Abstract

This study was conducted to assess the physicochemical characteristics of urban forest soil contaminated by copper and the dynamics of macro- and micronutrients uptake by Schinus terebinthifolia and Eugenia uniflora seedlings. The seedlings received 0 (control), 60, 120, 180 or 240 mg Cu kg^-1 soil applied to urban forest soil within São Paulo City, Brazil. Our results showed that K was reduced in Cu-contaminated soil used for S. terebinthifolia cultivation and that organic matter was higher in Cu- contaminated soil used for E. uniflora cultivation. Other physicochemical properties of soil remained unaltered. S. terebinthifolia presented nutritional imbalances in N, K and Mg on leaves, while E. uniflora presented nutritional imbalances in K on leaves and in S on roots. It can be concluded that copper contamination can negatively affect chemical and nutritional characteristics of urban forest soil, as well as the nutritional dynamics of S. terebinthifolia and E. uniflora.

Keywords:
Schinus terebinthifolia; Eugenia uniflora; pollution; heavy metal; nutrition

1. INTRODUCTION AND OBJECTIVES

Copper (Cu) is an essential micronutrient for plant growth and development, playing key roles in several physiological processes, such as cell respiration, photosynthesis, ATP generation (Marques et al., 2018Marques DM, Júnior VV, Silva AB, Mantovani JR, Magalhães PC, Souza TC. Copper toxicity on photosynthetic responses and root morphology of Hymenaea courbaril L. (Caesalpinioideae). Water, Air, & Soil Pollution 2018; 229:138. doi:10.1007/s11270-018-3769-2.
https://doi.org/10.1007/s11270-018-3769-... ), participation in redox reactions and in oxidative stress protection (Rehman et al., 2019Rehman M, Liu L, Wang Q, Saleem MH, Bashir S, Ullah S, Peng D. Copper environmental toxicology, recent advances, and future outlook: A review. Environmental Science and Pollution Research 2019; 26:18003-16. doi: 10.1007/s11356-019-05073-6.
https://doi.org/10.1007/s11356-019-05073... ). Despite its necessity, copper in high concentrations can be toxic to plants, inducing adverse effects on plant physiology and biochemistry, such as excessive formation of reactive oxygen species (ROS) (Saleem et al., 2020Saleem MH, Fahad S, Khan SU, Din M, Ullah A, Sabagh AE, Liu L. Copper-induced oxidative stress, initiation of antioxidants and phytoremediation potential of flax (Linum usitatissimum L.) seedlings grown under the mixing of two different soils of China. Environmental Science and Pollution Research 2020; 27:5211-5221. doi:10.1007/s11356-019-07264-7.
https://doi.org/10.1007/s11356-019-07264... ), nutritional imbalance (Zeng et al., 2019Zeng Q, Ling Q, Wu J, Yang Z, Liu R, Qi Y. Excess copper-induced changes in antioxidative enzyme activity, mineral nutrient uptake and translocation in sugarcane seedlings. Bulletin of Environmental Contamination and Toxicology 2019; 103:834-840. doi:10.1007/s00128-019-02735-6.
https://doi.org/10.1007/s00128-019-02735... ), reduced growth and morphological alterations (Hossain et al., 2020Hossain MS, Abdelrahman M, Tran CD, Nguyen KH, Chu HD, Watanabe Y, Hasanuzzaman M, Mohsin SM, Fujita M, Tran LSP. Insights into acetate-mediated copper homeostasis and antioxidant defense in lentil under excessive copper stress. Environmental Pollution 2020; 258:113544. doi:10.1016/j.envpol.2019.113544.
https://doi.org/10.1016/j.envpol.2019.11... ).

Many factors influence the uptake of nutrients by terrestrial plants, such as temperature, pH and aeration of soils, organic matter, and soil contaminated with heavy metals (Hu et al., 2018Hu C, Liu L, Li X, Xu Y, Ge Z, Zhao Y. Effect of graphene oxide on copper stress in Lemna minor L.: evaluating growth, biochemical responses, and nutrient uptake. Journal of Hazardous Materials 2018; 341:168-176. doi:10.1016/j.jhazmat.2017.07.061.
https://doi.org/10.1016/j.jhazmat.2017.0... ). In general, excess copper in soil can disrupt plant nutritional metabolism by inhibiting the uptake of nutrients from soil and interfering with nutrient translocation from roots to shoots (Kumar et al., 2020Kumar V, Pandita S, Sidhu GPS, Sharma A, Khanna K, Kaur P, Bali AS, Setia R. Copper bioavailability, uptake, toxicity and tolerance in plants: a comprehensive review. Chemosphere 2020; 262:127810. doi:10.1016/j.chemosphere.2020.127810.
https://doi.org/10.1016/j.chemosphere.20... ). However, the degree of nutritional imbalance caused by Cu toxicity in plants is dependent on environmental factors, including the concentration and bioavailability of Cu in soil, length of exposure to heavy metal, and environmental growth conditions, as well as the morphophysiological characteristics of each plant species (Adrees et al., 2015Adrees M, Ali S, Rizwan M, Ibrahim M, Abbas F, Farid M, Zia-ur-Rehman M, Irshad MK, Bharwana SA. The effect of excess copper on growth and physiology of important food crops: a review. Environmental Science and Pollution Research 2015; 22:8148-8162. doi:10.1007/s11356-015-4496-5.
https://doi.org/10.1007/s11356-015-4496-... ).

In recent years, the accumulation of copper in soils has become a worldwide environmental concern. Human activities like mining, excessive use of copper-based pesticides, chemical and organic fertilizers, untreated sewage sludge and combustion of fossil fuels increase copper levels in soils, reaching concentrations that present risks for the biota and human health (Farias et al., 2018Farias JG, Nunes ST, Sausen D, Nunes MA, Neis FA, Garlet LC, Nunes PAA, Dressler VL, Schetinger MRC, Rossato LV, Girotto E, Brunetto G, Nicoloso FT. Agricultural contamination: Effect of copper excess on physiological parameters of potato genotypes and food chain security. Journal of Applied Botany and Food Quality 2018; 91:249-259. doi:10.5073/JABFQ.2018.091.033.
https://doi.org/10.5073/JABFQ.2018.091.0... ; Shabbir et al., 2020Shabbir Z, Sardar A, Shabbir A, Abbas G, Shamshad S, Khalid S, Natasha, Murtaza G, Dumat C, Shahid M. Copper uptake, essentiality, toxicity, detoxification and risk assessment in soil-plant environment. Chemosphere 2020; 259:127436. doi:10.1016/j.chemosphere.2020.127436.
https://doi.org/10.1016/j.chemosphere.20... ). Toxic concentration of Cu can be observed in highly urbanized regions, such as large cities, compared to more forested areas or areas with native vegetation, such as urban forest fragments (Argyraki et al., 2018Argyraki A, Kelepertzis E, Botsou F, Paraskevopoulou V, Katsikis I, Trigoni M. Environmental availability of trace elements (Pb, Cd, Zn, Cu) in soil from urban, suburban, rural and mining areas of Attica, Hellas. Journal of Geochemical Exploration 2018; 187:201-213. doi:10.1016/j.gexplo.2017.09.004.
https://doi.org/10.1016/j.gexplo.2017.09... ; Li et al., 2019Li C, Zhou K, Qin W, Tian C, Qi M, Yan X, Han W. A review on heavy metals contamination in soil: effects, sources, and remediation techniques. Soil and Sediment Contamination: An International Journal 2019; 28:380-394. doi:10.1080/15320383.2019.1592108.
https://doi.org/10.1080/15320383.2019.15... ). Urban forest fragments are remnants of native and secondary forests inserted in the urban environment. These fragments play a specific role in the existence and long-term maintenance of ecosystem services, conservation of biodiversity and habitat for several plant species (Dislich & Pivelo, 2002Dislich R, Pivello VR. Tree structure and species composition changes in an urban tropical forest fragment (São Paulo, Brazil) during a five-year interval. Boletim de Botânica da Universidade de São Paulo 2002; 20:1-12.). However, the sheer proximity of various pollutants places urban forest fragments at risk of particulate material from the emissions of automotive fleets and industry. These emissions contain heavy metals and can contaminate both soil and ground water (Nakazato et al., 2021Nakazato RK, Lourenço IS, Esposito MP, Lima ME, Ferreira ML, Campos OAR, Rinaldi MCS, Domingos M. Trace metals at the tree-litter-soil-interface in Brazilian Atlantic Forest plots surrounded by sources of air pollution. Environmental Pollution 2021; 268: 115797. doi:10.1016/j.envpol.2020.115797.
https://doi.org/10.1016/j.envpol.2020.11... ; Ferreira et al., 2019Ferreira ML, Andrade NGV, Costa MCLD, Araujo DM, Côrtes PL, Quaresma CC, Conit DM, Camargo PB. Soil fertility and litterfall assessment in a peri-urban forest of São Paulo, SP: understanding for urban green areas manegement. Holos 2019; 35:1-16. doi:10.15628/holos.2019.8290.
https://doi.org/10.15628/holos.2019.8290... ). Air pollution and deposition of particulate matter from human activities can also negatively affect the nutritional status of trees, reducing their development in urban forest remnants (Bulbovas et al., 2020Bulbovas P, Camargo CZ, Ferreira ML, Domingos M. Anthropic interferences in the nutritional status of tree species growing in urban and peri-urban Atlantic forest remnants. Urban Forestry & Urban Greening 2020; 50:126642. doi:10.1016/j.ufug.2020.126642.
https://doi.org/10.1016/j.ufug.2020.1266... ).

Studies with tree species have already reported that excess copper in soil can negatively affect plant growth (Marco et al., 2016Marco R, Silva RF, Andreazza R, Ros CO, Scheid DL, Bertollo GM. Copper phytoaccumulation and tolerance by seedlings of native Brazilian trees. Environmental Engineering Science 2016; 33(3):176-184. doi:10.1089/ees.2015.0307.
https://doi.org/10.1089/ees.2015.0307... ) and biomass production (Siqueira et al., 2021Siqueira MC, Kanashiro S, Domingos M, Rinaldi MCS, Tavares AR. Physiological and biochemical changes in tree seedlings growing in urban forest soil contaminated with copper in São Paulo, Brazil. Plant and Soil 2021; 464:149-163. doi:10.1007/s11104-021-04948-3.
https://doi.org/10.1007/s11104-021-04948... ). It can also lower CO₂ assimilation by reduced efficiency of the electron transport chain (Li et al., 2019Li Q, Chen HH, Qi YP, Ye X, Yang LT, Huang ZR, Chen, LS. Excess copper effects on growth, uptake of water and nutrients, carbohydrates, and PSII photochemistry revealed by OJIP transients in Citrus seedlings. Environmental Science and Pollution Research 2019; 26(29):30188-30205. doi:10.1007/s11356-019-06170-2.
https://doi.org/10.1007/s11356-019-06170... ). However, the direct effects of copper contamination on urban forest fragment soils, as well as the nutritional consequences observed in Brazilian native tree species from the Atlantic Forest cultivated in polluted soil, remain under investigated. It is herein hypothesized that the addition of increasing concentrations of copper in the soil of an urban forest fragment will alter its nutritional characteristics, thereby affecting the bioavailability of other elements, such as K or Mg, triggering nutritional imbalance in tree species. Therefore, this study aimed to validate if copper contamination changes the physicochemical properties of experimental plots of urban forest fragment in a manner that directly affects the nutritional dynamics of two tree species native to the Atlantic Forest.

2. MATERIALS AND METHODS

2.1. Characterization of plants and soil

Soil used in the experiment (Table 1) was a red-yellow latosol (LVA) (Santos et al., 2018Santos HG, Jacomine PKT, Anjos LHC, Oliveira VA, Lumbreras JF, Coelho MR, Almeida JA, Araujo Filho JC, Oliveira JB, Cunha TJF. Sistema brasileiro de classificação de solos. Brasília. Embrapa; 2018.) collected between 0.0 and 40.0 cm in depth in a forest area in the Parque Estadual Fonte dos Ipiranga (PEFI), São Paulo City, São Paulo State, Brazil. The experiment was carried out in a greenhouse at the Institute of Botany (23º30’S and 46º40’W; 770 m altitude) located inside the PEFI. Seedlings of the pioneer tree species Schinus terebinthifolia Raddi. (aroeira-vermelha) and the non-pioneer tree species Eugenia uniflora L. (pitanga), both native to the Atlantic Forest, were used in the experiment.

2.2. Experimental conditions and design

The seedlings were transplanted into 2.6 L vases containing natural PEFI soil and remained there for 30 days to allow acclimatization. The experiment consisted of the application of 75 ml Hoagland and Arnon solution n.1. (Hoagland & Arnon, 1950Hoagland D, Arnon DI. The water culture method for growing plants without soil. Berkeley. California Agricultural Experiment Station Press; 1950.) modified with 0 (control), 60, 120, 180 or 240 mg Cu (CuSO₄ ^.5H₂O) kg^-1 soil dry weight (DW) (Table 2). Copper concentrations were based on the Environmental Agency of São Paulo State (CETESB, 2016CETESB (Companhia Ambiental Do Estado De São Paulo) Valores orientadores para solo e água subterrânea no estado de São Paulo. Diário Oficial Estado de São Paulo - Caderno Executivo I (Poder Executivo, Seção I) 2016; 126, 55-56.) prevention value for Cu (60 mg Cu kg^-1 DW soil). Ionic balance of the solutions was carried out in order to maintain the constancy of the other macro- and micronutrients, and pH was adjusted to 5.8. The plants were irrigated weekly with ¼ of the total concentration, repeating the procedure 4 times to obtain the total values of copper in the soil for each treatment. During the experimental period, distilled water was used to irrigate the plants whenever necessary. The experiment lasted 120 days after soil contamination with copper between the spring and summer seasons in Brazil (September to December 2019) with a mean temperature of 26 °C and relative humidity of 56% inside the greenhouse.

2.3. Analyses of soil and macro- and micronutrient content in plant tissues

After the experimental period, soil samples of each treatment were homogenized, fractioned and oven-dried at 60 °C until constant weight. The pH, total organic matter (O.M.), potential acidity (H+Al) and content of total macro- and micronutrients were determined following the method adopted and described by Raij et al. (2001Raij BV, Andrade JC, Cantarella H, Quaggio JA. Análise química para avaliação da fertilidade de solos tropicais. Campinas. IAC; 2001.). Soil pH was potentiometrically determined in 0.01 mol L^-1 CaCl_2. O.M. was determined by the colorimetric method after soil digestion with sulfochromic solution. H+Al was determined using 1 mol L^-1 calcium acetate at pH 7. Calcium (Ca) and magnesium (Mg) were determined by atomic absorption spectrophotometry (AAS); potassium (K) by flame spectrophotometry; and phosphorus (P) by Ultraviolet-visible (UV/Vis) spectrophotometry after extraction with ion exchange resin. Boron (B) was determined by the colorimetric method (azomethine-H) after extraction with barium chloride. Iron (Fe), manganese (Mn) and zinc (Zn) were determined by atomic absorption spectrometry (AAS) after extraction in DTPA solution at pH 7.3.

Plants were removed from the pots, and the leaves, stems and roots were sectioned. The roots were washed under distilled running water. After oven-drying at 60 °C until constant weight, the plant material was weighed and milled in a knife mill to obtain a homogeneous powder. The total contents of macro- and micronutrients in vegetable material were determined according to the methods described by Malavolta (1997Malavolta E Avaliação do estado nutricional das plantas. Piracicaba. POTAFOS; 1997.). Cu, Ca, Mg, Zn, Fe, and Mn were determined by atomic absorption spectrophotometry (AAS); P was determined by the ammonium metavanadate colorimetric method; K was determined by flame spectrophotometry; and sulfur (S) was determined by the turbidimetric method (BaCl₂.2H₂O) after nitric-perchloric acid digestion. Nitrogen (N) was evaluated by the Kjeldahl method after sulfuric digestion, and B was assessed by the colorimetric method (azomethine-H) after incineration.

2.4. Absolute copper content in tissues

The absolute copper content (mg Cu kg^-1 DW) in leaves and roots of S. terebinthifolia and E. uniflora was determined by multiplying the total copper content in each tissue by the tissue’s dry weight (DW) as Cu_{tissue *} DW_tissue.

2.5. Statistical analyses

The experimental design adopted completely randomized blocks, consisting of 5 blocks with 5 treatments and 20 plants per plot, totaling 100 plants of each species. Data were submitted to Shapiro-Wilk normality test and Brown-Forsythe homoscedasticity test (^{Tables S1} SUPPLEMENTARY MATERIAL The following online material is available for this article: Table S1 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S2 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S3 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia leaf nutritional data. Table S4 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional data. Table S5 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia root nutritional content. Table S6 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional content. , ^S2 SUPPLEMENTARY MATERIAL The following online material is available for this article: Table S1 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S2 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S3 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia leaf nutritional data. Table S4 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional data. Table S5 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia root nutritional content. Table S6 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional content. , ^S3 SUPPLEMENTARY MATERIAL The following online material is available for this article: Table S1 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S2 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S3 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia leaf nutritional data. Table S4 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional data. Table S5 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia root nutritional content. Table S6 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional content. , ^S4 SUPPLEMENTARY MATERIAL The following online material is available for this article: Table S1 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S2 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S3 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia leaf nutritional data. Table S4 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional data. Table S5 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia root nutritional content. Table S6 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional content. , ^S5 SUPPLEMENTARY MATERIAL The following online material is available for this article: Table S1 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S2 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S3 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia leaf nutritional data. Table S4 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional data. Table S5 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia root nutritional content. Table S6 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional content. and ^S6 SUPPLEMENTARY MATERIAL The following online material is available for this article: Table S1 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S2 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia. Table S3 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia leaf nutritional data. Table S4 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional data. Table S5 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia root nutritional content. Table S6 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional content. ) using the statistical software GraphPad, v 9.0. If the data were determined to meet assumptions of normality and homogeneity of variance, then they were submitted to analysis of variance (One-Way ANOVA), and means were compared by Tukey’s test at 5% probability (p ≤ 0.05). Otherwise, data were submitted to the Kruskal-Wallis non-parametric test, followed by Dunn’s multiple comparison test at 5% probability (p ≤ 0.05).

3. RESULTS

3.1. Soil

O.M. levels in the soil cultivated with E. uniflora showed an increase after treatment with 240 mg Cu kg^-1 when compared to 180 mg Cu kg^-1, while no change in O.M. levels was observed in the soil cultivated with S. terebinthifolia (Table 3). No difference was observed for pH and H+Al among treatments in soil cultivated with S. terebinthifolia and E. uniflora.

The K contents in soil cultivated with S. terebinthifolia decreased after treatment with 240 mg Cu kg^-1 compared to 120 mg Cu kg^-1, but copper-contaminated soil did not affect K content in the soil cultivated with E. uniflora (Figure 1). No difference was observed for Ca and Mg or the micronutrients B, Fe, Mn, and Zn among treatments in soil cultivated with S. terebinthifolia and E. uniflora.

3.2. Absolute concentration of copper content in tissues

The absolute concentration of copper in both species increased as the concentration of copper applied to the soil increased (Figure 2). The absolute concentration of copper in the leaves of S. terebinthifolia varied from 0.025 to 0.028 mg Cu kg^-1 leaf DW, while in roots, it increased from 0.222 to 0.537 mg Cu kg^-1 root DW (Figure 2a). E. uniflora showed variation in absolute concentration of copper in the leaves from 0.028 to 0.033 mg Cu kg^-1 leaf DW and in the roots from 0.040 to 0.105 mg Cu kg^-1 root DW (Figure 2b).

Macro- and micronutrient content in plant tissues

S. terebinthifolia showed a reduction in N levels in the leaves after treatment with 60 mg Cu kg^-1 when compared to the control, while E. uniflora showed no statistical differences for N (Figure 3). S. terebinthifolia leaves showed a reduction in K levels after treatment with 60 and 180 mg Cu kg^-1 when compared to the control. E. uniflora leaves also showed a reduction in K levels after treatment with 180 mg Cu kg^-1 compared to control and 60 mg Cu kg^-1. S. terebinthifolia leaves showed an increase in Mg levels after treatments with 180 and 240 mg Cu kg^-1 compared to 120 mg Cu kg^-1, but no difference compared to control. No difference was observed for P, Ca, S, Fe, B, Mn, Zn in S. terebinthifolia and E. uniflora leaves among treatments (Figure 3).

E. uniflora roots showed an increase in S levels after treatment with 120 mg Cu kg^-1 compared to 60 mg Cu kg^-1, while no alteration of macro- and micronutrient levels in the roots of S. terebinthifolia among treatments was observed (Figure 4).

4. DISCUSSION

Copper contamination can induce different effects on the physicochemical properties of soils (Seguel et al., 2019Seguel CG, Muñoz H, Segovia J, Ávalos B, Martín JR. Assessment of soil contamination in Caleta Vitor and surrounding areas, northern Chile, due to heavy metal enrichment caused by an abandoned copper mine. Interciencia 2019; 44:241-246.). Changes in soil characteristics caused by excess of copper result from the effects of copper on the chemical bonds between the soil and the aggregated particles that adhere strongly to O.M., preventing its decomplexation (Karkush & Ali, 2019Karkush MO, Ali SD. Effects of copper sulfate contamination on the geotechnical behavior of clayey soils. Journal of GeoEngineering 2019; 14:47-52. doi:10.6310/jog.201903_14(1).6.
https://doi.org/10.6310/jog.201903_14(1)... ). The difference in the contents of O.M. between species is likely related to the heterogeneity of soils from urban forest fragments, which have different levels of O.M. from soil microbiota activities and particulate matter deposition (Ferreira et al., 2019Ferreira ML, Andrade NGV, Costa MCLD, Araujo DM, Côrtes PL, Quaresma CC, Conit DM, Camargo PB. Soil fertility and litterfall assessment in a peri-urban forest of São Paulo, SP: understanding for urban green areas manegement. Holos 2019; 35:1-16. doi:10.15628/holos.2019.8290.
https://doi.org/10.15628/holos.2019.8290... ). More specifically, the release of exudates of each species and the chelation of metals in the soil surrounded by plant roots (rhizosphere) can also change nutrient contents and physicochemical characteristics of soils (Campillo-Cora et al., 2019Campillo-Cora C, Fernández-Calviño D, Pérez-Rodríguez P, Fernández-Sanjurjo MJ, Núñez-Delgado A, Álvarez-Rodríguez E, Arias-Estévez M, Nóvoa-Muñoz JC. Copper and zinc in rhizospheric soil of wild plants growing in long-term acid vineyard soils. Insights on availability and metal remediation. Science of The Total Environment 2019; 672:389-399. doi:10.1016/j.scitotenv.2019.03.301.
https://doi.org/10.1016/j.scitotenv.2019... ).

Decrease in total K content in soil caused by excess of copper can be related to competition for adsorption sites in soil particulates (Wyszkowski, 2019Wyszkowski M. Soil Contamination with copper and its effect on selected soil properties after applying neutralizing substances. Polish Journal of Environmental Studies 2019; 28:2465-2471. doi:10.15244/pjoes/90357.
https://doi.org/10.15244/pjoes/90357... ), as well as the negative influence of excess copper on the natural cycling of nutrients (Bulbovas et al., 2020Bulbovas P, Camargo CZ, Ferreira ML, Domingos M. Anthropic interferences in the nutritional status of tree species growing in urban and peri-urban Atlantic forest remnants. Urban Forestry & Urban Greening 2020; 50:126642. doi:10.1016/j.ufug.2020.126642.
https://doi.org/10.1016/j.ufug.2020.1266... ). However, the availability of nutrients in copper-contaminated soils is dependent on the plant species present and their uptake capacity (Vendruscolo et al., 2018Vendruscolo D, Santana NA, Souto KM, Ferreira PA, Melo GWBD, Jacques RJS. Differential behavior of the summer cover crops in the absorption and translocation of copper. Ciência Rural 2018; 48:e20180005. doi:10.1590/0103-8478cr20180005.
https://doi.org/10.1590/0103-8478cr20180... ), as well the release of exudates from each species, which may modify the availability of nutrients in soil (Campillo-Cora et al., 2019Campillo-Cora C, Fernández-Calviño D, Pérez-Rodríguez P, Fernández-Sanjurjo MJ, Núñez-Delgado A, Álvarez-Rodríguez E, Arias-Estévez M, Nóvoa-Muñoz JC. Copper and zinc in rhizospheric soil of wild plants growing in long-term acid vineyard soils. Insights on availability and metal remediation. Science of The Total Environment 2019; 672:389-399. doi:10.1016/j.scitotenv.2019.03.301.
https://doi.org/10.1016/j.scitotenv.2019... ). Therefore, the distinct responses in the nutritional status of soil with S. terebinthifolia or E. uniflora is a result of the difference between species uptake capacity and nutrient availability.

The copper we added to the soil was effectively uptake by S. terebinthifolia and E. uniflora roots and incorporated into plant tissues, mostly accumulated in the root system. Copper uptake by roots is carried out through several specific and non-specific carriers of heavy metals located in the plasma membrane of root cells (Printz et al., 2016Printz B, Lutts S, Hausman JF, Sergeant K. Copper trafficking in plants and its implication on cell wall dynamics. Frontiers in Plant Science 2016; 7:601. doi:10.3389/fpls.2016.00601.
https://doi.org/10.3389/fpls.2016.00601... ). Copper naturally has a strong affinity for groups of enzymes and proteins present in the apoplast and cell wall of root cells, promoting its retention in plant roots (Girotto et al., 2016Girotto E, Ceretta CA, Rossato LV, Farias JG, Brunetto G, Miotto A, Tiecher TL, Conti L, Lourenzi CR, Schmatz R, Giachini A. Nicoloso FT. Biochemical changes in black oat (Avena strigosa Schreb) cultivated in vineyard soils contaminated with copper. Plant Physiology and Biochemistry 2016; 103:199-207. doi:10.1016/j.plaphy.2016.02.030.
https://doi.org/10.1016/j.plaphy.2016.02... ). In addition, under situations of toxicity and excess of copper, several species of plants have physiological mechanisms of tolerance to heavy metals, such as release of exudates by the roots, as well as chelation, sequestration and compartmentalization of metal ions in the vacuoles of root cells (Kumar et al., 2020Kumar V, Pandita S, Sidhu GPS, Sharma A, Khanna K, Kaur P, Bali AS, Setia R. Copper bioavailability, uptake, toxicity and tolerance in plants: a comprehensive review. Chemosphere 2020; 262:127810. doi:10.1016/j.chemosphere.2020.127810.
https://doi.org/10.1016/j.chemosphere.20... ). The increase in copper concentration in tissues, with preferential accumulation in the roots of S. terebinthifolia and E. uniflora, is a defense response for these tree species, restricting heavy metal to the roots and preventing translocation to leaves and causing a negative impact on the photosynthetic efficiency of plants (Marques et al., 2018Marques DM, Júnior VV, Silva AB, Mantovani JR, Magalhães PC, Souza TC. Copper toxicity on photosynthetic responses and root morphology of Hymenaea courbaril L. (Caesalpinioideae). Water, Air, & Soil Pollution 2018; 229:138. doi:10.1007/s11270-018-3769-2.
https://doi.org/10.1007/s11270-018-3769-... ).

Heavy metal stress can cause disturbances in the regulation of N uptake by roots and its translocation to shoots, leading to a decrease in the total N levels in leaves (Hippler et al., 2018Hippler FWR, Mattos Jr. D, Boaretto RM, Williams LE. Copper excess reduces nitrate uptake by Arabidopsis roots with specific effects on gene expression. Journal of Plant Physiology 2018; 228:158-165. doi:10.1016/j.jplph.2018.06.005.
https://doi.org/10.1016/j.jplph.2018.06.... ). Toxicity produced by excess of Cu in plants reduces N uptake and translocation by decreasing the expression level of genes encoding NO₃ ^- transporters (Huo et al., 2020Huo K, Shangguan X, Xia Y, Shen Z, Chen C. Excess copper inhibits the growth of rice seedlings by decreasing uptake of nitrate. Ecotoxicology and Environmental Safety 2020; 190:110105. doi:10.1016/j.ecoenv.2019.110105.
https://doi.org/10.1016/j.ecoenv.2019.11... ). The changes in K levels could be triggered by an impairment in uptake and translocation of K from roots to leaves as a result of copper toxicity. Cu toxicity induces the reduction of macronutrient content in shoot (i.e., K) as a result of interferences from ion uptake and translocation from root to shoot (Marastoni et al., 2019Marastoni L, Sandri M, Pii Y, Valentinuzzi F, Brunetto G, Cesco S, Mimmo T. Synergism and antagonisms between nutrients induced by copper toxicity in grapevine rootstocks: monocropping vs. intercropping. Chemosphere 2019; 214:563-578. doi:10.1016/j.chemosphere.2018.09.127.
https://doi.org/10.1016/j.chemosphere.20... ), producing an imbalance in homeostasis and distribution of nutrients throughout the plant (Souza et al., 2014Souza VL, Almeida AAF, Souza JDS, Mangabeira PA, Jesus RM, Pirovani CP, Ahnert D, Baligar DC, Loguercio LL. Altered physiology, cell structure, and gene expression of Theobroma cacao seedlings subjected to Cu toxicity. Environmental Science and Pollution Research 2014; 21:1217-1230. doi: 10.1007/s11356-013-1983-4.
https://doi.org/10.1007/s11356-013-1983-... ; Zaouali et al., 2020Zaouali W, Mahmoudi H, Salah IB, Mejri F, Casabianca H, Hosni K, Ouerghi Z. Copper-induced changes in growth, photosynthesis, antioxidative system activities and lipid metabolism of cilantro (Coriandrum sativum L.). Biologia 2020; 75:367-380. doi:10.2478/s11756-020-00419-9
https://doi.org/10.2478/s11756-020-00419... ). Furthermore, since cationic transporters can act in the transport of different ions in a nonspecific way, a decrease in potassium concentration (K⁺) in the aerial part of plants may be related to K in competition with copper ions (Cu⁺ and Cu²⁺) in the translocation processes (Cao et al., 2017Cao Y, Ma C, Chen G, Zhang J, Xing B. Physiological and biochemical responses of Salix integra Thunb. under copper stress as affected by soil flooding. Environmental Pollution 2017; 225:644-653. doi:10.1016/j.envpol.2017.03.040.
https://doi.org/10.1016/j.envpol.2017.03... ; Kobayashi et al., 2019Kobayashi R, Kobayashi NI, Tanoi K, Masumori M, Tange T. Potassium supply reduces cesium uptake in Konara oak not by an alteration of uptake mechanism, but by the uptake competition between the ions. Journal of Environmental Radioactivity 2019; 208:106032. doi:10.1016/j.jenvrad.2019.106032.
https://doi.org/10.1016/j.jenvrad.2019.1... ). Excess of copper can induce an increase of Mg translocation from roots to leaves, thus maintaining the homeostasis of mineral composition in shoots (Zeng et al., 2019Zeng Q, Ling Q, Wu J, Yang Z, Liu R, Qi Y. Excess copper-induced changes in antioxidative enzyme activity, mineral nutrient uptake and translocation in sugarcane seedlings. Bulletin of Environmental Contamination and Toxicology 2019; 103:834-840. doi:10.1007/s00128-019-02735-6.
https://doi.org/10.1007/s00128-019-02735... ). With enough concentration of Mg in plant tissues, the effects of excess Cu caused by the ionic competition between elements can be mitigated, preventing excess Cu from binding to vital action sites and causing toxicity (Juang et al., 2014Juang KW, Lee YI, Lai HY, Chen BC. Influence of magnesium on copper phytotoxicity to and accumulation and translocation in grapevines. Ecotoxicology and Environmental Safety 2014; 104:36-42. doi:10.1016/j.ecoenv.2014.02.008.
https://doi.org/10.1016/j.ecoenv.2014.02... ).

Cu toxicity can also cause impairment in nutrient uptake by roots by the competition among ions for uptake and nutritional imbalance as a consequence of saturation of nonspecific ionic transporters (Freitas et al., 2015Freitas TA, França MGC, Almeida AAF, Oliveira SJR, Jesus RM, Souza VL, Silva JVS, Mangabeira PA. Morphology, ultrastructure and mineral uptake is affected by copper toxicity in young plants of Inga subnuda subs. luschnathiana (Benth.) TD Penn. Environmental Science and Pollution Research 2015; 22:15479-15494. doi:10.1007/s11356-015-4610-8.
https://doi.org/10.1007/s11356-015-4610-... ). However, these effects are highly dependent on copper concentrations and method of cultivation. For example, plants cultivated in nutritive solutions have higher copper toxicity by the elevated availability of elements and fast uptake by the roots (Mezzavilla & Neto, 2017Mezzavilla NV, Neto JJ. Avaliação do desenvolvimento de plântulas de aroeira (Schinus terebinthifolius R.) em diferentes concentrações de alumínio. Semioses 2017; 11:9-18. doi:10.15202/1981996x.2017v11n3p9.
https://doi.org/10.15202/1981996x.2017v1... ,), while plants grown in soils present lower copper uptake by the strong adhesion of copper to soil particles and a host of soil-system biotic and abiotic relationships that can lead to less adverse effects on nutritional balance (Kelepertzis et al., 2015Kelepertzis E, Paraskevopoulou V, Argyraki A, Fligos G, Chalkiadaki O. Evaluation of single extraction procedures for the assessment of heavy metal extractability in citrus agricultural soil of a typical Mediterranean environment (Argolida, Greece). Journal of Soils and Sediments 2015; 15:2265-2275. doi:10.1007/s11368-015-1163-x.
https://doi.org/10.1007/s11368-015-1163-... ; Chua et al., 2019Chua J, Banua JM, Arcilla I, Orbecido A, Castro ME, Ledesma N, Belo L. Phytoremediation potential and copper uptake kinetics of Philippine bamboo species in copper contaminated substrate. Heliyon 2019; 5:e02440. doi:10.1016/j.heliyon.2019.e02440.
https://doi.org/10.1016/j.heliyon.2019.e... ). Since our study was carried out with soil, it is possible that copper remained partially aggregated to the soil, suggesting that the concentrations uptake by both tree species were insufficient to cause nutritional imbalance in all measured elements. It is also possible that higher doses or longer exposure to Cu could have increased the amount uptake by roots, causing nutritional imbalances and negatively interfering with the homeostasis of more elements. In addition to factors related to experimental condition, copper effects on the nutritional imbalance in plants are associated with synergism and/or competition between copper and mineral nutrients for specific absorption sites and transporters (Printz et al., 2016Printz B, Lutts S, Hausman JF, Sergeant K. Copper trafficking in plants and its implication on cell wall dynamics. Frontiers in Plant Science 2016; 7:601. doi:10.3389/fpls.2016.00601.
https://doi.org/10.3389/fpls.2016.00601... ), and some nutrients have weak competition against copper as a result of different ionic characteristics and lack of affinity with the same carriers (Andrés-Bordería et al., 2017Andrés-Bordería A, Andrés F, Garcia-Molina A, Perea-García A, Domingo C, Puig S Peñarrubia L. Copper and ectopic expression of the Arabidopsis transport protein COPT1 alter iron homeostasis in rice (Oryza sativa L.). Plant Molecular Biology 2017; 95:17-32. doi:10.1007/s11103-017-0622-8.
https://doi.org/10.1007/s11103-017-0622-... ). Thus, the concentration and translocation of the unaffected elements may be a consequence of their weak competition with copper for specific absorption sites and carriers (Kobayashi et al., 2019Kobayashi R, Kobayashi NI, Tanoi K, Masumori M, Tange T. Potassium supply reduces cesium uptake in Konara oak not by an alteration of uptake mechanism, but by the uptake competition between the ions. Journal of Environmental Radioactivity 2019; 208:106032. doi:10.1016/j.jenvrad.2019.106032.
https://doi.org/10.1016/j.jenvrad.2019.1... ). The irregular responses in nutrient concentration of S. terebinthifolia and E. uniflora to copper stress can also be explained by the fact that different species grown in copper-contaminated soils present different dynamics of accumulation, transport, and nutritional homeostasis in relation to their uptake capacity and the rate of development for each tissue (Zeng et al., 2019Zeng Q, Ling Q, Wu J, Yang Z, Liu R, Qi Y. Excess copper-induced changes in antioxidative enzyme activity, mineral nutrient uptake and translocation in sugarcane seedlings. Bulletin of Environmental Contamination and Toxicology 2019; 103:834-840. doi:10.1007/s00128-019-02735-6.
https://doi.org/10.1007/s00128-019-02735... ; Zabotto et al., 2020Zabotto AR, França WS, Domingos M, Rinaldi MCS, Kanashiro S, Ferreira ML, Tavares AR. Copper accumulation and distribution in two arboreal species of the atlantic forest. Floresta e Ambiente 2020; 27:e20190027. doi:10.1590/2179-8087.002719.
https://doi.org/10.1590/2179-8087.002719... ). Furthermore, a variety of tolerance mechanisms and strategies are adopted by plants which can result in distinct nutritional imbalances for each species when cultivated in copper-contaminated soils (Yruela, 2009Yruela I. Copper in plants: acquisition, transport and interactions. Functional Plant Biology 2009; 36:409-430. doi:10.1071/FP08288.
https://doi.org/10.1071/FP08288... ).

In our study, we observed that excess copper can modify the chemical properties of urban forest fragment soil. However, the magnitude of these changes can be affected by the species inhabiting that soil. Moreover, when cultivated in soils with high concentrations of copper, we found that the level of nutrients of S. terebinthifolia and E. uniflora can be affected in different ways. For instance, while the pioneer species S. terebinthifolia presents nutritional imbalance only in the leaves, the non-pioneer species E. uniflora presents nutritional imbalance in both leaves and roots. During the experiment, a small number of characteristics of soil and plant nutrition were altered. Therefore, it can be assumed that the applied dose or time was insufficient to cause major nutrient imbalance, but that larger doses or longer cultivation time could possibly result in more significant nutritional imbalance. We conclude that the excess of copper can change the chemical and nutritional characteristics of soil, as well as the nutritional dynamics of Schinus terebinthifolia and Eugenia uniflora seedlings.

ACKNOWLEDGEMENTS

We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo [FAPESP] under Grant [proc. 2017/50341-0] and M.C.S Master’s scholarship was supported by FAPESP under Grant [proc. 2019/03105-4].

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Table S1 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia.

Table S2 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of chemical and nutritional data of soil cultivated with Schinus terebinthifolia.

Table S3 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia leaf nutritional data.

Table S4 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional data.

Table S5 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Schinus terebinthifolia root nutritional content.

Table S6 Shapiro-Wilk Normality test, Brown-Forsythe Heteroscedasticity test and ANOVA table of Eugenia uniflora leaf nutritional content.

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