Does sewage sludge amendment to soil enhance the development of Silver birch and Scots pine? 393 Hungarian Geographical Bulletin 59 (4) (2010) pp. 393–410. Does sewage sludge amendment to soil enhance the development of Silver birch and Scots pine? Dovilė Vaitkutė1, Edita Baltrėnaitė1, Colin A. Booth2 and Michael A. Fullen3 Abstract Sewage sludge can be used to improve forestry soil properties, because it is rich in phos- phorus, nitrogen and organic material and, thus, can enhance the growth of tree seedlings in poor quality soils. Our study was performed on a site amended with industrial sew- age sludge and aff orested with birch and pine seedlings. To evaluate the growth of tree seedlings, tree dry biomass, height, diameter, root/shoot ratio, specifi c root length, shoot and root length were calculated. Higher concentrations of heavy metals and no signifi cant increase in the biomass of trees on sewage sludge amended soil suggest an inhibitory eff ect of heavy metals on tree biomass growth. The site treated with sewage sludge had signifi cantly higher soil moisture content, soil pH, total copper and total lead concentrations and signifi cantly lower exchangeable acidity. Tree tissues at the sewage sludge treated site contain signifi cantly higher concentra- tions of copper and cadmium. Therefore, both positive and negative impacts of treatment are apparent. In terms of management strategies, it is recommended that the chemical quality of sewage sludge is analyzed prior to possible fi eld applications and only sewage sludges with toxic heavy metal concentrations below accepted safety limits are applied. Keywords: Betula pendula Roth., biomass, Pinus sylvestris L., heavy metals, root/shoot ratio, sewage sludge, specifi c root length. Introduction The potential utilization of sewage sludge as a fertilizer in forestry is much debated (Gradeckas, A. et al., 1998; Bojarczuk, K. et al., 2002; Bramryd, T. 2002; Katinas, V. et al., 2002; Pikka, J. 2005; Hermle, S. et al., 2006). Deforested 1 Department of Environmental Protection, Vilnius Gediminas Technical University, Saulėtekio al. 11, Vilnius, LT-10223, Lithuania. Corresponding author: dovile.vaitkute@vgtu.lt 2 School of Technology, The University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1LY, United Kingdom. 3 School of Applied Sciences The University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1LY, United Kingdom. 394 soils usually lack nutrients and are mainly acidic, especially in exploited peat areas (Gradeckas, A. et al., 1998). Sewage sludge contains components that are potentially benefi cial for soils (such as organic matt er, phosphorus, nitrogen, calcium and magnesium). However, sludges can have high concentrations of heavy metals (HMs), especially cadmium (Cd), lead (Pb), copper (Cu) and zinc (Zn), typically originating from industry. At high concentrations, HMs can be phytotoxic and cause reduced tree growth or even death (Kabata-Pen- dias, A. and Pendias, H. 2001). Toxic metal ions present in the substrate may also adversely aff ect trees by damaging roots, which leads to inhibition of the transport of water and nutrients to upper parts of the plant (Kupčinskienė, E. 2006). The distribution and mobility of HMs mostly depends on soil proper- ties, which controls their mobility within soil systems and their availability to trees. Specifi c soil properties (pH, exchangeable acidity (H+ and Al3+), soil moisture, soil texture and organic matt er (SOM)) are the key factors that de- scribe soil quality and tree growing conditions (ICP Forest Manual, 2005). The level of adsorption of HMs, and associated phytotoxicity, mainly depends on tree species. For example, Betula and Salix tree species are con- sidered as metal tolerant and accumulators (Eltrop, L. et al., 1991; Kahle, H. 1993). Experiments on two Salix clones failed to show inhibition eff ects on growth for any HM treatment (max. 41.4 Cd, 655 mg·kg-1 Pb) (Vandecasteele, B. 2004). However, another study revealed that in acidic subsoil Salix viminalis displayed a signifi cant growth reduction following the increased Zn and Cd accumulation (Hermle, S. et al., 2006). In contrast, analysis of birch and pine trees in a site treated with industrial sewage sludge (The Taruškos Forest site, Lithuania) did not reveal any negative eff ects for either tree species (Katinas, V. et al., 2002; Baltrėnaitė, E. and Butkus, D. 2007). Other investigations have shown that birch grown in metal polluted soil decreased above-ground biomass (Bojarczuk, K. et al., 2002). Silver birch (Betula pendula Roth.) grown in polluted substrate was characterized by high biomass allocation to roots (60% versus 30–40% in the control substrate). However, fertilization with sewage sludge, which mainly consists of nutritious organic material, can accelerate tree growth, and increase biomass allocation to foliage (Bojarczuk, K. et al., 2002). The heavy metals Cu, Cd and Pb have phytotoxic and synergistic eff ects (Breckle, S.W. and Kahle, H. 1991; Arduini, I. et al., 1994; Kabata-Pendias, A. and Pendias, H. 2001). For example, Cu is an essential metal for normal plant growth and development. Cu participates in numerous physiological processes and is an es- sential cofactor for many metalloproteins. However, excess Cu inhibits plant growth and disturbs important cellular processes (i.e. photosynthetic electron transport) (Bojarczuk, K. 2004; Yruela, I. 2005). The determined phytotoxic concentrations of Cu in the soil was 60–125 mg·kg-1 (Kabata-Pendias, A. and Pendias, H. 2001). 395 Lead is a general protoplasmic toxic metal, which is cumulative and slow- acting (Sharma, P. and Dubey, R.S. 2005). It has a wide range of negative eff ects on: hormonal status, membrane structure, water potential, electron transport and enzyme activation (Sharma, P. and Dubey, R.S. 2005). However, Pb becomes harmful to plants when concentrations in the soil reaches 100–200 mg·kg-1 (Bergman, W. 1986; Kabata-Pendias, A. and Pendias, H. 2001). When Pb is combined with other metals, it displays synergistic eff ects. For example, root elongation rates of beech (Fagus sylvatica) seedlings were signifi cantly reduced by ~30% by 44 mg·kg-1 plant-available Pb, but the same eff ect was observed with only 24 mg·kg-1 Pb when combined with 2 mg·kg-1 Cd (Breckle, S.W. and Kahle, H. 1991). Decrease in birch biomass was observed when Pb concentrations in soil reached 18 and Cd was 3.6 mg·kg-1. Investigations on diff erent tree species showed that the concentration of metals in soil decreased the growth of shoots and roots by ~50% when Pb concentrations were in the range of 519 to >1280 (285–445) mg Pb kg-1 dry soil and Cu were 48–232 (<40–110) mg Cu kg-1 dry soil, respectively (An, Y.J. 2006). Typically, Cu is more toxic than Pb, and root growth is more sensitive to the toxicity endpoint than shoot growth in Cu or Pb amended soils. Cadmium disturbs the uptake, transport and use of Ca, Mg, P and K and water uptake by plants. Cadmium decreases nitrate absorption and its transport from roots to shoots, by inhibiting nitrate reductase activity in shoots (Balestrasse, K.B. et al., 2003). Arduini, I. et al., (1994) found tap-root elongation of stone pine (Pinus pinea) and maritime pine (Pinus pinaster) was drastically reduced by 5 μm·kg-1 Cd2+ and Cd2+ + Cu2+ treatments. Burton, K.W. et al., (1986) showed a Cd concentration of 2.5 mg·kg-1 signifi cantly decreased the biomass of shoots and roots of Picea sitchensis. The objectives of the investigations were to determine the infl uence of sew- age sludge with high concentrations of heavy metals aft er 10 years from applica- tion on: i) the forest soil properties; ii) growth of Silver birch and Scots pine (Pinus sylvestris L.) trees (biomass, stem diameter, and height), iii) growth traits (root/shoot ratio, specifi c root length, and root/shoot maximum lengths), and iv) heavy metal concentrations in tree components (root, shoots, leaves and needles etc.). Materials and methods Site description The experimental site is located in Gitėnai Forest, near Panevėžys town (Lithuania) (Figure 1). Panevėžys is a Mid-lowland Climatic region and is part of the Mūšos-Nevėžis subregion (Climatic regionalism 2010). The aver- age precipitation in this subregion is 500–600 mm annually, and the prevailing winds are south-westerly. 396 In 1998, ~300 t/ha of industrial sewage sludge was spread on the 2-ha ex- perimental site and aft er one year birch (Betula pendula) and pine (Pinus sylvestris) seedlings were planted. The experiment was started in order to defi ne the indus- trial sludge impact on soil chemical composition aft er 9–10 years of application. A more detailed experimental description is available (Katinas, V. et al., 2002). Table 1 shows the HM concentrations of the industrial sludge (from Panevėžys town) and HM background concentrations of the site soil before experiment started. According to the Lithuanian regulation ‘LAND 20-2005’, the sewage sludge is Category II and can be used in forestry or agriculture only once every three years. High concentrations of Cu and Pb in sewage sludge were due to industrial activities in Panevėžys, typically electroplating and refrigerator manufacturing. Fig. 1. Experimental site of industrial sewage sludge utilization in the Gitėnai Forest within the Taruškos Forest, located in Panevėžys region (Lithuania), (55°44’ N; 24°33’ E) Table 1. Mean concentrations of HMs and phosphorus in Panevėžys industrial sewage sludge and background concentrations in experimental site before trees were planted in 1998 (published in Katinas, V. et al. 2002) HMs Background concentration of experimental site (mg·kg-1) Concentration in industrial sewage sludge (mg·kg-1) Cu Pb Cd P 3.1–9.9 11.0–15.0 2.2–5.0 490.0–273.0 291 1,456 6.2 21,764 397 Soil sampling Sampling was carried out in May 2007. Sites measuring 50x50 m were chosen at the site where the sewage sludge was applied (Site S) and in the adjacent forest area (~200 m from the contaminated plot), which was chosen as the background control (Site C). Before soil sampling the litt er layer was removed. From both the C and S sites, six composite soil samples (mix of fi ve subsamples) were taken (at 0–10 and 20–30 cm depths). Samples were transported at 4°C to the laboratory and then air-dried. For further analysis, soil samples were oven- dried at 105°C and fractionated through a 2.0 mm sieve (Retsch, As 2006). Tree sampling Ten year-old Betula pendula and Pinus sylvestris trees were sampled from both the C and S sites (three birch and pine trees per plot). Tree height and the diam- eter at 30 cm height (±1 mm) above the ground were measured. Leaves or nee- dles, shoots, stem and roots (coarse >2.0 mm and fi ne <2.0 mm diameter) were shredded. Each component was weighed (±0.05 g) and measured (±1 mm). Soil properties Soil moisture content was determined in 15 g of each sample that was dried at 105°C to constant mass. Soil pH was measured by agitating air-dried soil in a mechanical shaker (Gerhardt, Rotoshake RS 12) in 0.01 M CaCl2 solution for 1 hour, and waiting for another hour prior to pH measurement using a calibrated digital pH meter (pH 538 WTV). For total carbon determination, air-dried soil was fractionated through a 2.0 mm sieve (Retsch, As 2006), milled, homogenized and 100 mg soil samples were taken. Total C content was ana- lyzed by dry combustion using a Total Organic Carbon Analyzer (TOC-V by SHIMADZU) at 900°C. Exchangeable acidity was determined in 0.1 mol·l-1 BaCl2 soil solution. Aft er two hours of shaking the soil extract was titrated with a 0.05 mol·l-1 NaOH solution at pH ≤7.8 (ICP Forest Manual, 2006). Total and mobile Cd, Cu and Pb Mobile Cd, Cu and Pb were measured in extraction of neutral salt 0.01 M CaCl2, at 1:10 ratio. The solution was mixed and shaken for 16 h, at 20°C. Wet digestion was employed. Each soil sample (weighing 0.5 g, within 10 ml of HNO3 and 2 ml of HCl solution) was digested for 31 minute in Mileston 398 ETHOS digester (Soon, Y.K. and Abboud, S. 1993). Total metal concentrations in solutions were analyzed using a Buck Scientifi c 210 VGP Atomic Absorption Spectrophotometer (FAAS and GFAAS). Cd, Cu and Pb concentrations in tree seedlings Each tree component (roots, stem, shoots and needles/leaves) was shredded and then incinerated at 400°C to ash. Before HM analysis, tree component ashes were powdered and pressed. Metal analysis was performed using the X-Ray Fluorescence Spectrometer in the University of Wolverhampton (UK), using pulverized samples embedded in a wax base. Statistical analysis Each sample was measured in duplicate. T-test analysis was performed to determine signifi cant diff erences between the two investigation sites (p<0.05). Data analysis was carried out using Statistica (version 7.0) soft ware. Results Soil properties Soil moisture content was higher at Site S than at Site C (Table 2). At Site C soil moisture content varied signifi cantly (p<0.05) and was greater in the upper soil layer than in deeper soil and the diff erence of moisture content values was signifi cant between sites (p<0.05). Site C was signifi cantly (p<0.05) more acidic (topsoil and subsoil) than Site S (Table 2). Exchangeable acidity at Site S was signifi cantly less than at Site Table 2. Selected soil properties (n=6, mean±1SD) in the soil from the control site (C) and the soil amended with sewage sludge (S) Site, soil depth, (cm) Moisture % ±1SDa pH ±1SD a Exchang. acidity, cmol/kg ±1SDb TC, mg·kg-1 ±1SD 0–10 (C) 20–30 (C) 0–10 (S) 20–30 (S) 1.87±0.79 0.50±0.14 3.49±0.44 6.79±0.32 3.15±0.07 3.69±0.11 6.27±0.17 6.13±0.41 1449±85 1199±76 61.3±22.6 73.3±39.6 5.35±0.97 1.33±0.27 5.03±0.90 3.45±0.56 signifi cance between sites ap<0.05, bp<0.01. 399 C (Table 2). However, at Site S the diff erence was not signifi cant with depth, (p>0.05), but it was remarkable at Site C (p<0.05). Between the two sites this diff erence was signifi cant (p<0.01) in both soil layers. Total carbon (TC) varia- tion was similar in both sites and was higher in the surface soil layer (Table 2). However, between the two sites a considerable diff erence (p<0.05) was only found in the 20–30 cm soil layer. Metal contamination of soil Total Cu concentrations were higher at Site S than at Site C (Table 3). Copper concentrations within sites C and S did not vary signifi cantly between soil layers (p>0.05). However, between both sites, Cu concentration diff erences between upper soil layers were signifi cant (p<0.05). Total Cd concentration was signifi cantly less at Site C than at Site S (p<0.05) (Table 3). However, Cd concentration varied with depth insignifi cantly (p>0.05) at both sites. Pb concentrations were signifi cantly (p<0.05) higher at Site S than at Site C. As was the general case with HMs, Pb did not vary sig- nifi cantly between soil layers (p>0.05). The mobile fraction of HMs was distributed in the sequence: Cd>Cu>Pb (Table 3). Contamination of tree tissue Copper concentrations in roots and shoots of both tree species were signifi - cantly higher (p<0.05) at Site S than at Site C (Figure 2). Copper concentrations in the birch tree from Site S was 5.3±0.2 in shoots and 2.5±0.2 mg·kg-1 in roots. At Site C the concentrations of Cu in the birch tree components was 3.3±0.02 in shoots and 2.4±1.3 mg·kg-1 in roots. Table 3. Total concentrations and mobile fraction (mean value ±1SD, n=6) of Cu, Cd and Pb in two soil layers (0–10 cm and 20–30 cm) at the site amended with sewage sludge (S) and at the control site (C) Site, soil depth, (cm) Cu Cd Pb Total a, mg·kg-1 Mobile, % Total a, mg·kg-1 Mobile, % Total a, mg·kg-1 Mobile, % 0–10 (C) 20–30 (C) 0–10 (S) 20–30 (S) 4.00±1.04 4.53±1.92 9.9±0.07 9.35±4.41 4.4±0.8 2.5±0.37 2.1±0.3 1.5±0.2 0.85±0.11 0.75±0.07 1.33±0.18 1.15±0.24 36.1±13.1 47.5±16.5 13.8±0.8 18.7±0.8 24.78±0.63 23.00±1.18 38.83±8.72 42.92±2.42 1.6±0.1 1.6±0.5 0.7±0.2 0.6±0.2 Signifi cance between sites ap <0.05. 400 In pine tree components Cu concentrations from Site S were 4.4±0.4 in shoots and 1.2±0.1 mg·kg-1 in roots. At Site C, higher Cu concentrations were measured in shoots (3.2±0.2 mg·kg-1) than in roots (0.3 ±0.1 mg·kg-1). Diff erences between Cu values in shoots and between Cu values in roots from both sites were signifi cant (p<0.05). In birch tree shoots and roots, Cd concentrations were lower at Site C (Figure 3). In the components of birch trees from Site S, Cd concentrations were 1.7±0.2 in shoots and 1.3±0.1 mg·kg-1 in roots. At Site C the concentra- tions of Cd in birch tree components varied from 1.6±0.05 in shoots to 0.6±0.5 mg·kg-1 in roots. However, Cd concentration diff erences between trees from both investigation sites are insignifi cant (p>0.05). In pine tree components, Cd concentrations at Site S were higher than Site C: 1±0.1 in roots and 0.7±0.01 mg·kg-1 in shoots. At Site C, higher Cd con- centrations were also found in roots: 0.8±0.01 and in shoots 0.7±0.1 mg·kg-1. Cadmium concentrations in roots between investigation sites were signifi - cantly diff erent (p<0.05). Lead concentrations in birch tree components were lower at Site S than at Site C (Figure 4). At Site S the concentration was 0.6±0.03 in shoots and 0.4±0.06 mg·kg-1 in roots. At Site C the concentrations were 1.6±0.1 in shoots and 0.6±0.2 mg·kg-1 in roots. The lead concentration in shoots between inves- tigation sites are signifi cantly diff erent (p<0.05). Fig. 2. Copper concentrations in roots and shoots of both tree species in the site amended with sewage sludge (Birch S; Pine S) and in the control site (Birch C; Pine C). Bars represent mean values of three samples ±1SD 401 Fig. 3. Cadmium concentrations in roots and shoots of both tree species in the site amended with sewage sludge (Birch S; Pine S) and in the control site (Birch C; Pine C). Bars represent mean values of three samples ±1SD Fig. 4. Lead concentrations in roots and shoots of both tree species in the site amended with sewage sludge (Birch S; Pine S) and in the control site (Birch C; Pine C). Bars represent mean values of three samples ±1SD 402 In pine tree components, Pb concentrations were lower at Site C than at Site S. The concentration was 0.4±0.2 in shoots and 0.1±0.01 mg·kg-1 in roots at Site S, Pb concentrations were similar in shoots (0.06±0.03) and roots (0.06±0.04 mg·kg-1) at Site C. Lead concentrations in shoots are signifi cantly diff erent between investigation sites (p<0.05). Tree biomass and growth Total and diff erent tree components dry mass The total biomass of birch and pine trees was less at Site S than at Site C, but these diff erences were not signifi cant (p>0.05) (Table 4). Dry mass of diff erent trees components were greater in the control site, but these diff erences are insignifi cant (p >0.05). However, pine root mass was signifi cantly (p<0.05) less at Site S (16±2 g) than at Site C (30±1 g). Stem diameter and height The diameter (at 30 cm height) of birch trees varied from 1.0–1.8 cm at Site C and from 1.0–1.4 cm at Site S (Table 5). The diameter of pine tree varied from 2.7–3.4 cm at Site C and from 2.0–2.9 cm at Site S and no signifi cant diff erences were detected between stem diameter mean values at both sites (p>0.05). The stem height of both tree species was insignifi cant (p>0.05) between both sites. Table 4. Total dry mass and mass of diff erent components of birch and pine tree in the site amended with sewage sludge (Birch S; Pine S) and in the control (Birch C; Pine C), g/tree; mean value ±1SD, n=3 Tree species Leaves/needles Shoots Stem Roots Total biomass Pine S Pine C Birch S Birch C 111±26 175± 8 14± 4 19± 8 122±18 144± 7 19± 7 28± 3 458±41 528±84 53± 7 70±18 16±2.0* 30±1.0* 12±2.5 24±2.0 749±156 821±21 101±12 112± 5 *diff erence signifi cant, p <0.05. Table 5. The diameter (cm) at 30 cm height and stem height (cm) of birch and pine trees at the site amended with sewage sludge (S) and at the control site (C), mean, ±1SD, n = 3 Tree species Diameter Stem height Birch C Birch S Pine C Pine S 1.2±0.2 1.4±0.4 2.6±0.5 3.0±0.3 188±0.7 223±13 168± 8 190±19 403 Tree development traits The root/shoot ratio of birch tree was signifi cantly (p<0.05) larger at Site C (Table 6). The birch root biomass was even greater (1.78±0.07) at Site C than the mass of shoots. At Site S the ratio was also high (0.80±0.12), compared with the ratios of pine trees. These were 0.13±0.01 at Site S and 0.21±0.02 at Site C, but the diff erence was insignifi cant (p>0.05). The specifi c root length (SRL) of birch tree was signifi cantly shorter at Site S than at Site C (p<0.05). In the case of pine trees, SRL was also signifi cantly shorter at Site S than at Site C (p<0.05). The maximum root and shoot lengths of birch and pine trees were longer at Site C than at Site S. The maximum lengths of pine tree seedlings roots and shoots were also less at Site S. The number (branching) of roots and shoots is an important factor that indicates environmental nutrient status (Figure 5). The branching of birch roots was signifi cantly (p<0.05) less at Site C (11±1) than Site S (22±4). Shoot branching varied from 20±4 at Site C to 25±1 at Site S. The branching of pine shoots and roots was not signifi cantly greater for the pine trees at Site S. The number of roots was 23±9 at Site C and 23±3 at Site S (p<0.05). The number of shoots was: 27±1 and 31±6, respectively (p<0.05). Discussion More favourable growth conditions in soil amended with sewage sludge Soil is important to plants as a source of nutrients and water and has an inher- ent potential to resist (stability) and recover (resilience) from environmental stresses (Griffiths, B.S. et al., 2005). Plant growing conditions depend on many soil properties, including pH, texture, moisture and aeration. It is known that soil moisture, carbon content, exchangeable acidity and pH are good indicators of conditions for vegetation growth (ICP Forest Manual, 2006). Table 6. Root/shoot ratio, Specifi c root length (SRL) (m·g-1), Shoots and roots max. length (cm) of birch (Betula pendula) and pine (Pinus sylvestris) tree at the site amended with sewage sludge (S) and at the control site (C), n=3 ±1SD Tree species Tree development traits Root/shoot ratio SRL Shoot max Root max Birch C Birch S Pine C Pine S 1.78±0.07 0.80±0.12 0.21±0.02 0.13±0.01 0.06±0.01 0.30±0.10 0.19±0.04 0.26±0.08 47.1–77.1 47.5–70.0 47.5–73.5 42.0–67.5 20.4–81.0 13.4–53.0 22.0–47.5 17.5–35.1 404 Soil moisture infl uences the transportation of soil solutions through roots. Lower moisture content can also indicate suppression of the diff usion and the mass fl ow from soil to plants. Moisture content was higher in soil amended with sewage sludge. These results reveal bett er moisture capacity in the site amended with sewage sludge. Changes in soil carbon infl uence physical properties (Denef, K. et al., 2001) and due to higher soil carbon contents soils are physically more stable than non-amended soils (Sort, X. and Alcaniz, J.M. 1999). Soil carbon content was higher at Site S than at Site C. Carbon content of the upper layer was ap- proximately equal at both sites, as it was the case with organic compounds too. However, in deeper layers carbon content was signifi cantly greater at Site S. More acidic soil conditions can increase Al+3 availability, which in turn can disturb the normal development of tree roots and minimize the uptake of macro-nutrients, such as Ca2+ or Mg2+ (Kupčinskienė, E. 2006). Low pH also increases the mobility of toxic HMs, which can be taken up more easily by plants (Kabata-Pendias, A. and Pendias, H. 2001). As soil in the control site is more acidic, it could inhibit the growth of tree seedling. Exchangeable acidity (Al+3, H+) indicates soil disturbances due to high Al+3 concentrations which, as discussed previously, are toxic to plants and Fig. 5. Number of shoots and roots of both tree species in the site amended with sewage sludge (Birch S; Pine S) and in the control site (Birch C; Pine C). Bars represent mean values of three samples, ±1SD.0 405 soil organisms (Sparks, D. 1995). Toxic eff ects of aluminium (acidic, pH <5.5) increase the thickness and stunt root fi bres, leading to decreased assimilation of nutrients from the soil and slowing down plant development (Göransson, A. and Eldhuset, T.D. 1995; Bojarczuk, K. et al. 2002). The results illustrate signifi cant diff erences between the two investi- gated sites indicating 1.60–1.99 times higher pH values, 2.59 times more SOM in deeper soil layers and 1.9–13.5 times higher moisture content. Exchangeable acidity (Al+3, H+) was 23.7 times less in the upper and 16.35 times less in the lower layers of sewage sludge amended soil. This reveals that in the latt er the conditions for the trees are bett er than in that of the control site. Variation of Cu, Cd and Pb in soil Our results reveal Pb is the least mobile heavy metal and Cd tends to eluviate to deeper soil. Furthermore, the mobile fraction of HMs is strongly related to soil pH, as it is one of the main factors infl uencing HM migration (Eckert, D. and Sims, J.T. 1995; Kabata-Pendias, A. and Pendias, H. 2001). HMs in sewage sludge can inhibit tree biomass development. For ex- ample, only 0.005 mg·kg-1 Cd reduced spruce tree root elongation (Arduini, I. et al., 1994) and 0.005 mg·kg-1 Cu in solution can reduce pine biomass (Arduini, I. et al., 1998). In our study plant available Cd was 0.16 mg·kg-1 in soil amended with sewage sludge, which may have had a negative infl uence on pine root elongation, as 0.22 mg·kg-1 Cu have in pine biomass. Furthermore, possible synergistic eff ects should be considered (Arduini, I. et al. 1994). The infl uence of Pb is hard to predict, because it is very stable in the soil and is probably only available in the very acidic soil of the control site. In addition, plant available Pb in the site amended with sewage sludge was ≤0.3 mg·kg-1, which is much lower than the 18 mg·kg-1 that is known to inhibit tree growth (Breckle, S.W. and Kahle, H. 1991). Variation of Cu, Cd and Pb in tree Higher metal concentrations were determined in birch trees than in pine trees, which accords with the hypothesis that birch tends to extract more HMs from soil than pine (Eltrop, L. et al., 1991; Kahle, H. 1993). However, there were minor diff erences between their concentration in trees from sites both C and S. Normal contents of Cd and Cu in plants are 0.1–1.0 and 1–10 mg·kg-1, re- spectively (Kabata-Pendias, A. and Pendias, H. 2001; Kupčinskienė, E. 2006. Copper toxicity in plants may occur when the tissue concentration is >20–30 Cu mg·kg-1. Decreased rates of plant growth occur at tissue concentrations of 406 >3 Cd mg·kg-1 (Pais, I., and Jones, J.B. Jr. 1997). In our study, the concentra- tion in birch tree tissues at Site S was >5 Cd and >4.9 mg·kg-1 Cu and these concentration in pine tree were ~3.9 and 7.0 mg·kg-1, respectively. In addition, no signifi cant relationship between tree growth and HMs accumulation in tree tissues was observed. However, some tendencies were highlighted. For example, higher Cd and Cu concentrations were determined in shoots and roots in trees from Site S, these diff erences being particularly evident in the Cd content of birch roots and in the Cu content of birch shoots. In the case of pine trees, concentrations of both of these HMs were higher in shoots and roots, more signifi cantly in roots. Copper and Cd are inhibitors of tree biomass development, especially tree root systems (Arduini, I. et al. 1994). Tree biomass indications Tree biomass was expected to be less in the control site considering positive infl uence of sewage sludge on soil properties (Pikka, J. 2005). Tree biomass is strongly related to root systems, because poor soil conditions (low pH and high exchangeable acidity (Al+3, H+)) have a negative infl uence on the develop- ment of the root system (Kupčinskienė, E. 2006). In our study, biomass did not signifi cantly change by the application of sewage sludge with the exception of reduced pine root mass (by 50.0%) with- out signifi cant change of root/shoot ratio, which is in contrast to a signifi cant reduction of the latt er in birch. Bett er root system development at Site C is associated with the relatively poor nutritional environment and this tendency is remarkable for birch trees (Påhlsson, A.B. 1991; Bojarczuk, K. et al. 2002; Gradeckas, A. et al. 1998; Katinas, V. et al. 2002; Pikka, J. 2005). Tree growth traits Specifi c root length (SRL) is an important indicator to determine carbon al- location into the root system and indicate a nutritious soil environment. In our study, the roots at Site C had greater mass density, as their SRL value was lower (Eissenstat, D.M. 1991), which indicates the decreased ability of plants to uptake nutrients (Hartikainen, H. et al. 2001). Higher SRL values indicate soils richer in nutrients (Ryser, P. 1996) and exhibits high hydraulic conduc- tivity of roots (Eissenstat, D.M. 1997). The larger the SRL the more eff ective is the strategy of allocation of assimilates to the development of short roots (Lõhmus, K. et al 1989; Ostonen, I. et al. 1999; Wahl, S., and Ryser, P. 2000). However, the length and branching of roots and shoots of birch trees were greater at Site S than at Site C. In addition, roots at Site C tended to elongate 407 more than branches, for example, the maximum length of birch root varied from 20–80 cm and at Site S comparative values were 13–54 cm. Moreover, infertile soils produce root systems with long, poorly branched surface roots; whereas, fertile soils produce well-branched roots that may penetrate deeper into the soil (Crow, P. 2005). In the case of pine trees the branching of roots and shoots was slightly longer at Site S, but their mean length was shorter than at Site C. These diff erences among species can be explained by diff erences in root physiology, for example, the highest concentration of birch coarse roots accumulates in deeper layers (13–16 cm) than pine trees (5–15 cm) (Laitakari, E. 1934). In fertile soil, roots penetrate easier into deeper layers. The spread sewage sludge was contaminated with HMs, but the leaching of HMs into deeper layers may have been suppressed by organic matt er from sewage sludge, the surface peat layer and the Fe-Mn geochemical barrier (Katinas, V. et al. 2002). The production of longer, thinner roots may be an important mecha- nism to compensate for reduced carbon allocation to, and dry matt er accumu- lation by, roots of trees exposed to pollution. These factors may also help ex- plain diff erences of root/shoot ratio between sites. To adapt to less favourable conditions at Site C, trees had greater propensity to develop root systems at the expense of above-ground biomass. The roots at Site S, which have greater absorptive surfaces, could more easily transport nutrients and water to sur- face parts and expand above-ground biomass. However, the roots were thin and long, and carbon accumulation was less. This could lead to weaker root systems and increased risk of mechanical disturbance (e.g. by strong winds and fl oods). The production of longer, thinner roots may be an important mechanism to compensate for reduced carbon allocation to, and dry matt er accumulation by, roots of trees exposed to pollution. Site S had more favourable conditions for tree growth. However, plant biomass and tree growth trends (except root branching) did not support this tendency. This suggests that sewage sludge might have additional constituents (e.g. heavy metals) that inhibited tree development. Conclusions The results reveal that soil amended with sewage improves soil quality. How- ever, higher concentrations of metals and no signifi cant increase in the biomass of trees in soil amended with sewage sludge suggest an inhibitory eff ect of heavy metals on tree biomass growth. During a 10 year period, pine trees produced 87% more biomass and accumulated ~60% more heavy metals (Cu, Cd and Pb) than birch trees. 408 For indication of soil nutritious environment it is recommended to use the following tree functional traits: specifi c root length (SRL), root/shoot ratio, root branching; and for possible toxicity eff ect of heavy metals or other harmful compounds: tree height and stem diameter (if trees are of the same age) and tree biomass (dry mass). Acknowledgements: Scientific research was carried out under the implementation of projects funded by the Agency for International Science and Technology Development Programmes in Lithuania under COST Action 639 (Greenhouse gas budget of soils under changing climate and land use (BurnOut)) and COST Action 859 (Phytotechnologies to promote sustainable land use and improve food safety). Finally, all the authors would like to express their gratitude to the technical staff at both universities for the assistance with this research. REFERENCES An, Y.J. 2006. Assessment of comparative toxicities of lead and copper using plant assay. Chemosphere, 62. 1359–1365. Arduini, I., Godbold, D.L. and Onnis, A. 1994. Cadmium and copper change root growth and morphology of Pinus pinea and Pinus pinaster seedlings. Physiologia Plantaru, 92. (4): 675–680. Arduini, L., Godbold, D.L., Onnis, A. and Stefani, A. 1998. Heavy metals infl uence mineral nutrition of tree seedlings. Chemosphere, 36. 739–744. Balestrasse, K.B., Benavides, M.P., and Gakkego S.M. 2003. Eff ect of cadmium stress on nitrogen metabolism in nodules and roots of soybean plants. Functional Plant Biology, 30. 57–64. Baltrėnaitė, E. and Butkus, D. 2007. Accumulation of heavy metals in tree seedlings from soil amended with sewage sludge. Ekologij a, 53. (4): 68–76. Bergman, W. 1986. Farbatlas Ernährungsstörungen bei Kulturpfl anzen. (In English: Atlas of nutritional disorders in culture plants.) Fischer, Jena. Bojarczuk, K. 2004. Eff ect of toxic metals on the development of Poplar (Popus tremula×P. alba) cultured in vitro. Polish Journal of Environmental Studies 13. (2). 115–120. Bojarczuk, K., Karolewski, P., Oleksyn, J., Kieliszewska-Rokicka, B., Zytkowiak, R., and Tjoelker, M.G. 2002. Eff ect of polluted soil and growth and physiology of silver birch (Betula pendula) seedlings. Polish Journal of Environmental Studies 11. (5): 483–492. Bramryd, T. 2002. Impact of Sewage Sludge Application on the Long-Term Nutrient Balance in Acid Soils of Scots Pine (Pinus Sylvestris, L.) Forests. Water, Air and Soil Pollution. 140. (1–4): 381–399. Breckle, S.W. and Kahle, H. 1991. Eff ects of toxic heavy metals (Cd, Pb) on growth and mineral nutrition of beech (Fagus sylvatica L.). Plant Ecology, 101. (1): 43–53. Burton, K.W., Morgan, E.A. and Roig A. 1986. Interactive eff ects of cadmium, copper and nickel on the growth of Sitka spruce and studies of metal uptake from nutrient solutions. New Phytologist, 103. (3): 549–557. Climatic regionalism. 2010. Available at: htt p://www.meteo.lt/klim_rajonavimas.php (Looked in: 2010 05 18) 409 Crow, P. 2005. The infl uence of soil and species on tree root depth. Forestry Commission Information Note. Available at: www.forestresearch.gov.uk (Accessed 24 November 2008). Denef, K., Six, J., Paustian, K. and Merckx, R. 2001. Importance of macroaggregate dynam- ics in controlling soil carbon stabilization: short-term eff ects of physical disturbance induced by dry-wet cycles. Soil Biology and Biochemistry, 33. 2145–2153. Eckert, D., Sims, J.T. 1995. Recommended Soil pH and Lime Requirement Tests. Chapter 3. In: Recommendation for Soil Testing Procedures. For North Eastern United States. Second edition. Eissenstat, D.M. 1991. On the relationship between specifi c root length and the rate of root proliferation: a fi eld study using citrus rootstocks. New Phytologist, 118. (1): 63–68. Eissenstat, D.M. 1997. Trade-off s in root form and function. In: Jackson L.E., ed. Ecology in Agriculture. San Diego (173–199), CA, USA: Academic Press. Eltrop, L., Brown, G., Joachim, O. and Brinkmann, K. 1991. Lead tolerance of Betula and Salix in the mining area of Mechernich, Germany. Plant and Soil, 131. 275–285. Göransson, A. and Eldhuset, T.D. 1995. Eff ects of aluminium ions on uptake of calcium, magnesium and nitrogen in Betula pendula seedlings growing at high nutrient sup- ply rates. Water, Air and Soil Pollution, 83. 351–361. Gradeckas, A., Kubertavičiene, L. 1998. Utilization of wastewater sludge as a fertilizer in short rotation forests on cut away peatlands. Baltic Forestry, 4. (2): 7–13. Griffiths, B.S., Hallett, P.D., Kuan H.L., Pitkin, Y. and Aitken, M.N. 2005. Biological and physical resilience of soil amended with heavy metal-contaminated sewage sludge. European Journal of Soil Science, 56. 197–205. Hartikainen, H., Pietola, L., Simojoki, A. and Xue, T. 2001. Quantifi cation of fi ne root responses to selenium toxicity. Agricultural and Food Science in Finland, 10. 53–58. Hermle, S., Günthardt-Goerg, MS. and Schulin, R. 2006. Eff ects of metal-contaminated soil on the performance of young trees growing in model ecosystems under fi eld conditions. Environmental Pollution. 144. (2). 703–14. ICP Forest Manual. 2006. Part III. Sampling and Analysis of Soil, available at: (Accessed 2 September 2008). Kabata-Pendias, A. and Pendias, H. 2001. Trace elements in soils and plants. Third edition. ISBN 0849315751. CRC Press. Kahle, H. 1993. Response of roots trees to heavy metals. Environmental and Experimental Botany, 33. (1): 99–119. Katinas, V., Kadūnas, V., Radzevičius, A. and Zinkutė, R. 2002. Processes of chemical element dispersion and redistribution in the environment with wastewater sludge used for recultivation of woodcutt ing areas. Geologij a, 38, 3–11. Kupčinskienė, E. 2006. Latentiniai paprastosios pušies pakitimai lokalios taršos aplinkoje (Latent injuries of Scots pine (Pinus sylvestris L.) under the infl uence of local pollution] Kaunas: Lututė. Laitakari, E. 1934. Koivun juuristo (Summary: The root system of birch, Betula verrucosa and odorata). Acta Forestalia Fennica, 40. 853–901. Lõhmus, K., Oja, T. and Lasn, R. 1989. Specifi c root area: A soil characteristic. Plant and Soil, 119. 245–249. Ostonen, I., Lõhmus, K. and Lasn, R. 1999. The role of soil conditions in fi ne root ecomor- phology in Norway spruce (Picea abies (L.) (Karst). Plant and Soil, 208. 283–292. Påhlsson, A.B. 1991. Infl uence of aluminium on biomass, nutrients, soluble carbohydrates and phenols in beech (Fagus sylvatica). Physiologia Plantarum, 78. (1): 79–84. Pais, I., and Jones, J.B. Jr. 1997. The Handbook of Trace Elements. St. Lucie Press, Boca Raton, Florida. 410 Pikka, J. 2005. Use of wastewater sludge for soil improvement in aff oresting cutover peat- lands. Metsanduslikud uurimused/Forestry Studies, 42. 95–105. Ryser, P. 1996. The importance of tissue density for growth and life span of leaves and roots: a comparison of fi ve ecologically contrasting grasses. Functional Ecology, 10. 717–723. Sharma, P. and Dubey, R.S. 2005. Lead toxicity in plants. Brazilian Journal of Plant Physiology, 17. (1): 35–52. Soon, Y.K. and Abboud, S. 1993. Total Heavy Metals. In Soil Sampling and Method of Analysis. Ed.: Carter, M.R. Lewis Publishers, USA. Sort, X. and Alcaniz, J.M. 1999. Eff ects of sewage sludge amendment on soil aggregation. Land Degradation and Development, 10. 3–12. Sparks, D. 1995. Environmental Soil Chemistry. San Diego, USA, Academic Press. Vandecasteele, B., Meers, E., Vervaeke, P., De Vos, B., Quataert, P. and Tack, F.M.G. 2004. Growth and trace metal accumulation of two Salix clones on sediment-derived soils with increasing contamination levels. Chemosphere, 58. 995–1002. Wahl, S., and Ryser, P. 2000. Root tissue structure is linked to ecological strategies of grasses. New Phytologist, 148. 459–471. Yruela, I. 2005. Copper in plants. Brazilian Journal of Plant Physiology, 17. 145–156. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.3 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize false /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. Stvoreni PDF dokumenti mogu se otvoriti Acrobat i Adobe Reader 5.0 i kasnijim verzijama.) /ITA /JPN /KOR /LTH /LVI /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /POL /PTB /RUM /RUS /SKY /SLV /SUO /SVE /TUR /UKR /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) /HUN >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /ConvertToCMYK /DestinationProfileName () /DestinationProfileSelector /DocumentCMYK /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice