of the metals within the stem differed depending on the level of the addition. Further, the elevated levels of Ca, Cu and Zn, but not Cd, expected after spreading of wood ash, are suggested to be enough to enhance the accumulation of these metals in wood and/or bark of young Norway spruce. This suggestion is in agreement with the results found in the field study of young Norway spruce where waste products from the forest industry had been applied (V). The higher metal concentrations found in inner phloem + cambium (Figure 6) can probably be explained by a higher need of mineral nutrients, such as Ca, Mn and Zn, in the cambial region than the rest of the stem, since this is an active region where new cells are formed. A similar explanation was suggested for the high Ca content in phloem and cambium of Picea abies (Kuhn et al., 1997) and for the high Zn content in the wood near the cambium in Fagus sylvatica (Hagemeyer and Schäfer, 1995). In a study where the subcellular Ca content was determined in phloem, cambium and xylem cells of Picea abies, an extremely high Ca content was found in specialised sieve cells of the inner phloem (Dünish

31 et al., 1998). These cells were assumed to store Ca, probably for cambial cell development and periderm formation. The lack of a Cu concentration peak at the inner phloem + cambium may be explained by the need of Cu in cellular processes being very low, and if this level is only slightly exceeded Cu becomes toxic and can disturb the metabolic processes (Fernandes and Henriques, 1991).

Table 5. Increase in accumulation of metals in bark, new wood (NW) and old wood (OW) of 2-year-old Norway spruce treated with a low or a high elevated addition of Ca, Cd, Cu or Zn for 3 months (II). Enhanced accumulation of the treatment metal is marked with a + sign, and the highest increase in accumulation in the different parts of the stem is marked with two + signs. No effect is marked with 0. The metal additions are given times the control, except for Cd, where the low Cd addition is 3.3 x 10-7 and the high 0.009 given as Cd:K-ratios Treatment Bark NW OW Ca x 4 ++ 0 Ca x 17 ++ + +

Cd low 0 0 0 Cd high +++++

Cu x 1.5 0 ++ + Cu x 30 ++ + +

Zn x 3 ++ + Zn x 100 +++++

One explanation of the higher metal concentrations found in pith (Figure 6) may be that it has a higher binding capacity than the rest of the wood. The cation exchange capacity (CEC) has been shown to be highest at the pith and decreases towards the cambium in stems of mature Picea rubens (Momoshima and Bondietti, 1990). This explanation may also be suitable for the high increase in the accumulation of Cd in the old wood found at elevated additions (III). Accumulation of Cd in the old wood may also be a way of detoxifying the cambial region. In older trees, translocation of toxic substances via the rays into the heartwood has been suggested to be a possible mechanism for detoxification of the live part of the stem (Stewart, 1966). Hagemeyer and Lohrie (1995)

32 suggested similar explanations for the higher concentrations of Cd found towards the pith in stems of five-year-old Norway spruce treated with elevated levels of Cd. The high accumulation of Ca in bark and/or new wood at elevated Ca addition (Table 5) may reflect a higher need of Ca near the cambial region, where new cells are formed, since Ca had a positive influence on growth, increasing the stem diameter (III). Further, the results suggest that elevated Ca addition increases the amount of strongly attached Ca, or more likely, a non-soluble salt, e.g., Ca-oxalate, in outer bark and wood, since significantly more Ca remained in these parts of the Ca-treated plants compared with the control after treatment with EDTA (IV). Spruce trees grown on Ca-rich sites have been shown to contain more Ca-oxalate in the secondary phloem than on Ca-poor sites (Kartusch et al., 1991). Precipitation of Ca in the form of Ca-oxalate may be a way of detoxifying Ca in excess (Webb, 1999). Thus, elevated bioavailability of Ca can increase the accumulation of the non-soluble form of Ca in bark and wood of Norway spruce. The lack of a Cu concentration peak at the pith (Figure 6; IV) and the high increase in accumulation of Cu in new wood (Table 5; II; III) may show that Cu, due to its high binding affinity to polygalacturonic acids in cell walls (Ernst et al., 1992) and low mobility within the wood (McClenahen et al., 1989), foremost is accumulated in the wood closest to the cambial region where new cells are formed and to which the main translocation occurs due to the need of minerals. This explanation may also account for the high accumulation of Cu found in bark (II, III). Another explanation for the high accumulation of Cu in bark and new wood may be, as in the case of Ca, a higher need of Cu close to the cambial region, since it had a positive influence on growth, increasing the stem diameter (III). At higher Cu levels, the high accumulation of Cu in bark may be, as in the case of Ca, a way for the plant to detoxify Cu in excess. A negative correlation was found between the concentration of Ca, Mn, Zn and Cu in the stem, and density of the stem, of young Norway spruce, mainly due to a lower density and higher concentration of metals in bark and pith (Figure 6; IV). A negative correlation was also found in the wood between the density, and the Ca and Mn concentration, indicating that there are higher concentrations of Ca and Mn in early wood than in late wood. Similarly, Lövestam et al. (1990) found higher concentrations of Ca in early wood than in late wood of pine. In addition, Saka and Goring (1983) found that in all analysed morphological regions of the wood in Picea mariana the total inorganic content was higher in early wood compared with late wood. The main function of early wood tracheids is to transport solutes, whereas the main function of late wood tracheids is to provide mechanical support (Sjöström, 1993). Thus, the higher metal content in early wood than late wood can probably be explained by the

33 early wood tracheids being formed during spring and early summer when the growth activity is high, and thereby also the translocation of water and nutrients, whereas late wood tracheids are formed during late summer when growth activity is low and thereby also the translocation of water and nutrients. A similar explanation was suggested by Saka and Goring (1983). The metal accumulation was not confined to the wood formed during the treatment time (Table 5; II; III). This may be explained by all the annual rings of these young spruce trees receiving an input of minerals from the root, since it has been found that the translocation of water in the stem wood of Picea abies is not confined to the outermost rings (Cermak et al., 1992). Another explanation is the radial translocation of metals through xylem tissue via ray cells (Stewart, 1966). Because of these properties, coniferous species are generally not thought to be suitable for monitoring historical changes in trace metal deposition (Hagemeyer, 1993). Thus, even though a change in metal bioavailability is temporary, such as after spreading of wood ash, it will not only affect the metal content of the wood formed during the changes but also the rest of the wood.

Interactions between metals For optimal growth, adequate amounts and proportions of nutrients have to be taken up by the plant. This is a finely tuned system, depending on several factors, one of which is interactions with other ions. If one or another metal ion is supplied in excess, metal ions will compete with each other for binding sites in the tissue, thereby affecting the accumulation of each other. Competition for binding sites occurs particularly between ions with similar physicochemical properties, i.e. charge, valence and ion diameter (Marschner, 1995). Depending on the binding affinity of the metal cations for the binding sites in the plant tissue, they will be more or less competitive. The affinity of metal ions to polygalacturonic acids in cell walls decreases in the order Pb > Cr > Cu > Ca > Zn (Ernst et al., 1992). Further, the affinity of Cd to cell walls has been found to be similar to Zn (Williams and Rayson, 2003). As was hypothesised, metals were found to interact with each other, negatively affecting the accumulation of each other in the stem and/or roots of young Norway spruce (Table 6, II – IV). Calcium was found to decrease the accumulation of Cd, Cu, Mn and Zn, and Cu and Cd to decrease the accumulation of Ca and Zn in wood, bark and/or roots. Even at low elevated Ca or Cu addition, comparable to wood ash application, interactions were found (Figure 7; II). Calcium reduced the accumulation of Cd in bark and Cu reduced the accumulation of Ca in new wood. The accumulation of Cd in bark decreased with increasing addition and accumulation of Ca (Figure 7A), and the

34 accumulation of Ca decreased with increasing addition and accumulation of Cu (Figure 7B). Calcium has earlier been found to reduce the absorption, uptake, translocation and/or accumulation of Cd, Cu, Zn and Mn in other plants (Wallace et al. 1980; Chaudhry and Loneragan, 1972; Jarvis et al., 1976; Tyler and McBride, 1982; Kawasaki and Moritsugu, 1987). Copper and Cd have earlier been found to reduce the uptake, translocation and/or accumulation of Ca and Zn in plants (Gussarsson, 1994; Ouzounidou, 1994; Arduini et al., 1998; Kim et al., 2003; Wu et al., 2003).

Table 6. Summary of changes in accumulation of metals in roots, bark and wood of 2- year-old Norway spruce treated with elevated concentrations of Ca (0.3mM), Cd (0.5µM) and Cu (1.5µM) in indicated combinations, compared with a control (III – IV). Enhanced accumulation of the metal in question is given as a + sign, and decreased accumulation of a metal is given as a – sign. No effect is marked with 0. n.d. = no data Root Bark Wood Treatment Ca Cd Cu Ca Cd Cu Zn Mn Ca Cd Cu Zn Mn Ca +–0 + –––0 +––0 –

Cd 0 + n.d. 0 + 0 – 0 –+000 Cd + Ca ++n.d. 0 + 0 – 00+ 00–

Cu – n.d. + 0 n.d. +–0 – n.d. +–0 Cu + Ca + n.d. ++n.d. 0 – 00n.d. 0 ––

The results found suggest that an elevated bioavailability of Ca in forest soils, due to spreading of liming material such as wood ash, may decrease the accumulation of Cd, Cu, Mn and Zn in the stem and/or roots. A reduced accumulation of heavy metals in roots and stem will probably affect the trees positively if the bioavailability of heavy metals is high, as in the case of acidic soils (Mannings et al., 1996). Further, a reduced accumulation of heavy metals, like Cu and Mn, in the wood can be positive for pulp production, since these ions are known to catalyse the decomposition of the bleaching chemicals (Colodette et al., 1988; Brooks et al., 1994). However, if the accumulation of essential trace elements, like Mn and Zn, are decreased too much it may negatively affect the growth of the trees, since they are involved in the metabolism of the trees. The results found also suggest that an elevated bioavailability of Cu or Cd, due to acidification, deposition of air pollutions and spreading of wood ash, may

35 reduce the accumulation of Ca and Zn in the stem and/or roots. A reduced accumulation of Ca and/or Zn in the stem due to an enhanced accumulation of Cu or Cd may negatively affect the growth, including wood formation, of Norway spruce (II, III).

A B Ca in newCa (mg/g wood DW)

16 0.4 DW)Cd in bark (µg/g 60 1.2 Ca Ca Cd Cu 12 0.3 40 0.8 8 0.2 20 0.4 4 0.1

Ca in bark (mg/g DW) 0 0 0 0 051015 20 (µg/g DW) wood Cu in new 0102030 Addition of Ca Addition of Cu

Figure 7. The Ca and Cd contents in bark (A), and the Cu and Ca contents in new wood (B), of 2-year-old Norway spruce treated with elevated additions of Ca or Cu for 3 months. The addition of Ca is given times the control. Values are means of 5-6 replicates ± SE. Based on data from paper II.

The influence on growth, and accumulation of metals in the stem, was found to be more complex when the addition of two metals was enhanced (Table 6; III; IV). This shows the importance of understanding the interactions between metals before spreading new vitalising and fertilising agents in the forest to make sure that no negative effects will appear on the growth of trees. In all cases, except one, Ca and Cd or Cu is suggested to act synergistically on the Mn and Zn contents of the stem. This was apparent, since the combined treatment with Ca and Cd or Cu increased the negative effect on the accumulation of Zn and/or Mn in the stem compared with adding Ca, Cd or Cu alone (IV). As an exception, Ca and Cu are suggested to act antagonistically on the Mn content of the stem, since the combined treatment with Ca and Cu decreased the negative influence of Ca on the accumulation of Mn in the wood. Calcium and Cd are suggested to act antagonistically on growth, since treatment with Ca and Cd in combination gave a lower fresh weight increase and root dry weight compared with when only the addition of Ca was enhanced (III). The reductive influence of Cd on growth can probably be explained by its negative influence on the accumulation of Ca in the stem. Thus, an enhanced availability of both Ca and Cd may depress the positive growth influence by Ca, due to a negative influence of Cd on the accumulation of Ca in the stem.

36 Enhanced Ca and Cu addition in combination had a larger negative effect on the accumulation of Cu in the stem, compared with when only the addition of Ca was enhanced, and a lower negative effect on the accumulation of Ca in the stem compared with when only the addition of Cu was enhanced (Table 6; III). Thus, if the bioavailability of Cu and Ca are increased at the same time, which may be the initial response in the forest soil after application of wood ash, the results found suggest that accumulation of Ca is less affected by Cu and accumulation of Cu is more depressed by Ca. As was hypothesised, the results from the metal attachment studies showed that the reducing influence of Ca, Cu or Cd on the accumulation of Ca, Cu, Zn and/or Mn in the stem mainly was on the exchangeably bound fraction and not the irreversibly bound fraction of the metal in question. This was apparent, since after treatment of the stem sections with EDTA, in almost all cases, there were no differences in the remaining Ca, Cu, Mn or Zn concentrations of the metal- treated plants compared with the control (IV). As an exception, Cd is also suggested to decrease the firmly bound fraction of Zn in the stem, since the Cd- treated plants had a significantly lower remaining concentration of Zn in outer bark and inner phloem + cambium and the reduction of Zn was higher in these parts compared with the control after treatment with EDTA. The results found suggest that the binding affinity of the competing cations to the binding sites in the tissue decides where in the plant and how much the competing cations will influence each other. Calcium is suggested to be a stronger competitor than Zn, since elevated Ca addition negatively affected the accumulation of Zn in the stem, but not vice versa (Table 4; II). Further, calcium is suggested to be a stronger competitor than Cd, since elevated Ca addition negatively affected the accumulation of Cd in the root, but not vice versa (Table 4; III). Finally, Cu is suggested to be a stronger competitor than Ca, since elevated Cu addition negatively affected the accumulation of Ca in the root, but not vice versa (Table 4; III). These results imply that the competitive ability of the metals tested increases in the order Zn, Cd < Ca < Cu. These findings are in agreement with the affinity of metal ions to cell walls (Ernst et al., 1992; Williams and Rayson, 2003). The main reason for the negative influence found of metals on each other in our studies is suggested to be due to metals with higher affinity reducing the amount of metals with lower affinity to binding sites in the cell wall (apoplast), thereby negatively affecting the uptake, translocation and accumulation of metals in plants. Similarly, Saleh et al. (1999) suggested that the decreased level of Cu and Zn in roots of sugar beet plants (Beta vulgaris) at increased Ca addition was due to Ca reducing the amount of these metals bound in the apoplast. Kawasaki and Moritsugu (1987) showed that Ca can reduce the

37 absorption of Cd, Zn and Mn, and the translocation of Zn and Mn, in excised barley roots. Our results also suggest that even though a metal does not have a higher affinity than other metals to binding sites in the cell wall it can still, if supplied in excess, affect the accumulation of more competitive metals, probably due to mainly an effect on translocation. The negative influence of Cd on the accumulation of Ca in the stem, and of Ca on the accumulation of Cu in the stem, are suggested to be an affect on the translocation, since Cd and Ca did not influence the accumulation of Ca and Cu, respectively, in roots (Table 4; III). Many cations are transported in the xylem via ion-exchange and divalent ions are known to exchange each other in the xylem and to affect each others translocation (Bell and Biddulph, 1963; Shear and Faust, 1970; Fergusson and Bollard, 1976; Wolterbeek, 1987). Cadmium has earlier been suggested to interfere with the internal transport of Ca in trees, since the Ca content of the shoot but not roots of three tree species was reduced by Cd treatment (Arduini et al., 1998). The decreased Ca content of wood and bark at the high Cu and Cd addition in paper II may also depend on the toxic effect on the root growth (not shown), which will affect the uptake of Ca (Marschner, 1995). A similar explanation was suggested for the decreased content of Ca in needles of Picea abies trees treated with lead (Godbold and Kettner, 1991). Thus, at high heavy metal concentrations the Ca content of the wood is probably decreased indirectly by toxic effects on the root and in lower concentrations by competition for binding sites in cell walls.

Influence of waste products from the forest industry on the Ca and heavy metal content in wood and bark Spreading of wood ash from the forest industry will change the bioavailability of metals in forest soil (Bramryd and Fransman, 1995; Rumpf et al., 2001; Arvidsson and Lundkvist, 2003) and thereby probably also the metal content in forest trees. The effect of the ash will vary with the type of soil it is applied onto, the amount spread, and the properties of the ash, e.g. metal content and degree of stabilisation (Demeyer, 2001). The properties of the ash are mainly determined by type of fuel and combustion method used (Etiégni and Campbell, 1991; Someschwar, 1996). Generally, application of wood ash is thought to increase the content of potassium and Ca in plants (Demeyer, 2001), however, the influence of wood ash on the heavy metal content of forest trees is an area that has been scarcely investigated.

38 As suggested by the hypothesis and in agreement with the findings from the nutrient solution studies in paper II, changes in the contents of Ca, Cu, Zn and/or Cd of wood and bark of young Norway spruce trees were found 3 and 6 years after spreading of 3000 kg ha-1 pellets of wood ash (ash) or a mixture of wood ash and green liquor dregs (ash+GLD) on a felling site (Table 7; V). As was also expected, the effect of the waste products on the heavy metal content decreased over time. This is in agreement with the assumption of Eriksson (1998), that wood ash application will only initially increase the release of heavy metals from the mor layer.

Table 7. Changes in accumulation of metals in bark and wood of young Norway spruce 3 and 6 years after application of 3000 kg ha-1 pellets of wood ash (ash) or a mixture of wood ash and green liquor dregs (ash+GLD) on a felling site. Enhanced accumulation of a metal compared with the control is marked with a + sign, and decreased accumulation of a metal compared with the control is marked with a – sign. No change in accumulation is marked with 0. P ≤ 0.1 Bark Wood Treatment Years Ca Cd Cu Zn Ca Cd Cu Zn 30–+0000+ Ash 60– 00 ––00

3 +–0 – 0 – 0 – Ash+GLD 60– 00 0– 00

Treatment with ash increased the accumulation of Zn and Cu, in wood and bark, respectively, 3 years after application (Table 7; V). These findings are in agreement with results from the nutrient solution study in paper II, which showed that elevated levels of Cu and Zn, comparable to wood ash application, would enhance the accumulation of these metals in bark and/or wood of young Norway spruce. The enhanced accumulation of Zn in wood after spreading of ash (V) was similar to the enhanced accumulation of Zn found in new wood in the nutrient solution study at low elevated addition (II). The enhanced accumulation of Cu and Zn after treatment with ash (V) should not negatively affect the growth of Norway spruce (II). Treatment with ash+GLD did not increase the heavy metal content of the stem 3 or 6 years after application (Table 7), implying that it had a lower effect on the heavy metal content of bark and wood in young Norway spruce than

39 treatment with only ash. This may be explained by a lower salt effect, leaking less potassium and sodium, of pellets of ash+GLD, compared with pellets of ash, thereby releasing less heavy metals from the mor layer. In addition, it may also be explained by a higher leakage of Ca from pellets of ash+GLD, compared with pellets of ash, thereby decreasing the accumulation of heavy metals in trees due to competition with Ca for binding, uptake and translocation (II, III, IV). Finally, it may also be explained by a release of humic acids from pellets of ash+GLD, since green liquor dregs have been shown to increase the release of humic acids into the leakage water and humic acids can decrease the uptake of heavy metals, like Cd, in trees (Greger et al., 1998). Thus, looking in a short- term perspective, wood ash pellets should therefore be spread in amounts lower than 3000 kg ha-1, earlier suggested by Greger et al. (1998), or as a mixture with green liquor dregs to decrease or eliminate these effects. However, after 6 years treatment with the waste products there were no increases in heavy metal contents (Table 7; V). Thus, in a longer perspective, spreading of pellets of waste products from the forest industry does not seem to enhance the accumulation of heavy metals, like Cd, Cu and Zn, in wood and bark of Norway spruce. The only treatment that increased the Ca content compared with the control was ash+GLD (Table 7; V). It increased the Ca content of bark after 3 years of treatment, but after 6 years this effect tended to disappear. The accumulation of Ca in bark is in agreement with earlier findings that showed that Ca foremost is accumulated in the outer bark (II, III, IV), and it may reflect that Ca is supplied in excess. However, an increased accumulation of Ca in the stem may positively influence the growth and wood formation of young Norway spruce if the bioavailability of Ca is limited (III; Österås and Greger, 2000). In an earlier study, young birch trees grown for 18 months in a soil treated with a hardened and crushed mixture of wood ash and green liquor dregs, corresponding to 6000 kg ha-1, got an increased Ca content in wood (Greger et al., 1998). Further, it has been found that application of 3000 kg ha-1 crushed hardened wood ash in a young Norway spruce stand increased the concentration Ca in needles five years after application (Arvidsson and Lundkvist, 2002). In contrast to the ash+GLD treatment, the ash treatment did not influence the Ca content in wood and bark after 3 years of treatment and after 6 years it decreased the Ca content of wood compared with the control (Table 7; V). This negative effect of the ash treatment on the Ca content in wood may be explained by competition between Ca and Cu, since the accumulation of Cu in bark was increased 3 years after spreading of ash. Due to its stronger binding affinity to polygalacturonic acids in cell walls, Cu can exchange Ca at its binding sites (Van Cutsem and Gillet, 1982; Ernst et al., 1992), thereby negatively affecting the uptake, translocation and accumulation of Ca (II, III). Only 1.5 times higher

40 addition of Cu, comparable to what can be found in the soil after spreading of ash, was found to decrease the accumulation of Ca in the newly formed wood of young Norway spruce (Figure 7B; II). However, the increased accumulation of, e.g. Cu in wood and bark after ash application, is low compared with trees growing on a Cu mine tailing (V). Further, the Cu content of the bark drops to the same level as the control six years after ash treatment (Table 7). Thus, Cu should not be a problem for the Ca content of the spruce plants in the future. The decrease in accumulation of Ca in the wood by 13 % (Table 7; V) after treatment with ash is only about half of the decrease found in new wood (22 %) or wood (27 %) of young Norway spruce treated with Cu in nutrient solution that did not reduce growth (II; III). In the nutrient solution study in paper II, toxic effects by Cu on wood formation were found when the Ca content of new wood was reduced by 44 % (II). Thus, the decreased accumulation of Ca in the wood after spreading of ash will probably not affect growth in a short-term perspective. However, it ought to be followed up, since if it continues to drop it may cause a deficiency of Ca in the wood, which may negatively affect the wood formation of young Norway spruce. Both treatments decreased the content of Cd in the stem (Table 7; V), which is a positive effect, since Cd is known to be very toxic to plant growth and Norway spruce is very sensitive to Cd compared with other forest tree species (I). A decreased accumulation of Cd in the stem after treatment with ash was probably mainly due to a reduced solubility of Cd due to an increased soil pH (V). A similar effect was suggested for the decreased Cd content of needles in young Norway spruce 5 years after application of crushed hardened wood ash (Arvidsson and Lundkvist, 2002). Another explanation of the decreased Cd content of the stem after treatment with ash may be the increased Zn content of the wood 3 years after the treatment (Table 7; V), since Cd and Zn are competing ions that can adversely affect each others uptake, translocation and binding (Clarkson and Lüttge, 1989; Wu et al., 2003; IV). The decreased Cd content in the stem after treatment with ash+GLD was probably mainly caused by competition with Ca, since the bioavailable fraction of Ca in the forest soil tended to be higher five years after application (V), and the accumulation of Ca in bark was enhanced 3 years after treatment (Table 7; V). A low elevated level of Ca in nutrient solution was found to negatively affect the accumulation of Cd in bark of young Norway spruce (Figure 7A; II). Calcium was also found to reduce the accumulation of Cd in wood and root (Table 6; III). Similar to Cd, the reduced accumulation of Zn found in bark and wood 3 years after spreading of ash+GLD is probably explained by competition with Ca, since the nutrient solution studies showed that Ca could negatively affect the accumulation of Zn in the stem of young Norway spruce (Table 6; II, IV).

41 CONCLUSIONS

Small changes in the bioavailability of Ca, Cu, Cd and Zn in forest soils, such as after spreading pellets of wood ash (ash) or a mixture of wood ash and green liquor dregs (ash+GLD) from the forest industry in the proposed amount of 3000 kg ha-1, will be reflected in an altered accumulation of metals in wood and bark of Norway spruce. It will not only be reflected in changed accumulation of those metals in which bioavailability in the soil has been enhanced, but also of other metals, probably partly due to interactions between metals. These small alterations in accumulation of metals should not affect the growth of Norway spruce, especially since the changes in accumulation of metals are low, and further since they decrease over time. However, as an exception, one positive and maybe persistent effect of the waste products is the decreased accumulation of Cd in wood and bark of Norway spruce, which partly may be explained by competition with Ca for uptake, translocation and binding. A decreased accumulation of Cd in Norway spruce will probably affect the trees positively, since Norway spruce is one of the most sensitive species to Cd of the forest trees in Sweden. Thus, if the bioavailability of Cd is high in the soil, spreading of waste products from the forest industry may be a solution to decrease the accumulation of Cd in Norway spruce. This will, in a long-term perspective, decrease the risk that Cd will alter the tree species composition of the forest ecosystem. An elevated bioavailability of Ca in forest soils, such as after spreading of ash+GLD will, in addition to Cd, probably also decrease the accumulation of other loosely bound heavy metals, like Zn and Mn, in the stem. Further, the accumulation of firmly bound Ca in the stem may also be increased. A decreased accumulation of heavy metals in the wood may be positive for the production of paper pulp, since heavy metals can catalyse the decomposition of the bleaching chemicals, thereby increasing the consumption of chemicals and production cost. Further, if the bioavailability of Ca is limited, an enhanced accumulation of Ca in Norway spruce may increase the growth and wood formation. However, if the leakage of Ca from the waste product pellets is lower, and the salt effect releasing heavy metals of the mor layer is higher, probably as in the case of application of pellets of ash, it may increase the accumulation of Cu and Zn in bark and/or wood initially and temporarily after application. Further, it may also decrease the accumulation of Ca in wood, probably due to competition with Cu, since Cu can reduce the accumulation of Ca in roots, wood and bark of spruce. This decrease in accumulation of Ca should not be enough to affect the growth, but if it continues to drop it might cause a deficiency of Ca in wood of Norway spruce and in the long run negatively affect the wood formation.

42 Mixing wood ash with green liquor dregs seems to eliminate this effect of the wood ash. In a short-term perspective, wood ash pellets should be spread at lower level than 3000 kg ha-1or as a mixture with green liquor dregs to decrease or eliminate the effects on the heavy metal content in wood and bark of Norway spruce. However, in a long-term perspective spreading of wood ash pellets in the amount of 3000 kg ha-1 may be suitable in a sustainable forestry, since the influence of the waste products on the Ca and heavy metal content decreases or disappears over time, and should not affect the growth of Norway spruce.

RGA

Att ge ut vissa doktorsavhandlingar är som att släppa ett rosenblad i Grand Canyon och vänta på ekot (Fritt efter Don Marquis).

To publish certain doctoral thesis is like dropping a rose petal in the Grand Canyon and wait for the echo.

43 ACKNOWLEDGEMENTS

I want to express my sincere gratitude to:

My supervisor Associate Prof. Maria Greger for guiding me trough my time as a PhD student, giving me scientific support and encouragement.
Members of the "Metal group", Tommy Landberg, Åsa Fritioff, Eva Stoltz, Clara Neuschütz, Johanna Nyquist, Wang Yaodong and Agneta Göthberg, for friendship, scientific support, interesting discussions and encouragement.
Prof. Lena Kautsky (assistant supervisor) for very valuable comments on the manuscripts and this thesis.
The Department of Botany (SU), Prof. O. Eriksson (administrative head) and Prof. B. Bergman (head of plant physiology) for providing all facilities for my research and training.
Former members of the metal group, Lars Ekvall, Lisa Ekstig and Ingela Sunnerdahl, for friendship and scientific support.
Robert Berg for help with the metal analysis.
Lars Bengtsson, who sadly passed away, for friendship and help with metal analysis.
Patrik Dimetz for always taking the time to explain statistical problems.
Anders Löfgren and Johan Klint for help with the computers.
The staff of the Department of Botany for being kind and helpful.
Catrin Johansson for the German translation in paper II.
Bengt-Arne Wexell for help with sampling spruce plants in the field.
Bengt Stoklassa for all the help with XRF-analysis and creating images.
Other friends and colleagues for support and friendship, especially my sister, Dr Maria Bergman, and my parents, Marianne and Rolf Gunnar.
Lugnets tree nursery, Assidomän AB, which provided us with plant material.
The journals "Canadian Journal of Botany" and "Journal of plant nutrition and soil science" for permission to print paper I and II.

Financial support from the Swedish Waste Research Council, ÅF foundation and The Swedish Research Council for Environment, Agricultural Science and Special Planning (FORMAS) is gratefully acknowledged.

Last but not least, I want to thank my husband Thomas and my children, Johanna and Harald, for always being there for me, and for making my life special, filled with happiness and love.

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