Peat influence on Zn tolerance , bioconcentration and bioaccumulation in Eucalyptus grandis Hill ex Maiden

1 Department of Agronomic and Environmental Sciences, Universidade Federal de Santa Maria, Campus Frederico Westphalen, City of Frederico Westphalen, State of Rio Grande do Sul, Brazil. 2 Post-Graduation in Agronomy, Universidade Federal de Pelotas, City of Pelotas, State of Rio Grande do Sul, Brazil. 3 Department of Environmental Engineering, Centro Universitário Franciscano, City of Santa Maria, State of Rio Grande do Sul, Brazil. 4 Post-Graduation in Soil Science, Universidade Federal de Santa Maria, City of Santa Maria, State of Rio Grande do Sul, Brazil.


INTRODUCTION
Zinc is a micronutrient, normally present in the soil in concentrations of up to 70 mg kg -1 plant-available Zn; however, concentrations above 100 and up to 500 mg kg increase zinc content in the soil.Brazilian law, through Resolution CONAMA 420 2009, fixed a reference limit of 450 mg Zn kg -1 for intervention in agricultural areas where strategies and techniques are needed to mitigate the deleterious effect of zinc accumulation.The use of tree species is considered an important technique for the recovery of metal-contaminated areas, because trees typically have large biomass production and a long growth cycle (Domínguez et al., 2009).Among tree species, E. grandis is favored due to its rapid growth and high biomass production (Souza and Fiorentin, 2013).However, the accumulation of heavy metals and nutrients in eucalyptus roots has been little studied, especially due to difficulty in determination of its content (Robinson, 2003).Silva et al. (2015), working with Corymbia citriodora, Eucalyptus saligna, Eucalyptus grandis and Eucalyptus dunnii seedlings showed a reduction of, respectively, 35, 56, 60 and 81% in shoot dry mass with increasing doses of Zn.No differences in response of plant species to Zn level were found.In environments where heavy metal contents in the soil are high, establishment of plants is restricted, and the use of other techniques that mitigate the effects of contamination is required.
The possibilities for reducing contamination levels include the use of both organic and inorganic materials.One consequence of an application of organic amendments is a decrease in the availability of metals via adsorption and complexing reactions in the soil, thus reducing absorption by plants (Park et al., 2011).One example of such an amendment is peat, which, once applied to the soil, tends to make Zn less bioavailable (Pichtel and Bradway, 2008).Peat is described by George et al. (2010) as a natural organic material, stabilized and recognized for its superior ion exchange capacity.According to Santos and Rodella (2007), peat reduced available Zn by up to 12% in comparison with that in the control treatment.
Phytoremediation is the use of plant species as an alternative for the recovery of contaminated areas.Tree species, specifically young plants, are more sensitive to soil contaminated by metals (Souza et al., 2012), which facilitates studies focused on the growth of seedlings in contaminated soil.However, some questions still remain regarding the use of peat in ameliorating soil contaminated with Zn and its ability to enable greater growth, increase tolerance, and lower absorption of Zn by eucalyptus.Therefore, the hypothesis underlying the present study is that peat has an ameliorating effect in zinc-contaminated soils with the concurrent development of E. grandis.In this context, this study aimed to evaluate the influence of peat on the development, tolerance, bioconcentration and bioaccumulation of Zn in E. grandis seedlings.

MATERIALS AND METHODS
The experiment was conducted in a greenhouse belonging to the da Silva et al. 321 Agricultural College of Frederico Westphalen, Rio Grande do Sul, south region of Brazil, between the months of May and September, 2015.The soil is characterized as an oxisol and was collected in the agricultural area in the Federal University of Santa Maria, campus Frederico Westphalen, Rio Grande do Sul.Green® peat was used as the organic amendment, and was characterized according to the methodology of Tedesco et al. (1995); results are presented in Table 1.Experimental units were composed of 600 cm 3 soil in polyethylene plastic bags containing one seedling each.E. grandis Hill ex Maiden seeds was provided by the Forestry Research Center of the State Foundation for Agricultural Research (FEPAGRO -RS), in Santa Maria, RS.The seeds were sown in seedbeds and transplanted into experimental units upon presentation of a pair of true leaves.The experimental design was completely randomized in a factorial arrangement (2 x 6), with and without the addition of peat (200 mL L -1 soil (v:v)) and six doses of Zn (zero, 200, 400, 600, 800 and 1000 mg kg -1 ) with 15 repetitions.Zn doses were applied 30 days prior to transplanting in the form of zinc acetate dihydrate solution (C4H6O4Zn.2H2O)diluted in 50 ml of water.In order to homogenize the soil, the plastic bag was shaken.After 15 days of soil contamination, peat was added and the soil left for another 15 days prior to transplanting seedlings.A sample from each treatment was taken for the determination of pseudototal Zn content according to the USEPA method 3050b (USEPA, 1996).
The experiment was conducted for 120 days after transplanting.During this period, daily irrigations were performed, based on the weight of the experimental units, keeping the moisture content at 80% of field capacity.Base fertilization was performed, applying the equivalent of 150 g of N, 700 g of P2O5 and 100 g of K2O per cubic meter of soil.Covered fertilization was done using 100 g of N and 30 g of K2O diluted in 10 L of water.Fertilization after transplanting was carried out in three stages: 30 days with the application of N and K; 60 days only N; and 90 days applying N and K, following the recommendations of Gonçalves and Benedetti (2005).In order to meet the requirements of design, experimental units were rotated weekly.
At the end of the experiment, the following parameters were evaluated: plant height (H), measured with graduated ruler (from seedling lap to the stem apex); stem diameter (SD), measured with a digital caliper with a precision of 0.01 mm; dry mass of the root system (DMR) and shoot (DMS), both fractions having been separated at the cervical region, dried in an oven at 60 ± 1°C to constant weight, and weighed on an analytical balance accurate to 0.0001 g.The total dry matter (TDM) was obtained as the sum of DMR and DMS.Specific surface area (SSA) of the roots was estimated using the methodology of Tennant (1975).
After weighing the root and aerial dry mass, the material was ground in a Wiley-type grinder (10 mesh sieve) to determine the concentration of Zn in the plant tissue through nitric-perchloric acid digestion (3:1) followed by determination by atomic absorption spectrophotometry as described by Miyazawa et al. (2009).
The index of tolerance (Toi) was calculated according to Equation 1 using the following parameters: TDM and zinc concentrations (mg kg -1 ) of the root system (ZnR) and shoot (ZnS), accumulated amounts of zinc (μg plant -1 ) in the root system (ZnAR) in shoot (ZnAS), and total accumulated zinc in seedlings (ZnAT) in the zero dose treatment (d0) and at doses of 200 to 1000 mg kg -1 (dn).The index of tolerance measures the ability of seedlings to grow in environments with high metal concentrations (Wilkins, 1978).The translocation index (Tri) was calculated according to Equation 2, and indicates is the total percentage of absorbed zinc which was transported to the shoot (Abichequer and Bohnen, 1998).Also, estimates were bioconcentration factor, by the ratio of metal concentrations in the roots (mg kg -1 ) and the pseudototal concentration in soil (mg kg -1 ), and the bioaccumulation factor, determined as the ratio of the metal concentration in shoots (mg kg - 1 ) and the pseudototal concentration in soil (mg kg -1 ).(1) (2) The results were subjected to variance analysis and when significant interactions were obtained, quantitative factor regression analysis was performed within each level of the qualitative factor, using the SISVAR program (Ferreira, 2011).

RESULTS AND DISCUSSION
Soil analysis after contaminant addition showed increases in the amount of zinc (Zn) in the soil with increasing doses, independent of peat application (Figure 1).The National Environmental Council, through Resolution CONAMA 420 2009, sets the reference limits for intervention in different areas, with 450 mg kg -1 , as the intervention threshold for Zn.In this experiment, artificial conditions of Zn contamination were efficiently created by addition of zinc acetate solution, thus enabling the realization of this work.
Using the analysis of variance, a significant interaction (p ≤ 0.05) is evident among Zn and peat treatments for all variables measured in this study (Figures 2, 3 and 4).The height of E. grandis seedlings was reduced significantly by increasing doses of Zn, both with or without the use of peat.With 200 ml L -1 of peat in the soil, seedling height was higher as compared to the treatment without peat (Figure 2A).This effect may be attributed to the supply of additional essential nutrients provided to the plants from peat as shown in Table 1.However, Magalhães et al. (2011) studied the phytostabilization of soil contaminated with Zn in two species of Eucalyptus (E.urophylla and E. saligna), and observed that the application of alleviation materials to the substrate with high metal concentrations positively affected the development of the two species studied, reducing the contaminant metal effect.A high Zn content in soil greatly reduces plant growth, as compared to plants grown in uncontaminated soils (Gichner et al., 2006).In pinion pine plants, Chaves et al. (2010) also observed a height reduction in treatments with Zn, confirming the detrimental effect of Zn contamination on plant growth.
Similar to seedling height, stem diameter was significantly reduced with increasing doses of Zn, but with less intensity in the treatments with peat (Figure 2B).This effect was also observed by Chaves et al. (2010), who also showed a significant reduction of stem diameter in treatments with copper and zinc, a reduction that was less intense in the presence of peat.This increased stem diameter with the addition of peat, in spite of the presence of metal contaminants, which may be associated with additional elements essential for plant growth (Taiz and Zeiger, 2013), in this case, P and K as shown in Table 1; this effect is confirmed in the zero Zn treatment.
The dry shoot weight, root weight and specific surface area were higher in treatments with peat, and showed reductions in both treatments with increasing doses of Zn (Figure 2C, D and E).According to Pereira et al. (2010), increased root and shoot dry mass due to the addition of alleviation materials is an important feature in phytostabilization strategies of heavy metals, contributing to a greater accumulation of metals in these plants.According to Carneiro et al. (2002), the toxicity of Zn in plants reduces the production of dry matter of shoot and root biomass.On the other hand, the addition of peat can promote development of the radicular system, because in addition to the nutritional aspect, it also improves the physical properties of the soil (Franchi et al., 2003).
The root specific surface area was reduced quadratically in the treatment without peat and reached a maximum at an estimated dose of 83.33 mg Zn kg -1 soil with the use of peat, being significantly higher with the addition of peat and 200 mg of Zn (Figure 2F).High concentrations of Zn in the soil generally reduce root length (Hooda, 2010), directly affecting specific surface area.Carneiro et al. (2002), working with herbaceous species in soils with different contaminants, reported that the stimulation of root growth is important as part of the phytostabilization of areas contaminated with heavy metals; furthermore, greater root growth gives more protection to the soil against erosion, reduces leaching, enhances aggregation and stimulates microorganism activity.The present data show that peat stimulated root growth, therefore confirming the possibility that these associated benefits will also be observed in similar situations.
Zn content increased linearly in roots with Zn dose (Figure 3A), whereas in the shoot, there was a quadratic trend, with an increase in Zn doses of 886.93 and 908.46  mg kg -1 soil, with and without peat respectively, being significantly lower in treatments with peat (Figure 3B).Other research has shown that increasing amounts of metals in soil increase the levels of these metals in the shoots and roots of the tree species, Salix humboldtiana Willd (Gomes et al., 2011), in the leaves of Myracrodruon urundeuva Fr.Allem (Gomes et al., 2013) and in the shoots of E. grandis (Silva et al., 2015).In addition, there are reports that humified organic materials reduce the availability of cations such as Zn (Jacundino et al., 2015), and Zn becomes less bioavailable with peat application (Pichtel and Bradway, 2008).This confirms the observed reduction in Zn levels in the roots and shoots of E. grandis seedlings upon application of 200 ml peat L -1 soil.The accumulation of Zn in roots was significantly higher with the use of peat as compared to treatments without peat, reaching a maximum at a Zn application of 660 mg kg -1 soil, in contrast to treatments without peat, in which a Soil+peat (200 mL of peat L -1 of soil) Soil decreasing linear tendency was observed (Figure 3C).The highest accumulation of Zn in roots in the presence of peat is related to the greater amount of root biomass produced in this treatment (Figure 2E) and is estimated as a 55.2% increase in accumulation of Zn with the use of peat and a reduction of 66.6% without peat, when the highest dose is compared to the zero dose.Plants have various mechanisms to tolerate excess metals; zinc can be immobilized onto cell walls or complexed with nondiffusible proteins in plant roots (Kabata-Pendias, 2010).Zinc accumulation in shoots was significantly higher in the treatment with peat, however, regression equations did not produce a suitable fit (Figure 3D).This is an indication that peat did not act as ameliorating factor to zinc contamination in the soil, but rather stimulated growth and plant biomass production, leading to greater Zn content in shoots.Santos and Rodella (2007) found that peat was less efficient than a mineral-humic compound in reducing the availability of Cu, although peat did favor greater plant development.
The Zn translocation ratio observed in the present study was significantly higher in peat treatments, whereas treatment without peat reached a maximum at 389 mg kg -1 Zn in soil (Figure 3E).The peat had a stimulating effect on the growth of E. grandis and thus may have provided greater absorption of nutrients and Zn.Silva et al. (2015), in contrast, working in a Latossolo soil contaminated with Zn, found a maximum Zn translocation of 515.4 kg -1 mg in E. grandis seedlings.Branzini et al. (2012) studied the absorption and translocation of Cu, Zn and Cr in Sesbania virgata, and commented on the existence of mechanisms that prevent translocation of Zn to shoots in order to avoid damage to aerial parts.Thus, the translocation index values indicate low translocation of this metal in E. grandis seedlings, suggesting that this species may possess a physiological mechanism that prevents translocation of Zn to shoots of seedlings.The tolerance was significantly higher with peat and showed minimal points at 986 and 784 mg kg -1 of soil Zn with or without peat, respectively (Figure 3F).
Tolerance values between 35 and 60% are generally considered as moderate tolerance (Lux et al., 2004).Thus, in the presence of peat, E. grandis showed a relatively high tolerance to doses less than 451 mg kg -1 Zn, while without peat low tolerance is observed at dose higher than 315 mg kg -1 Zn.It is known that tolerant species can reduce metal toxicity by immobilization or compartmentalization in the roots (Saraswat and Rai, 2011).Thus, the addition of peat, in part due to its stimulation of root growth, provides greater tolerance to E. grandis seedlings in soil contaminated with Zn.
The bioconcentration factor shows a minimum in the treatment with peat at an estimated dose of 632 mg kg -1 Zn and a decreasing linear effect without peat, although values for bioconcentration factor without peat remained mostly above those of plants grown with peat (Figure 4A).The bioconcentration factor is greater than one at Zn doses above 387 and 679 mg kg -1 with and without peat, respectively.McGrath and Zhao (2003) determined that plants showing a bioconcentration factor lower than one are not recommended for phytoextraction.Thus, it is possible to use E. grandis as a phytoextracting plant in soils with appropriately low concentrations of Zn.However, the use of peat must be evaluated in each specific situation, as it may lower the phytoextracting potential of this species at the intermediate soil Zn levels used in the present study (approximately 300 to 800 mg kg -1 ).The bioaccumulation coefficient was significantly higher in the treatments without peat as compared to treatments with peat above 400 mg kg -1 Zn, with a maximum point at 299 mg kg -1 without peat and 20 mg kg -1 with peat (Figure 4B).The coefficient of bioaccumulation was lower than the bioconcentration factor in all treatments because all treatments were below the classificatory value, indicating that E. grandis has only a limited ability to absorb soil Zn and translocate it to shoots.

Conclusions
Addition of 200 mL L -1 of peat to soil provides increased tolerance and a stimulating effect on morphological parameters of E. grandis seedlings.This species, however, does not meet the necessary criteria to be considered a zinc bioconcentrating and bioaccumulating plant.

Figure 1 .
Figure 1.Zinc content in the soil due to zinc doses applied with and without addition of peat (200 mL peat L -1 soil).

Figure 2 .Figure 3 .
Figure 2. Regression equations for the height (A); stem diameter -SD (B); dry matter of shoots -DMS (C); dry matter of roots -DMR (D); Total dry matter -TDM (E) and surface area specific -SSA (F) in Eucalyptus grandis seedlings subjected to increasing Zn doses in the absence and presence of peat (200 mL L -1 peat soil).LSD = least significant difference.

Figure 4 .
Figure 4. Regression equations for bioconcentration factor (A) and bioaccumulation factor (B) in Eucalyptus grandis seedlings subjected to zinc doses in the absence and presence of peat (200 mL L -1 soil).LSD = least significant difference.

Table 1 .
Chemical analyses of soil and soil plus peat used for the Eucalyptus grandis seedling growth trial.