Effects of phosphorus and nitrogen amendments on the growth of Egeria najas

Aquatic Botany 86 (2007) 191–196 www.elsevier.com/locate/aquabot

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Effects of phosphorus and nitrogen amendments on the growth of Egeria najas Sidinei Magela Thomaz a,*, Patricia A. Chambers b, Sandra Andre´a Pierini c, Gabrielli Pereira d a Universidade Estadual de Maringa´, Nupelia, Av. Colombo 5790, 87020-900 Maringa´, PR, Brazil National Water Research Institute, P.O. Box 5050, 867 Lakeshore Blvd., Burlington, Ont. L7R 4A6, Canada c Faculdade Integrado de Campo Moura˜o, Rodovia BR – 158, km 207, Jardim Batel, CEP 87 300-970, Campo Moura˜o, PR, Brazil d Programa de Po´s-Graduac¸a˜o em Ecologia de Ambientes Aqua´ticos Continentais, Universidade Estadual de Maringa´, Departamento de Biologia, Nupelia, Av. Colombo 5790, 87020-900 Maringa´, PR, Brazil b

Received 16 February 2006; received in revised form 16 October 2006; accepted 23 October 2006

Abstract The effect of nutrient addition on the growth of E. najas was evaluated in a dose response experiment using sand amended with phosphorus (P) and nitrogen (N), and in enrichment trials with N and P amendments to natural sediments. Plants, water and sediment came from lagoons of the Upper Parana´ River Floodplain and from Itaipu Reservoir (Brazil). Relative growth rates (RGRs) of E. najas shoots, based on dry mass (DM), varied from 0.03 to 0.060 d1 for both nutrients. Root:shoot biomass ratios were related to sediment exchangeable P (r = 0.419; P = 0.03) and N (r = 0.54; P = 0.006), however root RGR was not related to sediment nutrient concentrations. When natural sediments were amended with N and P, neither shoot nor root RGRs differed among treatments for substrata from either the reservoir or the floodplain lagoons (P > 0.05). Comparison of nutrient concentrations measured in natural sediments collected from several sites in both the Upper Parana´ River Floodplain (range 49– 213 mg P g1 DM; 36–373 mg N g1 DM) and Itaipu Reservoir (range 43–402 mg P g1 DM; 7.9–238 mg N g1 DM) showed that sediment N and P from these systems usually exceeded minimum requirements necessary for E. najas growth, as measured in the dose response experiment. Together, these results indicate that E. najas, at least in early stages of development, responds to sediment nutrient amendments and relies upon bottom sediments to meet its N and P requirements and that for at least two Brazilian ecosystems, growth of this species is not limited by insufficient sediment N or P. Thus, reducing N and P in water is not enough to control E. najas growth in short time periods in these ecosystems. # 2006 Elsevier B.V. All rights reserved. Keywords: Egeria najas; Phosphorus; Nitrogen; Sediment; Brazil; Macrophyte

1. Introduction Availability of nutrients is considered one of the main factors affecting the abundance and composition of aquatic plant assemblages (Van et al., 1999). Of the many nutrients required for growth, nitrogen (N) and phosphorus (P) are the elements typically of shortest supply in aquatic ecosystems and therefore most likely to affect the growth of submerged species (Barko et al., 1991; Carr and Chambers, 1998). Aquatic macrophyte distribution has been found to be strongly determined by N and P, and different species may occur along

* Corresponding author. Tel.: +44 261 4617. E-mail addresses: [email protected] (S.M. Thomaz), [email protected] (S.A. Pierini). 0304-3770/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2006.10.004

nutritional gradients (Robach et al., 1995). Positive relationships between nutrient availability and aquatic macrophyte biomass have also been found both in laboratory experiments and in the field (Grane´li and Solander, 1988; Carr and Chambers, 1998). However, controversy remains as to whether sediment or water is the major source of nutrients for rooted aquatic macrophytes. Submerged plants exhibit great phenotypic plasticity and are adapted to absorb and translocate nutrients from sediment to leaves, especially in limiting conditions (Twilley et al., 1977; Barko et al., 1991; Sutton and Portier, 1995). Although several studies have emphasized the importance of root uptake (Carignan and Kallf, 1980; Barko and Smart, 1983, 1986; Smart and Barko, 1985; Chambers et al., 1989; Barko et al., 1991; Barko and James, 1998), others (such as Robach et al., 1995; Eugelink, 1998; Madsen and Cedergreen, 2002) argue the importance of shoot uptake.

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Nutrient absorption from sediment or water may therefore be a function of the relative availability of N and P in each compartment (Carignan and Kallf, 1980) and on the ability of the species in question (Barko and Smart, 1986). Investigations of nutrient uptake by aquatic macrophytes from tropical and sub-tropical environments are rare. Even when basic ecological aspects of tropical and sub-tropical species are investigated (e.g., Champion and Tanner, 2000; Kahara and Vermaat, 2003), specimens are usually obtained from ecosystems that they have invaded and are tested under those ecosystems conditions. Tropical and sub-tropical macrophytes exhibit a life history that is distinctly different from temperate aquatic plants due to a year-round growing season with its consistent, and higher, temperatures and irradiance. For these reasons, it is important to investigate plant nutrient relations of sub-tropical/tropical macrophyte species under native conditions. Investigations into the effect of nutrient addition on tropical macrophytes are now particularly timely due to emerging problems of excessive submerged plant growth in multiple-use tropical waters (e.g., navigation, recreation and hydro-energy production). In Brazil, excessive growth of tropical macrophytes has become a relatively frequent problem, especially since the 1980s when impoundment of rivers to produce hydroelectric reservoirs resulted in shallow embayments that now support dense macrophyte beds (Thomaz and Bini, 1998). Planning and implementation of strategies for macrophyte control must be supported by scientific evidence as to the cause of extensive submerged plant growth. In this study, we evaluated the effect of P and N additions on the growth of E. najas. First, using artificial substrate, we established a dose response of E. najas to nutrient addition. Second we tested if plants from a native habitat (lagoons of the Upper Parana´ River Floodplain) and from a man-made habitat (Itaipu Reservoir) were growing under limiting conditions of one or both nutrients. Finally, we measured exchangeable N and P in sediment collected from several sampling stations in both the floodplain and the reservoir in order to extend our experimental results and test the generality of nutrient limitation or sufficiency of E. najas under sub-tropical conditions. This allowed us to assess if this species was growing under limiting N and P concentrations in its natural habitats. 2. Materials and methods 2.1. Microcosm experiments Microcosm experiments were conducted to: (1) establish a dose response of E. najas to sediment P and N, and (2) determine whether E. najas was growing under nutrient limiting conditions in either of two habitats (the Parana´ River floodplain or Itaipu Reservoir). In the dose response experiment, E. najas was grown in sand amended with P or N (75, 100, 150, 250, 300, 350, 400, 450, 500, 550 [except for N amendments], or 600 mg g1 wet mass). Potassium dihydrogen phosphate and ammonium chloride were used as P and N

sources, respectively. Each treatment was duplicated. E. najas was also grown in non-amended conditions (N = 5 for N and N = 6 for P). Thus, there were in total 25 and 28 observations for the N and P experiments, respectively. Prior to set up, the sand was acid washed to remove exchangeable P and N and had initial concentrations of 7 mg g1 DM exchangeable N and 11 mg g1 DM exchangeable P. At the end of the experiment, a sediment sample from every flask for all treatments was analyzed for exchangeable N and P. Exchangeable P was extracted by shaking the sediments for 16 h with 0.1 N NaOH– NaCl and exchangeable N was extracted from sediments by shaking for 1 h with 2M KCl. Exchangeable P and N were measured spectrophotometrically as PO43 and NH4 following the methods of Stainton et al. (1977) and Bremner (1965), respectively. In the sediment amendment experiment, plants were grown in natural sediment (collected from beneath macrophyte stands from each of the two habitats) that was either unamended (control treatment) or amended with P, N or both P and N. Bottom sediments were collected beneath E. najas stands along the east shore of Itaipu Reservoir (BrazilParaguay border; 548 300 W, 258 000 S) and from lagoons of the Upper Parana´ River floodplain (Brazil; 538 150 W, 228 450 S). Sediments were transported to the laboratory where debris were carefully removed, and then homogenized. Treatments were obtained by adding 100 mg P g1 of potassium dihydrogen phosphate (+P), 200 mg N g1 of ammonium chloride (+N) or 100 mg P g1 of potassium dihydrogen phosphate plus 200 mg N g1 of ammonium chloride (+PN) to natural sediments. These additions were based on wet sediment mass. Control and treatments were replicated seven times. Exchangeable P and N were measured at the start and end of the experiment, for every replicate from each treatment as in the first experiment. For both experiments, ramets of E. najas were collected between May and November 2001 from stands along the east shore of Itaipu Reservoir and from lagoons of the Upper Parana´ River floodplain. Details about limnological conditions of these sites can be found in Thomaz et al. (1999, 2004). At the start of each experiment, an 8 cm (Experiment 1) or 5 cm (Experiment 2) apical portion of E. najas was weighed and planted in 50 mL flasks containing either c. 80 g of sand (Experiment 1) or natural sediment (Experiment 2). In the first experiment, flasks were wrapped with PVC film to reduce ionic exchange at the water-sediment interface. Each flask was then placed in 2.8 L aquaria (one flask per aquaria) that was filled with water that had been collected from inside plant stands and filtered through Whatman GF/C membranes. Experiments started within 72 h of collecting water, sediments and plants. For both experiments, temperature was maintained at 25 8C and underwater photosynthetic active radiation was 259–320 mmol m2 s1 at the water surface, which is above the light requirements of this species (Tavecchio and Thomaz, 2003). Photoperiod was 12 h. After 15–20 days, the ramets were removed and mass was measured for both shoots and roots after drying at 90 8C for 48 h. Biomass at the start of the experiment was estimated from a fresh:dry weight ratio determined for 10 ramets of similar size as the ones used in the experiments. Final concentrations of

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exchangeable P and N were measured in the sediment using the method described earlier. Relative growth rates (RGR), based on shoot dry mass, were calculated using the following equation (Ricker, 1979): RGR ¼

ðW 2  W 1 Þ ; W 1 ðt2  t1 Þ

where W2 is the final dry mass, W1 the initial mass and t days of growth. Given the absence of roots at the beginning of the experiments, relative growth rate (r) for roots, based on root dry mass, was calculated as: r¼

ðln W 2  ln W 1 Þ ðt2  t1 Þ

For the dose response experiment, the Michaelis-Menten equation (Carr et al., 1997) was first used to model the response of shoot RGR to nutrient additions. However, the N treatment did not follow the Michaelis-Menten kinetics and deviation from linearity was found at the higher nutrient concentrations (see Fig. 1). Thus, we applied the Hanes plot conversion of the Michaelis-Menten equation to give a straight line: S 1 km ¼  RGR þ RGR V max V max where S is the concentrations of P or N in the substrate, Vmax is the maximum growth attained by E. najas, and km is a measure of the affinity of the uptake process in E. najas for N or P. Since the growth of plants is a complex process different from kinetics for enzyme reactions, the terms ‘‘apparent Vmax’’ and ‘‘apparent km’’ are used. The relationship between root RGR and sediment N and P, and between the ratio of root:shoot

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biomass and sediment N and P, were tested by Pearson correlation. Residual analyses were undertaken and, when necessary, data were log-transformed to meet the analyses requirements. For the sediment amendment experiment, to test the effects of N and P (both with two levels: control and enriched), a twoway ANOVA was used. Reservoirs were considered blocks in this analysis in order to increase power and to take into account limnological differences between them. 2.2. In situ observations Sediment was collected from nine sites distributed across four arms of the east shore of Itaipu Reservoir and from nine lagoons in the Upper Parana´ River floodplain. Some sites sampled were colonized by E. najas, but others were not. Samples were frozen and later analyzed, as described previously, for exchangeable N and P. 3. Results 3.1. Dose response experiment RGR of E. najas shoots showed a clear response to addition of N and P to low nutrient sand substratum (Figs. 1a, b). In general, shoot RGRs were in the same range, spanning 0.03– 0.07 d1 for both nutrients. In the case of the P amendment trial, shoot RGR was well fit by the Hanes plot (r2 = 0.97; P < 0.001; mean square error = 32,186,685), giving a maximum RGR (apparent Vmax) of 0.070 d1 that was approached at 100 mg P g1 DM. In the case of N trials, shoot RGR was also fit by Hanes plot (r2 = 0.98; P < 0.001; mean square

Fig. 1. (a and b) Hanes plot showing the substrate concentrations (sediment N and P) divided by shoot RGR (based on dry mass) against substrate concentrations; (c and d) relationship between root RGR and nutrient N and P concentrations. Relationships between RGR and nutrient concentrations in sediment are shown inside figures (a) and (b) to better visualize the effects of sediment nutrients upon E. najas growth.

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error = 79,039,441) and the maximum RGR (apparent Vmax) was of 0.050 d1. Affinity of E. najas for its substrate (represented by apparent km) was higher when substrate was fertilized with P (apparent km = 16.9 mg P g1 DM) than with N (apparent km = 0.05 mg N g1 DM). In contrast with shoot RGR, root RGR was not correlated with sediment exchangeable P (r = 0.27; P = 0.168; N = 27) or N (r = 0.31; P = 0.127; N = 25)(Figs. 1c, d). Root:shoot biomass ratios varied widely from 0.02 to 0.20. These ratios were significantly related to sediment exchangeable P (r = 0.41; P = 0.03; N = 28) and N (r = 0.54; P = 0.006; N = 25), although relationships between root:shoot ratios and sediment nutrients were not as strong as those for shoot RGR.

Table 2 Concentrations (mg g1 DM) of exchangeable P and N in sediments before and after incubation. ‘‘Before’’ measurements are concentrations for homogenized sediments prior to start of experiment; standard error for the analyses are <3%. ‘‘After’’ measurements are means (with S.E. in brackets) for samples from every replicate for each treatment (N = 7)

Before After Control P N N+P

Parana´ floodplain

Itaipu reservoir

P (mg g1 DM)

P (mg g1 DM)

34.7 19.7 75.5 29.2 55.5

N (mg g1 DM) 10.1

(1.5) (2.4) (2,8) (2.3)

2.7 (0.3) 6.3 (0.1) 15.6 (0.6) 1771 (14.9)

27.0 14.3 62.6 20.7 53.0

N (mg g1 DM) 11.0

(0.6) (2.4) (1.4) (4.0)

1.2 (0.02) 11.5 (0.9) 24.5 (6.4) 184 (26.2)

3.2. Sediment amendment experiment Growth of E. najas shoots in natural unamended sediment varied considerably, ranging from 0.012 to 0.050 d1 in the Parana´ floodplain (mean = 0.030  0.005 d1 S.E.) and from 0.020 to 0.104 d1 in Itaipu (mean = 0.056  0.011 d1 S.E.), despite constant light and temperature conditions and use of homogenized sediments (Table 1). Results of ANOVA showed that for shoot RGR there was an effect of local (F = 9.827; P = 0.003), and values were higher at the Itaipu treatments (Table 1). However, the effects of sediment N, P and the interaction N*P were not significant (P > 0.05). Concerning root RGR (values ranging from 0.00022 to 0.00036 d1; Table 1), the effects of local (Itaipu and floodplain), treatment (sediment N or P) and the interaction N*P were not significant (P > 0.05). Root:shoot biomass ratios varied widely (from 0.029 to 0.062; Table 1) but were not related to either N or P amendment (P > 0.05), although the effect of local was significant (F = 16.631; P < 0.001). For the control treatments, sediment nutrient concentrations decreased over the incubation period (Table 2). For the Parana´ floodplain sediments, exchangeable P decreased by 43% and exchangeable N by 73%. For the Itaipu trial, exchangeable P decreased by 47% and exchangeable N by 89%. At the end of the experiment, sediment nutrient concentrations were still four times higher for the P treatment and six (Parana´) or 20 (Itaipu) times for the N treatment compared to control sediments. Table 1 Shoot and root RGR, and root:shoot ratios found in the amendment experiments. Means (with S.E. in brackets) are shown Shoot RGR (d1)

Root RGR (d1)

Root:shoot ratios

Floodplain Itaipu

Floodplain Itaipu

Floodplain Itaipu

Control 0.030 (0.005)

0.056 0.00027 (0.011) (0.00005)

0.00022 (0.0004)

0.029 (0.006)

0.054 (0.009)

P

0.032 (0.005)

0.054 0.00030 (0.006) (0.00004)

0.00032 0.031 (0.00004) (0.005)

0.062 (0.010)

N

0.052 (0.007)

0.069 0.00033 (0.009) (0.00008)

0.00022 0.031 (0.00003) (0.008)

0.048 (0.008)

N+P

0.043 (0.007)

0.052 0.00036 (0.010) (0.00004)

0.00026 0.034 (0.00006) (0.004)

0.059 (0.012)

3.3. Concentrations of N and P at sediment of Itaipu and floodplain habitats Nutrient concentrations measured in natural sediments varied widely. In the Upper Parana´ lagoons, mean P was 108 mg g1 DM (49–213; S.D. = 58.5) and mean N was 147 mg g1 DM (36–373; S.D. = 130). At Itaipu sites, mean P concentrations were 137 mg g1 DM (43–402 S.D. = 113) and mean N was 57.0 mg g1 DM (7.9–238; S.D. = 71.4). 4. Discussion Our investigation about nutrient limitation is probably the first undertaken with E. najas, a native submerged species widespread in Brazilian lakes and reservoirs (Thomaz and Bini, 1998). Results of our dose response experiments showed that the growth of young shoots of E. najas was related to both N and P when these nutrients were scarce in the sediment. Although underwater radiation has long been considered the principal determinant of submerged plant colonization and growth (Spence, 1982), nutrient availability has emerged as an important factor affecting submerged macrophyte performance (Barko and Smart, 1983; Chambers and Kalff, 1985; Barko et al., 1988). Sediment fertility has been shown to directly affect the biomass and productivity of macrophyte species, such as Hydrilla verticilata, Myriophyllum spicatum and Thalassia testudinum such that higher availability of N, P or both cause incremental increases in biomass (Barko and Smart, 1986; Barko et al., 1991; Chambers et al., 1989; Lee and Dunton, 2000). Conversely, in some circumstances, it appears that water may be the main nutrient source (Robach et al., 1995; Madsen and Cedergreen, 2002). The reasons for such discrepancies may be due to differences among plant species in use of water versus sediment as a nutrient source, differences in availability of N and P in sediments versus water (Carignan and Kallf, 1980), water flow (Madsen and Cedergreen, 2002) or even the age of plants used in the experiments. Although we did not measure nutrients in the water during our experiments, N and P concentrations in both study sites are consistently low and typical of oligo-mesotrophic habitats (Wetzel, 2001): mean of 310 mg N–NO3 l1 (S.D. = 31) and 25 mg P–PO43 l1

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(S.D. = 6) for Itaipu Reservoir sites (L.M. Bini, unpublished) and of 85 mg N–NO3 l1 (S.D. = 55) and 19 mg P–PO43 l1 (S.D. = 16) for the upper Parana´ sites (S.M. Thomaz, unpublished). Our observations of increased growth with increasing sediment nutrients (up to about 100 mg P g1 DM and 60 mg N g1 DM) and depletion of nutrients in unamended control sediments (Table 2), combined with naturally low water-column concentrations of N and P in our study sites, are evidence that young shoots of E. najas rely upon sediments, rather than water-column, nutrients for growth. This finding is consistent with results from experiments on other species of Hydrocharitaceae: Hydrilla verticillata and Elodea nuttallii (both eloideids and Hydrocharitaceae, similar to E. najas) showed positive relationships between sediment fertility and growth under temperate conditions (Spencer and Ksander, 1995; James et al., 2006). Moreover, Feijoo et al. (1996) found a positive relationship between E. densa biomass and sediment N in Argentinian streams. Together, these results indicate that, at least under conditions of low concentrations of water-column nutrients, Hydrocharitaceae rely upon the bottom sediments for their nutrient supply in a variety of ecosystems. The affinity for substrate (indicated by apparent km values) estimates the nutrient concentration at which the plant reaches its maximum growth rate or productivity (Carr et al., 1997). In the case of E. najas, km values were low (ca. 16 mg P g1 DM and 0.05 mg N g1 DM), indicating that the species is well adapted to incorporate nutrients when they are in low concentration. It is difficult to make comparisons with data from the literature because of different methods employed during incubation and in nutrient analytical determination (e.g., total versus exchangeable nutrient in sediments). Using the same method for nutrient determination as ours, Carr and Chambers (1998) found that Potamogeton pectinatus biomass did not saturate over a wide range of exchangeable P concentrations (10–1000 mg P g1 DW). Our observation of a lower km value for N compared to P suggests a greater affinity of E. najas for the first nutrient. The fact that growth of E. najas saturates at low nutrient (particularly N) concentrations may give E. najas a competitive advantage in relatively unfertile environments over species typical of richer, more eutrophic environments. One of the strategies that enable E. najas to grow under low nutrient concentrations is the increase in root in relation to shoot mass, as indicated by the negative relationship between root:shoot ratios and sediment nutrient observed in the dose response experiment. Although our dose response experiment showed that growth of E. najas was related to both N and P when these nutrients were scarce in the sediment, E. najas RGR was not affected by either P or N when these nutrients were added to natural substrata. Other researchers have observed mixed responses by aquatic macrophytes to nutrient addition. For example, P enrichment had no significant effect on the growth of Acorus calamus whereas high sediment N caused a significant reduction in growth (Vojtı´sˇkova´ et al., 2004). Madsen and Cedergreen (2002) showed that Elodea canadensis and Callitriche cophorcarpa were not affected by enrichment of

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N and P in water and/or sediment. Conversely, Sosiak (2002) inferred from field monitoring that a decline in biomass of Potamogeton pectinatus was due to depletion in river sediment N. The lack of response of E. najas RGR to N and P amendments in natural substrata is explained by the fact that background concentrations of N and P in natural sediment were similar to or exceeded half-saturation constants measured in the dose response experiment. This fact, along with the variable growth rates shown by E. najas, resulted in no effect of added N or P for these sediments. Moreover, our survey of 18 sites in the Upper Parana´ lagoons and Itaipu reservoir showed that N and P concentrations were always higher than half saturation constants measured in our dose response experiment. These results show that it is quite probable that young shoots of E. najas are not limited by nutrients in their native habitats. In fact, other studies have shown that light (Tavecchio and Thomaz, 2003) and inorganic carbon (Pierini and Thomaz, 2004) are limiting macrophyte growth and photosynthesis at these sites. Our finding that, under natural conditions, growth of young shoots of E. najas is not constrained by insufficient nutrients has direct implications for the management of this species. Programs aimed at improvement of water quality are usually directed at lowering inputs of nutrients and reducing their concentrations in water (Somlyody and Altanfin, 1992). Nevertheless, as was demonstrated through nutrient amendment and dose response experiments and from measurement of nutrient concentrations in situ, E. najas (and probably other submerged macrophyte species) are not nutrient limited in two major Brazilian systems. If nutrients are to be managed to reduce aquatic macrophyte abundance, then control of E. najas in our Brazilian systems will occur only if sediment become impoverished in P and N. Other researchers studying submerged plants in north-temperate lakes have also concluded that reductions in sediment nutrient concentrations are needed for macrophyte control (Carignan and Kallf, 1980; Carr and Chambers, 1998). However, a paucity of sediment nutrient data for Brazilian freshwaters makes it impossible to determine if sediment nutrient concentrations in Brazilian lagoons and reservoir have been enriched by anthropogenic inputs and would therefore respond to a reduction in nutrient loading. Further research is needed on nutrients concentrations (sediment and water) in Brazilian freshwaters and their relationship to anthropogenic activity. Acknowledgements We acknowledge Dr. Luis Maurı´cio Bini (University of Goiania) for helping in data analysis, technician Raul Ribeiro (University of Maringa´ – Nupelia) for sampling assistance, MSc. Renata Ribeiro de Arau´jo Rocha (PEA – University of Maringa´) for laboratory assistance and Edvaˆnia Bernardineli for the first English revision. We also thank Dr. G. Bowes (Aquatic Botany Editor) and two anonymous reviewers for important suggestions. Gabrielli Pereira is grateful to CAPES (Coordenadoria de Aperfeic¸oamento do Pessoal de Nı´vel Superior – MEC) for a scholarship. Funds were provided by Itaipu Binacional.

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