Copper and zinc fractions in the profile of an Inceptisol cultivated with apple in southern Brazil

ABSTRACT

The present study aimed to evaluate the accumulation and distribution of Cu and Zn fractions in the profile of an Inceptisol by application of fungicides with a previous history of apple cultivation in southern Brazil. Stratified soil samples (0.0 – 0.025 m, 0.025 –0.050 m, 0.05 – 0.10 m, 0.10 – 0.15 m, 0.15 – 0.20 m and 0.20 – 0.40 m depth) were collected in two apple orchards (8 years old and 18 years old), and in a native pasture area adjacent to the orchards. The soil was subjected to analysis of physical and chemical properties and to chemical fractionation of Cu and Zn. The fractions of soluble, exchangeable, organic, residual and total Cu and Zn accumulated in the topsoil (0.0 – 0.025 m) of the orchards, being higher in the orchard cultivated for 18 years. Most Cu, especially in the topsoil of the 18 years old orchard, accumulated in the less stable organic fraction, which can potentiate toxicity to plants and transfer Cu e Zn to ground water. In deeper layers, Cu was also distributed in the residual and mineral fractions, which are more stable. Most Zn in the soil of the apple orchards was distributed in the mineral and residual fractions, which have low mobility and low potential toxicity to plants.

Key words
Malus domestica; trace elements; toxicity; environmental contamination

INTRODUCTION

In the state of Santa Catarina, in southern Brazil, an area of approximately 18.000 ha is cultivated with apple crops (Malus domestica). It is the largest apple-producing area in Brazil, and Santa Catarina is the second largest producer, after Rio Grande do Sul state. Current apple production amounts to 531.000 t, equivalent to 44% of national production (Epagri 2013; Acate 2014). Nevertheless, apple trees are grown in regions with frequent rainfall, which stimulates the incidence of foliar fungal diseases and may reduce fruit yield and harm fruit quality. Therefore, foliar fungicides, e.g., the copperbased Bordeaux mixture (Ca(OH)₂ + CuSO₄) and zinc-based mixtures, such as those with the active ingredient Mancozeb (C₄H₆MnN₂S₄ × Zn), are applied throughout the year. Furthermore, fungicides containing Cu and Zn are applied after leaf senescence and during plant dormancy to promote dormancy breaking and branches disinfection.

The accumulation of Cu and Zn fractions, especially in the upper soil layers, can be stimulated by several factors: unidirectional fungicide applications; Zn and Cu being washed off the leaf surface by rainfall; the deposition of senescent leaves and pruned branches; the cycling of Cu and Zn by species of ground cover plants that co-inhabit the orchards; a low transfer of Zn and Cu to apples; and an absence of soil disturbance between orchard rows (Fernández-Calviño et al. 2010; Mackie et al. 2012; Ruyters et al. 2013).

In soil, Cu and Zn are retained by physical and chemical bonds, by mineral and organic components, and their availability depends on: the mineral contents, oxides and hydroxides of Fe, Al and Mn; carbonates (Bradl 2004); soil pH values (Toselli et al. 2009); cation exchange capacity (CEC); content; and composition of organic matter (Fernández-Calviño et al. 2010). However, when Cu and Zn contents increase in the soil, as a result, for example, of frequent fungicide applications, there can be accumulation and changes in the distribution of their fractions in such soil components. Adsorption of Cu and Zn in the soil occurs primarily in high affinity sites, usually forming inner sphere complexes. The remainder is adsorbed at sites with low binding energy and, consequently, it increases the availability and mobility of these elements in the soil profile (Brunetto et al. 2014). Thus, the increase of Cu and Zn in the soil over the years may increase the amount of soluble and exchangeable fractions of these elements, increasing toxicity to plants (Michaud et al. 2007; Nagajyoti et al. 2010; Yadav 2010; Fernández-Calviño et al. 2012) and water contamination through their transfer in the soil profile (Mileusnić et al. 2014; Marchand et al. 2014).

The fractions of Cu and Zn can be estimated by chemical fractionation, which removes Cu and Zn from the most labile to the most stable fractions (Tessier et al. 1979). The total amount of these elements in soils or sediments can be separated into bioavailable fractions (water-soluble and exchangeable), potentially bioavailable fractions (bound to clay minerals, oxides, carbonates and organic matter) and residual fractions (bound to the structure of minerals) (Casali et al. 2008; Frankowski et al. 2010; Brunetto et al. 2013; Duplay et al. 2014). However, in the Americas, particularly in subtropical and temperate regions, few studies exist on the accumulation and distribution of Cu and Zn fractions in the soil profile in apple orchards with a history of continuous fungicides application, whose compositions include Cu and Zn. The present study aimed to evaluate the accumulation and distribution of Cu and Zn fractions in the profile of an Inceptisoil on two plantations previously cultivated with apple trees in southern Brazil.

MATERIALS AND METHODS

Study location

This study was conducted using soil collected from two commercial orchards, one with eight years of cultivation (O8) and another with 18 years of cultivation (O18), and a soil from native pasture (NP), located nearby the orchards, in the county of Urubici, in the Planalto Serrano region, state of Santa Catarina, southern Brazil (Long 49°35’30”W, Lat 28°00’05”S). At the three sites, the soil was classified as an Inceptisoil, a Cambissolo Húmico in Brazilian classification (Embrapa 2013). The local relief was classified as slightly undulating, with a 4% slope. The climate is humid mesothermal with mild summers, Cfb type according to Köppen classification, with an average annual rainfall of 1360 – 1600 mm, average temperature of 15.4 °C, and between 20 and 29 frosts can occur throughout the year (Pandolfo et al. 2002).

Orchard O8 was implanted in 2003 with a planting density of 4,444 plants∙ha^–1 (4.5 m between rows and 0.5 m between plants), and orchard O18 in 1993, with a planting density of 1,482 plants∙ha^–1 (4.5 m between rows and 1.5 m between plants). The orchards had two commercial varieties, with Gala and Fuji accounting for 70% and 30% of the plants, respectively, with trees grown under a central-leader planting system. The plants of orchard O8 were grafted onto a Marubakaido rootstock, with a 20-cm M9 filter, while in orchard O18 the plants were grafted onto a Marubakaido rootstock without filter.

The implantation of the orchards was preceded by an application of lime to raise the soil pH in water to 6.0. Lime was applied on the surface and incorporated into the soil with a plow and then with a harrow to a depth of approximately 0.3 m. The plantation received 1000 kg∙ha^–1 of formula fertilizer (5-20-10; N-P₂O₅-K₂O), totaling 50, 200 and 100 kg∙ha^–1 of N, P₂O₅ and K₂O, respectively. The fertilizer was applied to the soil surface and also incorporated to approximately 0.3 m. Maintenance fertilizer was added annually: approximately 50, 30 and 150 kg∙ha^–1 of N, P₂O₅ and K₂O in the formulation 11-7-35. The fertilizer was applied on the soil surface without incorporation, in a row coincident with the canopy of the plants. Weed control in these rows was performed annually with the use of an herbicide (with glyphosate as the active ingredient). Between rows, weeds consisted primarily of Trifolium repens, Trifolium pratenses, Paspalum notatum and Chaptalia nutans. They were mowed throughout the apple tree cycle at approximately 0.10 m height from the soil surface. The native pasture area, which was used as reference, was located adjacent to the orchards, and natural vegetation was composed mainly of cover crops.

For fungal disease control during the production period of the orchard (October to April), was applied a spray volume of approximately 1000 L∙ha^–1 of fungicides N(4,6-dimethylpyrimidine-2-yl) aniline (PYRIMETHANIL) (45 mL 100 L); N-(trichloromethylthio) cyclohex-4-ene-1,2-dicarboximide (CAPTAN) (22 g 100 L); Sulfur (sulfur) (100 g 100 L); and especially Mancozeb (160 g 100 L). The fungicides were applied every seven days, always with rotation between the products. However, if it rained for more than nine consecutive hours, a new application was performed. During the winter period, the plant apple trees received 1000 L∙ha^–1 spray volume of a copper-based fungicide (Copper II + Oxychloride – Cu oxychloride), one application at the beginning of the production cycle, during the budding of the leaves, and other at the end of the cycle, after leaf senescence, with a total of around 22 fungicide applications per year.

Collection and preparation of soil samples

In October 2011, eight years after the implantation of orchard O8 and 18 years after the implantation of orchard O18, six trenches measuring 0.5 m depth and 0.5 × 0.3 m side were opened in random spots close to the planting rows in each orchard. Six trenches were opened at random points on the NP. Stratified samples were collected in each of the trenches in the following layers: 0.0 – 0.025 m, 0.025 – 0.05 m, 0.05 – 0.10 m, 0.10 – 0.15 m, 0.15 – 0.20 m and 0.20 – 0.40 m. Soil samples were air-dried, ground in a porcelain mortar, passed through a 2-mm mesh sieve, and reserved for analysis.

Physical and chemical characterization of soil

The soil samples were quantified for the levels of clay, silt and sand by the pipette method (Embrapa 1997). Total organic carbon was determined by the Walkley-Black method (Embrapa 1997), and the levels of soil organic matter were measured by multiplying the total organic carbon content by 1,724 (Tedesco et al. 1995). Analysis of soil pH was performed using a soil:water suspension of 1:1 v/v ratio (Tedesco et al. 1995). The levels of available phosphorus (P) and exchangeable potassium (K) were determined in a solution extracted by Mehlich^–1 solution (HCI 0.05 mol∙L^–1 + H₂SO₄ 0.0125 mol∙L^–1); P was determined by colorimetry (Murphy and Riley 1962) in a spectrophotometer; and K in a flame photometer. The contents of exchangeable Ca²⁺, Mg²⁺ and Al³⁺ were determined in a solution extracted by KCl (1 mol∙L^–1), whereas Ca and Mg were determined by atomic absorption spectrometry (AAS), and Al by titration with NaOH (Tedesco et al. 1995). Effective cation exchange capacity (CEC_effective) was calculated by summing the exchangeable cations of Al³⁺ + Ca²⁺ + Mg²⁺ + K⁺. Cation exchange capacity at pH 7.0 (CEC_{pH 7.0}) was calculated by the sum of exchangeable bases (Ca²⁺ + Mg²⁺ + K⁺) plus H + Al_SMP estimated from the equation proposed by Kaminski et al. (2002). Table 1 shows the determined physicochemical parameters.

Table 1
Physical and Chemical attributes of an Inceptisoil under native pasture (NP) and in orchard O8 (8 years old) and orchard O18 (18 years old), both cultivated with apple trees.

Area		Layer		Clay		Silt		Sand		OC		Al		Ca		Mg		P		K		pH		CECpH 7.0		CTCeffective
		(m)		(g·kg1)								(cmoc·kg1 )						(mgkg1)						(cmoc·kg1 )
NP		0 - 0.025		91		545		364		105.5		0.9		4.6		3.6		7.1		585		5.00		31.5		10.2
		0.025 - 0.05		106		560		334		73.1		3.2		2.4		2.4		3.9		266		3.96		32.9		7.2
		0.05 - 0.10		113		540		347		72.8		5.6		1.4		1.3		2.9		138		3.91		32.1		6.0
		0.10 - 0.15		111		537		352		69.8		7.2		0.8		1.0		1.9		112		3.80		38.4		5.6
		0.15 - 0.20		139		515		345		45.2		7.4		0.7		0.7		1.6		59		3.65		40.6		6.1
		0.20 - 0.40		151		483		366		41.5		8.2		0.5		0.6		0.6		49		3.74		33.6		5.7
O8		0 - 0.025		240		195		565		50.0		0.2		6.5		8.0		35		144		5.19		15.1		10.2
		0.025 - 0.05		257		182		561		47.1		0.1		6.8		3.1		19.7		81		6.11		12.8		10.3
		0.05 - 0.10		259		186		555		37.4		0.1		7.2		3.2		21.7		55		6.22		13.4		10.6
		0.10 - 0.15		272		171		557		38.1		0.1		6.0		4.5		8.6		38		6.41		13.5		10.7
		0.15 - 0.20		272		168		560		32.5		0.1		6.7		4.6		4.8		38		6.09		14.5		11.5
		0.20 - 0.40		213		192		595		13.2		0.2		5.4		3.7		1.5		24		5.29		14.4		9.4
O18		0 - 0.025		209		211		580		63.5		0.2		6.8		6.5		95.6		271		5.18		21.7		14.2
		0.025 - 0.05		216		199		585		55.4		0.1		7.7		2.4		95.3		115		4.93		18.7		10.5
		0.05 - 0.10		209		210		582		41.1		0.1		13.7		3.2		83.1		110		5.07		17.5		10.9
		0.10 - 0.15		212		201		587		27.1		0.2		8.2		3.1		83.3		65		5.37		16.7		11.7
		0.15 - 0.20		213		208		580		27.3		0.1		9.0		3.0		79.1		64		5.51		17.0		12.3
		0.20 - 0.40		241		185		574		29.6		0.2		6.7		3.4		51.8		40		5.47		14.3		10.4

Chemical fractionation of copper and zinc in soils

The chemical fractionation of Cu and Zn was carried out by adapting the methodology proposed by Tessier et al. (1979). Soil samples (1 g, in triplicate) were transferred to 50 mL polypropylene tubes to perform the following sequential extractions: the soluble fraction, extracted with deionized water (8 mL) (Cu_sol and Zn_sol); the exchangeable fraction, extracted with 1.0 mol.L^–1 MgCl₂ at pH 7.0 (8 mL) (Cu_E and Zn_E); the mineral-bound fraction, extracted with 0.04 mol.L^–1 hydroxylamine hydrochloride (NH₂OH.HCl) in 25% acetic acid (v/v) at pH 2.0 (20 mL) (Cu_Min and Zn_Min); and the organic-bound fraction extracted with 0.02 mol.L^–1 HNO₃ (3 mL) + 30% H₂O₂ (8 mL) + NH₄OAc in 20% HNO₃ (v/v) (5 mL) (Cu_OM and Zn_OM). The supernatant was separated by centrifugation at 3200 xg for 30 min, and an aliquot was set aside to determine the Cu and Zn levels. Between each extraction, the samples were agitated with deionized water and centrifuged, and the supernatant was discarded. At the end of the sequential extraction, the remaining residues were dried at 65 °C for 24 h, and then, 0.50 g of the dry residue was subjected to digestion with concentrated HF + HClO₄ to determine the residual Cu and Zn fractions (Cu_R and Zn_R). To determine the pseudototal levels, 30% H₂O₂ + concentrated HF + concentrated HClO₄ solution was added to 0.5 g samples of dry soil (not subjected to sequential extraction of Cu and Zn) to digest the samples (Cu_T and Zn_T). In conducting the study, all glassware was soaked in 10% HNO₃ for at least 24 h and then washed in deionized water three successive times before use. The Cu_Sum and Zn_Sum represent the sum of fractions Cu_Sol, Cu_E, Cu_Min, Cu_OM, and Cu_R and Zn_Sol, Zn_E, Zn_Min, Zn_OM, and Zn_R respectively.

Statistical analysis

The results of the Cu and Zn fractions were submitted to analysis of variance (ANOVA), and when the effects were significant between the layers of the same area, the means were compared by Tukey’s test (α = 5%). The differences between areas were evaluated using orthogonal contrasts: native pasture versus orchards and orchard O8 versus orchard O18 within each sampled soil layer. An analysis of variance was performed, and the differences in the proposed contrasts were tested at 5%, 1% and 0.1% probability of error for the F-test.

The fractions of Cu_Sol, Cu_E, Cu_Min, Cu_OM, Cu_R and Cu_Sum and the fractions of Zn_Sol, Zn_E, Zn_Min, Zn_OM, Zn_R and Zn_Sum were submitted to principal component analysis (PCA). For this purpose, the mean of six replicates was calculated for each layer and subjected to PCA. To minimize the discrepancy between the data, the values were normalized using Eq. 1:

$$(\mathrm{value}–\mathrm{minimum}\mathrm{value}/\mathrm{maximum}\mathrm{value}–\mathrm{minimum}\mathrm{value})\times 2–1$$

PCA is used to reduce data size and thus facilitate analysis by means of a correlation circle graph (Herlihy and McCarthy 2006).

RESULTS

Fractions of Cu in soil

In the native pasture (NP) soil, the highest levels of Cu_Sol, Cu_R, Cu_Sum and Cu_T were observed in the layer of 0.0 – 0.025 m and decreased at deeper levels (Table 2). The levels of Cu_E in the NP soil did not differ between layers. The highest levels of Cu_Min in the NP soil were found in the layers at 0.0 – 0.025 m and 0.025 – 0.050 m, whereas for the Cu_OM fraction, the higher content was observed in the layers at 0.0 – 0.025 m to 0.10 – 0.15 m. In the soil of O8 and O18 orchards, the highest levels of all Cu fractions were observed in the layer at 0.0 – 0.025 m and decreased in deeper layers.

The Cu_R fraction had higher levels compared with the Cu_Sol, Cu_E, Cu_Min, and Cu_OM fractions in all soil layers of NP (Table 2). The levels of Cu_Sol, Cu_E, Cu_Min and Cu_OM were similar to each other, except for the layer at 0.15 – 0.20 m, in which the Cu_E fraction had higher levels than the fractions of Cu_Sol Cu_Min and Cu_OM (Table 2). In all soil layers evaluated for O8, the levels of Cu_R were also higher than those found in the Cu_Sol, Cu_E, Cu_Min and Cu_OM fractions, with the exception of the layer at 0.0 – 0.025 m, where there were no differences in the level of Cu_OM. For the orchard O18, there were also higher levels of Cu_E compared with the Cu_Sol, Cu_E, Cu_Min and Cu_OM fractions only in the layers at 0.05 – 0.10 m and 0.10 – 0.15 m. In the soil layer from 0.0 – 0.025 m in orchard O18, the level of Cu_Org was higher than the levels of Cu_R, Cu_Min, Cu_E and Cu_Sol (Table 2).

Table 2
Cu fractions of an Inceptisoil under native pasture (NP) and in orchard O8 (8 years old) and orchard O18 (18 years old), both cultivated with apple trees.

Layers (m)		Cu fractions (mg-kg-1)
		CuSol		CUE		CuMin		CuOM		CuRes		CuSum		CuTotal		CV (%)
NP
0 - 0.025		0.17 Ad1		0.65 Ad		0.56 Ad		0.56 Ad		7.14 Ac		9.07 Ab		12.70 Aa		17.29
0.025 - 0.05		0.11 Bd		0.61 Ad		0.54 Ad		0.50 ABd		5.24 Bc		700 Bb		10.59 Ba		16.57
0.05 - 0.10		0.11 Bd		0.70 Ad		0.39 Bd		0.42 ABCd		3.85 BCc		5.48 Cb		793 Ba		17.26
0.10 - 0.15		0.07 Cd		0.63 Ad		0.35 Bd		0.41 ACd		3.36 CDc		4.82 CDb		7.99 Ba		26.07
0.15 - 0.20		0.07 Ce		0.65 Ad		0.27 Ce		0.31 CDe		2.50 CDc		3.79 DEb		7.28 Ba		20.40
0.20 - 0.40		0.05 Cd		0.63 Acd		0.14 Dd		0.18 Dd		2.23 Dbc		3.23 Eb		6.99 Ba		28.78
CV (%)		12.42		3.99		8.89		16.67		15.18		10.99		11.28
O8
0 - 0.025		0.50 Ae		1.98 Ae		11.89 Ad		18.43 Ac		21.64 Ac		54.44 Ab		86.04 Aa		7.16
0.025 - 0.05		0.27 Bf		0.92 Bef		3.54 Bde		4.80 Bd		17.30 Bc		26.83 Bb		71.01 Ba		8.66
0.05 - 0.10		0.26 Bf		0.60 Cef		2.66 Cde		3.39 Cd		13.01 Cc		19.93 Cb		58.24 Ca		7.57
0.10 - 0.15		0.25 BCe		0.42 CDe		2.46 CDd		2.19 Dde		9.72 Dc		15.05 Db		51.56 Da		8.85
0.15 - 0.20		0.22 Cd		0.26 DEd		2.07 DEd		1.81 Dd		7.64 Dc		12.01 DEb		43.22 Ea		9.49
0.20 - 0.40		0.15 Dd		0.09 Ed		1.79 Ed		1.47 Dd		7.13 Dc		10.62 Eb		28.67 Fa		13.43
CV (%)		5.83		14.15		6.04		5.93		11.05		6.11		5.04
O18
0 - 0.025		0.70 Af		1.15 Af		19.13 Ae		50.13 Ac		35.39 Ad		106.50 Ab		120.16 Aa		6.34
0.025 - 0.05		0.39 Be		0.97 Be		12.55 Bd		23.92 Bc		24.70 Bc		62.52 Bb		78.02 Ba		8.58
0.05 - 0.10		0.28 Cf		0.93 Bf		11.82 BCe		15.28 Cd		18.63 Cc		46.93 Cb		68.71 Ca		5.94
0.10 - 0.15		0.24 Ce		0.70 Ce		10.99 Cd		13.99 Cd		18.96 Cc		44.87 Cb		55.04 Da		8.10
0.15 - 0.20		0.24 Ce		0.68 Ce		8.64 Dd		13.92 Cc		12.14 Dcd		35.61 Db		40.50 Ea		9.95
0.20 - 0.40		0.23 Cd		0.66 Cd		6.59 Ec		5.84 Dc		5.53 Ec		18.85 Eb		22.52 Fa		12.09
CV (%)		7.86		6.36		3.91		4.55		11.67		4.47		5.39

• ¹Means followed by the same capital letter in a column and lower case letter in a row do not differ by Tukey’s test (p < 0.05). CV = coefficient of variation.

The means of the levels of all Cu fractions in the six evaluated layers (0.0 – 0.025 m, 0.025 – 0.05 m, 0.05 – 0.10 m, 0.10 – 0.15 m, 0.15 – 0.20 m and 0.20 – 0.40 m), except for the Cu_E fraction, were higher in the soil of orchards O8 and O18, compared with the levels of soil under natural pasture (Tables 2 and 3). This result was also observed when comparing the means of Cu levels in the six evaluated layers between the two orchards, where all fractions, except for Cu_E, were higher in the orchard O18 compared with O8 (Tables 2 and 3). Furthermore, the Cu_Min, Cu_OM, Cu_E and Cu_sum fractions were higher in the orchard O18 compared with O8 in the six layers (Tables 2 and 3). The exceptions were the levels of fractions Cu_E and Cu_Total(0.025 – 0.05 m), Cu_Sol (0.05 – 0.10 m) and Cu_Sol and Cu_Total (0.10 – 0.15 m and 0.15 – 0.20 m), which showed no differences (Table 3).

Table 3
Orthogonal contrasts between the Cu fractions of an Inceptisoil under native pasture (NP) and in orchard O8 (8 years old) and orchard O18 (18 years old), both cultivated with apple trees.

Contrasts	CuSol	CUE	CuMin	CuOM	CuR	CuSum	CuTotal
NP vs O8 and O181	***	Ns	***	***	***	***	***
O8 vs O181	***	Ns	***	***	***	***	*
Layer 0.0 - 0.025 O8 vs O18	***	***	***	***	***	***	***
Layer 0.025 - 0.05 O8 vs O18	***	Ns	***	***	***	***	Ns
Layer 0.05 - 0.10 O8 vs O18	Ns	***	***	***	***	***	***
Layer 0.10 - 0.15 O8 vs O18	Ns	***	***	***	***	***	Ns
Layer 0.15 - 0.20 O8 vs O18	Ns	***	***	***	**	***	Ns
Layer 0.20 - 0.40 O8 vs O18	***	***	***	***	*	***	***

• ¹
  Means of six evaluated layers.
• *Significant at 5%; ** significant at 1%; *** significant a 0.1%; ns = no significant.

The native pasture layer at 0.0 – 0.025 m had the highest percentage of Cu in the residual fraction (Cu_Res) (Figure 1), but in the soil of O8 and O18, the percentage of Cu_Res decreased compared with that of the native pasture soil. There was also an increase in the Cu_OM and Cu_Min fractions. In the soil of O18, the percentage of Cu_OM was higher than that of the Cu_E and Cu_Res fractions. In the layer at 0.025 – 0.050 m, the Cu_Res percentages decreased in the soil of O8 and O18, compared with that of the native pasture soil, because of an increase in the percentage of Cu_OM, especially in the soil of O18. In the layer at 0.05 – 0.10 m, the percentages of Cu_Res were similar between the soil under native pasture and that of O8. However, in the soil of O8, the percentages of Cu_OM and Cu_Min were higher compared to the soil under native pasture. The highest percentages of Cu_OM and Cu_Min were observed in the soil of O18, compared with the soils of the native pasture and O8. In the layers at 0.10 – 0.15 m, 0.15 – 0.20 m and 0.20 – 0.40 m, the percentages of Cu_OM and Cu_Min increased in the soil of O8, and especially in the soil of O18. In the latter, because of the increase in the Cu_OM and Cu_E fractions, a lower proportion of Cu_Res was present compared with NP and O8. For the Cu_E fraction, the soil under native pasture had the highest percentages in all evaluated layers.

Figure 1
Relative percentage of each Cu fraction obtained from a sequential extraction compared with the sum of the amount extracted from six layers of an Inceptisoil under native pasture and in orchards cultivated with apple trees (O8 and O18).

The principal component analysis (PCA) of the Cu fractions showed a relation between the main component 1 (factor 1) and the main component 2 (factor 2), which was split among the areas (NP, O8 and O18) (Figure 2a), accounting for 98.87% of the results. The areas of native pasture and O8 were separated from O18 by factor 1 of PCA, which helps to explain 95.4% of the results. The result was strongly influenced by the variables Cu_sol, Cu_E, Cu_Min, Cu_OM and Cu_Res. Factor 2 of the PCA, which helps explain only 3.4% of the results, separated the areas of orchards O8 and O18 from the native pasture area.

Figure 2
The ratio between the principal component 1 (factor 1)and principal component 2 (factor 2), mean levels of (a) Cu and (b) Zn in the layer at 0.0 – 0.40 m. Cu_sol and Zn_Sol – soluble fraction extracted with H₂O; Cu_E and Zn_E – exchangeable fraction extracted with MgCl₂ 1.0 mol·L^–1; Cu_Min and Zn_Min– mineral fraction extracted with NH₂OHHCl 0.04 mol L^–1 in 25% CH₃COOH; Cu_OM and Zn_OM – organic fraction extracted with HNO₃ 0.02 mol·L^–1 + 8 mL of 30% H₂O₂; Cu_Sum and Zn_Sum – sum of all fractions; and Cu_Res and Cu_Res– residual fraction extracted with HNO₃ + HCL + H₂O₂.

Zn fractions in soil

The highest levels of Zn fractions (Zn_Sol, Zn_E, Zn_Min, Zn_OM, Zn_R and Zn_Sum) and Zn_Total were found in the layer at 0.0 – 0.025 m in all areas assessed, with a subsequent decrease in the levels because of increasing depth (Table 4). The exception to this pattern was found in the soil of O8 and O18, where the Zn_OM and Zn_Total fractions did not differ between the layers at 0.0 – 0.025 m and 0.025 – 0.050 m. Furthermore, in the soil of O18, the Zn_Res fraction showed equal values between those two layers and the layer at 0.0 – 0.15 m.

Table 4
Zn fractions of an Inceptisoil under native pasture (NP) and in orchard O8 (8 years old) and orchard O18 (18 years old), both cultivated with apple trees.

Layer(m)		Zn fractions (mg-kg-1)
		ZnSol		ZnE		ZnMin		ZnOM		ZnR		ZnSum		ZnTotal		CV (%)
		NP
0 - 0.025		0.18 Ae1		3.21 Ae		9.38 Ad		1.89 Ae		16.24 Ac		30.90 Ab		39.57 Aa		9.06
0.025 - 0.05		0.10 Be		1.70 Be		5.04 Bd		1.78 Be		12.02 Bc		20.62 Bb		24.38 Ba		10.38
0.05 - 0.10		0.08 BCe		1.51 Bcd		4.03 Cc		1.26 Bcd		9.35 BCb		16.22 Cb		19.59 Ca		20.38
0.10 - 0.15		0.07 BCe		1.42 Be		3.46 Dd		1.03 Be		8.29 Cc		14.26 CDb		17.97 CDa		13.21
0.15 - 0.20		0.06 CDe		0.73 Cde		3.01 DEd		0.43 Ce		708 Cc		11.39 DEb		14.53 Da		17.68
0.20 - 0.40		0.03 De		0.66 Cde		2.77 Ed		0.27 Ce		6.76 Cc		10.48 Eb		14.05 Da		18.51
CV (%)		19.30		9.24		4.83		9.96		11.95		7.24		9.24
O8
0 - 0.025		0.16 Ae		3.56 Ae		14.65 Ad		1.83 Ae		48.55 Ac		68.74 Ab		75.70 Aa		6.48
0.025 - 0.05		0.12 Be		0.57 Be		6.63 Bd		1.42 Be		33.40 Bc		42.14 Bb		76.25 Aa		4.93
0.05 - 0.10		0.11 BCc		0.42 Cc		5.29 Cc		1.40 Bc		23.51 Cb		30.74 Ca		34.16 Ba		18.84
0.10 - 0.15		0.11 BCDd		0.39 Cd		3.47 Dd		1.22 BCd		18.66 CDc		23.85 Db		30.37 Ba		13.19
0.15 - 0.20		0.08 CDd		0.41 Cd		1.83 Ed		1.07 Cd		15.32 Dc		18.71 Db		28.86 BCa		10.35
0.20 - 0.40		0.08 Dc		0.13 Dc		1.41 Ec		1.00 Cc		16.22 Db		18.82 Db		22.82 Ca		15.87
CV (%)		13.58		3.24		4.94		9.83		9.92		7.72		6.46
O18
0 - 0.025		0.22 Ad		12.03 Ad		41.75 Ac		4.56 Ad		49.69 Ac		108.25 Ab		155.00 Aa		12.67
0.025 - 0.05		0.16 Be		6.49 Be		29.74 Bd		4.36 Ae		45.88 Ac		86.63 Bb		100.86 Ba		7.44
0.05 - 0.10		0.15 BCe		4.09 Ce		28.52 BCd		3.26 Be		43.71 Ac		79.73 BCb		96.99 Ba		5.77
0.10 - 0.15		0.12 BCDe		2.79 De		26.94 CDd		2.72 Be		41.54 Ac		74.11 Cb		93.25 Ba		11.86
0.15 - 0.20		0.11 CDd		2.13 Ed		25.42 Dc		2.63 Bd		30.70 Bc		60.98 Db		89.28 Ba		9.99
0.20 - 0.40		0.09 De		1.73 Fe		14.56 Ed		1.88 Ce		24.21 Bc		42.48 Eb		59.59 Ca		14.25
CV (%)		15.32		3.25		3.15		9.68		10.04		5.39		8.43

• ¹Means followed by the same capital letter in a column and lower case in a row do not differ by Tukey's test (p < 0.05). CV = coefficient of variation.

In all layers and areas assessed, the highest Zn levels were found in Zn_Total, except for the Zn_sum fraction, which showed values equal to those of the Zn_Total fraction in the layer at 0.05 – 0.10 m for O8. For the Zn_Res fraction, there were higher levels compared with the Zn_Sol, Zn_E, Zn_Min and Zn_OM fractions in all layers of the soil under natural pasture, O8 and O18. The level of the Zn_Min fraction in the soil under native pasture, O8 and O18 in all layers was lower than that of Zn_Res, but higher than the levels of Zn_Sol, Zn_E and Zn_OM. In the soil of O18, in all layers, the Zn_E levels were higher than the levels in the soil layers of the native pasture and O8.

Contrast orthogonal analysis showed that the mean Zn content of the six measured layers (0.0 – 0.025 m, 0.025 – 0.05 m, 0.05 – 0.10 m, 0.10 – 0.15 m, 0.15 – 0.20 m and 0.20 – 0.40 m) and of all fractions (Zn_E, Zn_Min, Zn_OM, Zn_R, Zn_Sum and Zn_Total) was higher in the soil of O8 and O18, compared with the soil under native pasture (Tables 4 and 5). This result was also found by comparing the means of Cu levels measured in the six layers between the two orchards, where all the fractions were higher in orchard O18 compared with O8. Except for the Zn_Sol fraction in the layers at 0.10 – 0.15 mand 0.20 – 0.40 m, and Zn_R in the layer at 0.0 – 0.025 m, all other fractions in all layers were higher in the soil of O18, compared with that of O8 (Tables 4 and 5).

Table 5
Orthogonal contrasts between Zn fractions of an Inceptisoil under native pasture (NP) and in orchard O8 (8 years old) and orchard O18 (18 years old), both cultivated with apple trees.

Contrasts		ZnSol		ZnE		ZnMin		ZnOM		ZnRes		Znsum		ZnTotal
NP vs O8 and O181		***		***		***		***		***		***		***
O8 vs O181		***		***		***		***		***		***		***
Layer 0.0-0.025 O8 vs O18		A A		***		* * *		* * *		ns		***		***
Layer 0.025-0.05 O8 vs O18		***		***		***		***		***		***		***
Layer 0.05-0.10 O8 vs O18		*		***		***		* * *		***		***		***
Layer 0.10-0.15 O8 vs O18		ns		***		***		***		***		***		***
Layer 0.15-0.20 O8 vs O18		**		***		***		* * *		***		***		***
Layer 0.20-0.40 O8 vs O18		ns				***		***				***		***

• ¹
  Means of six evaluated layers.
• *Significant at 5%; ** significant at 1%; *** significant a 0.1%; ns = no significant.

In the layers at 0.0 – 0.025 m, 0.025 – 0.050 m, 0.05 – 0.10 m, 0.10 – 0.15 m, 0.15 – 0.20 m and 0.20 – 0.40 m, the highest percentages of Zn in soil under the native pasture, O8 and O18 were found in the residual fraction (Zn_Res), being that the highest percentages were in the soil of O8 (Figure 2). However, in the soil of O18 was an increase of Zn content in the mineral fraction (Zn_Min) in all layers, compared with the soils of the native pasture and O8.

The PCA of Zn fractions showed the relation between the main component 1 (factor 1) and main component 2 (factor 2), which suggested separation of the areas assessed (Figure 2b), supporting the PCA of Cu fractions. Factors 1 and 2 accounted for 98.96% of the results. Factor 1, which explained 93.19% of the data, separated the areas of native pasture and O8 from that of O18. The Zn_Sol, Zn_E, Zn_Min, Zn_OM and Zn_Res fractions contributed greatly to this result, whereas factor 2 contributed only 5.7% to the result and separated O8 and O18 from the native pasture area.

DISCUSSION

The highest levels of Cu fractions (Cu_Sol, Cu_E, Cu_Min, Cu_OM, Cu_R, Cu_Sum and Cu_Total) were observed in the surface layer (0.0 – 0.025 m), especially in the soils of orchards O8 and O18. They can be attributed to years of applications of foliar fungicides, such as Mancozeb, Pyrimethanil and [CuSO₄∙5H₂O + Ca(OH)₂)], with the aim of preventing fungal diseases and also to promote dormancy breaking of plants (Verdisson et al. 2001; Komárek et al. 2010; Duplay et al. 2014). Thus, Cu may have accumulated on the soil surface because the applications were directional to the trees and Cu was washed off the surface of leaves by rainfall, and also by deposition of senescent leaves and pruned branches containing Cu derived from these applications (Brunetto et al. 2013). Furthermore, the higher level of Cu on the soil surface was also favored by the absence of soil disturbance in planting rows and between rows of apple trees over the years, by the cycling of Cu through deposition and subsequent decomposition of senescent leaves and pruned branches of apple trees, or even by the decomposition of roots in the soil (Amery et al. 2007; Yuan et al. 2007; Wang and Mulligan 2013; Pérez-Esteban et al. 2013). Therefore, the organic matter content in the upper layers of the soil are maintained and some Cu remains on the soil surface because its electron configuration is [Ar]3d¹⁰4s¹, thus making it highly reactive with the functional groups of organic matter that contain S and N, in addition to the carboxyl and phenolic functional groups (Croué et al. 2003). Furthermore, organic compounds provide Cu with high binding energy and low desorption (Lair et al. 2006; Casali et al. 2008; Brunetto et al. 2013).

In the case of the orchards, especially O18, the higher Cu content, represented by Cu_Total, can be attributed to the 22 annual fungicide applications throughout the years. In addition to the factors mentioned above, the fresh pulp of apple fruit is between 2 and 4 mg∙kg^–1 Cu (Stüpp et al. 2013). In this sense, an orchard with production of 60 t∙ha^–1 has exported 0.24 kg∙ha^–1 Cu, a fact that contributes to a higher accumulation of this trace element in the soil. Cu accumulation in the orchards was also favored because the relief at the site of the orchards is gently undulating, and cover crops are planted between the orchard rows, favoring the deposition of plant residues on the soil surface, which minimizes the transfer of Cu by the drained solution (Loures et al. 2006; Pinho et al. 2006).

The higher Cu content in the soil – mostly in the organic fraction (Cu_OM), compared with other fractions (Cu_Res, Cu_Min, Cu_E and Cu_Sol), especially in the surface layer (0.0 – 0.25 m), with increasing age of the orchards (eight years for O8 and 18 years for O18) and, therefore, a larger number of Cu-containing fungicide applications and higher nutrient-cycling time – can be attributed to high affinity and reactivity with the functional groups of the soil organic matter (Pérez-Novo et al. 2008). Therefore, because Cu is part of the organic fraction, the adoption of conservation management practices in orchards is crucial, for example, the deposition and maintenance of plant residues on the soil surface helps maintain the levels of soil organic matter and the absence of soil disturbance minimizes the mineralization of organic matter (Ferreira et al. 2013; López-Piñeiro et al. 2013). Nevertheless, the migration of the Cu_Total to a depth of 0.40 m in the soil of orchards O8 and O18 was observed through the level of Cu and Cu_OM, and Cu_Res fractions, (Table 3, Figure 1). This may have ^CuMinhappened due to the saturation of functional groups of organic matter (Besnard et al. 2001; Croué et al. 2003), by the saturation of functional groups of Fe and Mn oxides (Bradl 2004), and the silanol and aluminol groups of phyllosilicates (Sposito 1989; McBride 1994). Furthermore, the decrease in pH values in the soil profile (Table 1) may change the sorption capacity of the clay minerals, especially iron oxides (Sposito 1989). At acidic pH values, sorption is lower and desorption is facilitated (Brunetto et al. 2013).

The highest levels of Zn_Total, in particular, as has occurred with Cu in the most superficial soil layer (0.0 – 0.025 m), mainly in the soil of orchards O8 and O18, are due to the unidirectional annual applications of fungicides containing Zn in their composition, such as Mancozeb (Komárek et al. 2010), Zn being washed off the surface of leaves by rainfall, and deposition of Zn-containing leaves or pruned branches (Fernández-Calviño et al. 2012; Brunetto et al. 2014; Duplay et al. 2014). Furthermore, the higher accumulation of Zn_Total in the surface layers of the soil of the orchards, particularly orchard O18, is associated with a larger number of Zn-containing foliar fungicide applications for the preventive control of fungal diseases over the years (Brunetto et al. 2013). As seen for Cu, accumulation of Zn forms on the surface is also associated with an absence of soil disturbance in the rows and between the rows of apple trees, in addition to Zn cycling in the soil by the ground cover plants located in the rows and between rows of orchards (Couto et al. 2015; Hristovski et al. 2014).

In all soil layers of NP and orchards O8 and O18, higher levels of Zn were observed in the Zn_Res and Zn_Min fractions (Table 4 and Figure 3b), and these fractions are more stable and characterized by low availability and mobility in soil (Ramos 2006; Fernández-Calviño et al. 2012). Zn_Total and the Zn_E, Zn_Min and Zn_R fractions (Table 4 and Figure 3b) showed migration of Zn to 0.40 m depth. This may have occurred because of the migration of Zn by the flow of water in the soil because a fraction of Zn tends to remain in the soil solution in free form or as soluble ionic pairs (Citeau et al. 2003) and also by turbulent water flow in macropores and by soil cracks or even bioturbation (Girotto et al. 2010; Brunetto et al. 2013). In addition, Zn can bind to organic acids with low molecular weight, increasing its mobility in the soil profile (Wang and Mulligan 2013). This may occur because the formation of metal complexes with organic compounds with low molecular weight alters the balance between the metals in the labile form in the solid phase and in the soil solution, thus, dissolved organic compounds can also increase the release of Cu in the solid phase, and consequently increase its mobility in the soil (Girotto et al. 2010). In addition, Zn mobility may have been facilitated because of the decrease in water pH in the deeper layers of the soil, contributing to the formation of stable (innersphere) complexes between Zn and the functional groups of organic and mineral fractions of soil (Brunetto et al. 2013).

Figure 3
Relative percentage of each Zn fraction obtained from a sequential extraction compared with the sum of the amount extracted from six layers of an Inceptisoil under native pasture and in orchards cultivated with apple trees (O8 and O18).

The increase in the Zn_E fraction in the soil profile, which is a bioavailable fraction (Tessier et al. 1979) especially in the orchard O18 (Table 4), increases the potential for contamination of subsoil water (Banas et al. 2010; Fernández-Calviño et al. 2012). However, the increase in Zn_E in the upper layers, where the roots of apple trees and ground cover plant species are located in the rows and inter-rows of the orchards, may potentiate toxicity to plants (Nagajyoti et al. 2010; Fernández-Calviño et al. 2012). One example is the data obtained by Zhang et al. (2013) and Hansi et al. (2014). The first authors, studying the length of barley (Hordeum vulgare) roots grown in soil in a greenhouse with the addition of 0, 12.5, 25, 50, 100, 200, 400 and 800 mg Cu∙kg^–1, observed that root elongation drastically decreases with increasing Cu in solution.

Decreases of roots systems is not desirable because it reduces the nutrient cycling by ground cover plant species and the amount of plant residues deposited on the surface of soil, which are responsible for breaking the kinetic energy of raindrops, as well as reducing transportation of the solution on the soil surface. Moreover, it helps maintain the organic matter content in the soil (Brunetto et al. 2013). The increased Zn concentration in the soil can also cause toxicity to microorganisms, adversely affecting, for example, the decomposition of the crop residue deposited on the ground and the mineralization of soil organic matter (Gillera et al. 1998). Zn_E also increases in the most superficial soil layer, which can enhance the transfer of Zn on the soil surface by the solution drained or adsorbed in the sediment (Fernández-Calviño et al. 2008; Fernández-Calviño et al. 2012), which, in turn, enhances contamination of surface water adjacent to the orchards.

CONCLUSION

The Cu and Zn fractions increased in the surface soil layer (0.0 – 0.025 m) of apple orchards compared to native pasture cultivation, being associated with the time of cultivation.

Most Cu was distributed in the less stable organic fraction in the upper layers of apple orchards, which may potentiate toxicity to plants and transfer Cu to subsurface waters. In deeper layers, however, Cu was also distributed in the residual and mineral fractions, which are more stable.

Most Zn in the soil of apple orchards was distributed in the residual and mineral fractions, indicating low mobility, as well as potential toxicity to plants.

ACKNOWLEDGEMENTS

We are thankful to the CNPq (National Council for Scientifi c and Technological Development) for its fi nancial support and for the research productivity grant awarded to the fi rst author.

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Publication Dates

• Publication in this collection
  16 Apr 2018
• Date of issue
  Apr-Jun 2018

History

• Received
  31 Jan 2017
• Accepted
  18 Sept 2017