Calotropis Procera : a PRELIMINARY SURVEY on ITS PHYTOEX- TRACTION CAPABILITIES

2009 International Nuclear Atlantic Conference - INAC 2009 Rio de Janeiro,RJ, Brazil, September27 to October 2, 2009 ASSOCIAÇÃO BRASILEIRA DE ENERGIA NUCLEAR - ABEN ISBN: 978-85-99141-03-8

Calotropis procera : A PRELIMINARY SURVEY ON ITS PHYTOEX- TRACTION CAPABILITIES

Newton P. U. Barbosa 1, George Uemura 2, Maria Ângela B. C. Menezes 2, Ludmila V. S. Matos 2, Maria Aparecida da Silva 2, Geraldo W. Fernandes 1, Rômulo S. C. Menezes 3 and Jarcilene S. Almeida-Cortez 4

1 Laboratório de Ecologia Evolutiva e Biodiversidade - ICB Universidade Federal de Minas Gerais C.P. 486 301661-970, Belo Horizonte, MG [email protected]; [email protected]

2 Centro de Desenvolvimento da Tecnologia Nuclear Comissão Nacional de Energia Nuclear C.P. 941 30123-970, Belo Horizonte, MG [email protected]; [email protected]

3 Departamento de Energia Nuclear, Centro de Tecnologia Universidade Federal de Pernambuco Av. Prof. Luís Freire, 1000 - Cidade Universitária 50740-540 - Recife, PE [email protected]

3 Laboratório de Interação Planta-Animal, Centro de Ciências Biológicas Universidade Federal de Pernambuco Av. Prof. Moraes Rego s/n° - Cidade Universitária 50670-901, Recife, PE [email protected]

ABSTRACT

Calotropis procera (Apocynaceae) is an exotic species, from Africa and Asia, introduced in North-eastern Bra- zil at the beginning of last century; C. procera is a ruderal plant (adapted to poor soil), fit to survive in dry environments, that blooms and fructifies all year round, and its seeds germinate easily. With these characteristics, it is not surprising that its invasiveness has become a matter of concern: its presence has already been reported among native vegetation of caatinga, savannah and rain forest in Brazil. C. procera has medical uses, in India, for many ailments; other uses are as forage, textile and food applications, and as fuel; Furthermore, it is capable of accumulating heavy metals and metalloids; comparisons between leaf samples from polluted and non-polluted sites were already carried out by different authors. Due to the above mentioned, it was decided to analyse, through neutron activation analysis, k 0-method, samples of leaves of C. procera , from polluted and non-polluted sites from the State of Pernambuco, in order to verify which elements were present; samples of the soils from these regions were also analysed. The results that will be presented strongly suggest that C. procera might be an accumulator of Se, and, what has not been reported before, it is capable of absorbing Ba, Cs, La, Sc, Sm, Sr, Ta and Th; also, our results indicate that experiments under controlled conditions should be carried out, in order to ascertain that C. procera is really capable of accumulating the mentioned elements, and its possibilities in phytoremediation strategies. 1. INTRODUCTION

Calotropis procera (Aiton) W.T. Aiton (Apocynaceae) is a shrub, 2.5 m to 6.0 m tall [1], a ruderal plant (adapted to poor soil), with a robust root system, with a main root that can reach a depth from 1.7 to 3.0 m in sandy desert soils, that blooms and fructifies all year round, and its seeds germinate easily [in 2]. After pruning, news branches are quickly formed, as can be seen in Figure 1, that presents also some other features of this species; specimens obtained from seeds yields plants with fast growth, i.e., high biomass production [3]. C. procera was introduced in North-eastern Brazil, at the beginning of 20 th Century, as an ornamental plant [4], where, due to its resistance to drought, it is also used as forage [5]; it is known, in Brazil, by different popular names: flor-de-seda, leiteira, algodão-de-seda, queimadeira or ciúme.

b c f

Figure 1: Calotropis procera : a) Specimen circa 3 month after pruning, ~1.5 m high; b) Aspect of the trunk of a mature specimen; c) Latex (arrow) exuding after a leaf was cut; d) Inflorescence (width of a flower = ~3.0 cm); e) Green fruits (~6.0 cm diameter); f: Dehiscent fruit (arrow points to a seed, length = ~ 0.5 cm).

INAC 2009, Rio de Janeiro, RJ, Brazil. C. procera is original from Tropical Africa, India and Middle East [6], where it can be found even in arid areas, i.e., regions where water deficit, high thermal amplitude, nutritionally poor and sandy soils are some of the natural traits [7,8].

Considering its origin, it is expected that C. procera has the capacity of establishing itself in arid, degraded and deficient soils, like road edges, pastures and abandoned areas [5,9,10]. This capacity is due to morphological and physiological adaptations, that added to other traits, like fast growth, non-specialised pollination system, large amount of seed production, efficient dispersion by the wind, the latter three leading to fast dissemination over wide areas [3], plus others already mentioned, make C. procera a highly capacitated invasive species. And what makes it a matter of concern, a very successful invasive one, a category that is very difficult to eradicate [11,12].

To evaluate the aforementioned concern, biological invasions are considered the second ma- jor cause of the world biodiversity loss [13,14] and, even more worrying, is the irreversibility of the impacts caused by invasion, which worsens as time goes by, with the dissemination of the exotic species [15]. Biological invasions occur when one species is established in an area beyond its normal distribution, remaining with a viable population within the time, usually generating impacts [16-20].

Occupation and transformation of a habitat, alterations of ecological relations and evolution- ary processes, hybridisation with native species and extinction are some of the impacts caused by biological invasions [16]. Biological, geographical, and environmental barriers, as well as resource competition with native species are some of the main hindrances for the establishment of exotic species [17, 19, 21]. A successful establishment depends on the features of the exotic species, as well as on the susceptibility of the invaded habitat, which usually is already disturbed [13,16].

In Brazil, C. procera has been successfully established itself in some tropical rain forest areas [2], savannah areas of Brazil (Cerrado) and mainly in the Northeast seasonally dry forest (Caatinga) [22], especially in areas subjected to anthropogenic disturbances. Caatinga is a bi- ome exclusive of Brazil, rich in biological diversity [23-25], with a xeromorphic vegetation, highly adapted to arid conditions, like water deficit, uneven rainfall, high temperatures, and soil heterogeneity, differing in depth, fertility, salinity and mineralogical constitution [24, 26].

The main impacts to this ecosystem are intensive agropecuary and extrativism activities; these activities have been conducted in a predatory and inadequate way, generating impacts such as overgrazing, deforestation and disordered burnings practised to open space for the ag- riculture. All together, these impacts led to erosive processes, degraded areas, and desertifi- cation [23,27]. Considering the physical similarity of Caatinga areas to the arid environment origins of C. procera , it is no wonder its spread over the Caatinga is so successful [28]; also, it will continue spreading itself over wider areas, and fast.

Due to the habitats where C. procera is found, some authors have analysed its leaves, to verify if this species is adequate for biomonitoring polluted areas [29-31]; Table 1 presents some of these data. Also, not listed in Table 1, As [32] and Hg [33] were reported in leaves of C. procera ; As is also reported in another species of this genus ( C. gigantea ) [34]. In [35], bio-

INAC 2009, Rio de Janeiro, RJ, Brazil. sorptive removal of Cd from groundwater and effluents is proposed, using C. procera biomass.

Table 1: Elemental concentrations in leaves of Calotropis procera -1 Refe- Concentration (mg.kg ) ren- (standard deviation) ce Br Cd Cl Co Cr Cu Fe Mg Mn Ni Pb Rb Sc Se Zn 1.03 2.59 9.31 5.69 17.85 5.17 1.29 nd nd nd nd nd nd nd nd [29] ±0.0 ±0.0 ±0.73 ±1.46 ±1.1 ±1.46 ±1.1 3.11 68.4 12.68 4.63 287.94 18.08 30.05 * nd nd nd nd nd nd nd nd ±2.09 ±9.08 ±2.82 ±4.25 ± 3.39 ±2.5 ±3.63 [30] 3.11 68.4 12.68 4.63 287.9 18.08 30.05 nd nd nd nd nd nd nd nd * ±2.09 ±9.08 ±2.82 ±4.25 ±3.4 ±2.5 ±3.63 257 17300 0.57 0.74 65.4 8630 40 4.65 0.005 0.11 40.2 nd nd nd nd [31] ±11 ±25.0 ±14.0 ±12.0 ±12.0 ±14.0 ±11.0 ±16.0 ±32.0 ±15.0 ±8.0 362 19700 0.5 60 8620 50 5.13 0.09 49.8 ** nd 0.46 nd nd nd nd ±31 ±16.0 ±14.0 ±14.0 ±14.0 ±19.0 ±16.0 ±17.0 ±8.0 * = atomic absorption spectrometry; ** = instrumental neutron activation analysis; nd = non-determined

Another interesting feature of C. procera is its use as a medicinal plant, due to its anti- inflammatory, analgesic, anti-microbial and abortive properties [36]. All parts of this plant are used, including its latex; latex production is a common trait among many unrelated plant families [37], and contrary to popular view, its not always a white sap, it may also be yellow, orange, red, brown, or even colourless [38]. Latex composition is extremely variable, ac- cordingly to the species considered; it is basically considered as a plant defence mechanism, against herbivory; for a review in this topic, see [37,38].

A few examples of plants that produce latex, just to give a glimpse of their variety, are: hemp (Cannabis sativa ); lettuce ( Lactuca sativa ); milkweeds ( Asclepias spp. ); moonflower ( Ipo- moea alba ); opium poppy ( Papaver somniferum ); papaya ( Carica papaya ); para rubber tree (Hevea brasiliensis ), etc [37,38].

It is obvious, by some of the examples just listed, that latex might contain an infinity of or- ganic compounds of medical interest. Additionally, there might be metals in latex composition: in the case of a Sapotaceae native to New Caledonia, Sebertia acuminata , its latex has a nickel content of 25% of its dry weight [39]. It is also obvious, that it is always wise to analyse plants of medical use for metal and/or metalloid accumulation, specially the ones whose use is part of the lore in many countries.

Plants that present the accumulation described are also fit for Phytoremediation, which is a technology that can be considered as a relatively recent one, although it is based on knowl- edge that has been gathered and accumulated for a long time. According to the United States of America Environmental Protection Agency (EPA), Phytoremediation, is the direct use of living green plants for in situ risk reduction for contaminated soil, sludges, sediments, and ground water, through contaminant removal, degradation, or containment [40].

The necessity of accurate, multi-elemental analysis techniques in this kind of activity has already been discussed in [41], where the choice for Neutron Activation Analysis, k 0-method (NAA), has been thoroughly explained. Regardless of redundancy, it is never unnecessary to remind that it is a non-destructive procedure.

INAC 2009, Rio de Janeiro, RJ, Brazil. In Belo Horizonte, the Laboratory for Neutron Activation, located at Centro de Desenvol- vimento da Tecnologia Nuclear/Comissão Nacional de Energia Nuclear, CDTN/CNEN, has been applying the k 0-method as a routine procedure. At CDTN/CNEN, roughly 70% of the known elements can be determined by this analysis [42,43], as shown in [41].

The aim of this work is to verify which elements specimens of C. procera , growing in Brazil, are capable of absorbing, if this species can be indicator of pollution and has a possible use in phytoremediation activities, and to establish the proper methodology to analyse this species.

2. MATERIAL & METHODS

Twenty-four specimens of Calotropis procera were chosen from polluted and non-polluted sites from the State of Pernambuco, Brazil; one polluted site was on the roadside of BR 232, a federal road with heavy traffic, in the outskirts of the state capital, Recife.

The non-polluted site was located in a Caatinga area (S 7° 58’ 32’’, O 38° 17’ 18’’ – Unidade Acadêmica de Serra Talhada), belonging to Universidade Federal Rural de Pernambuco, in Serra Talhada, Pernambuco, Brazil.

In each site, four plots were randomly chosen; in each of them, three plants of approximately the same size were randomly selected, and from each of them 15 leaves, without visible signs of predation and healthy, were sampled. From each plot, one sample of soil was also collected, at a depth ~ 20.0 cm.

Soils samples were dried at room temperature, to avoid loss of more volatile compounds, sieved to five granulometric fractions and the finest-grained fractions (< 0.06 mm) were ho- mogenised by quartering, transferred to irradiation vials and weighed.

From each set of leaves, five were used for area measurement; the remaining ones were washed with distilled water, frozen, and sent to CDTN, where they were weighed, freeze- dried, hand ground, and transferred to irradiation vials and weighed.

The k 0-standardisation method was applied to determine the elemental concentrations in the samples. The irradiation was performed in the carrousel IC-7 of the TRIGA MARK I IPR-R1 reactor at CDTN/CNEN, under a thermal neutron flux of 6.35x10 11 cm -2 s -1 , 100 kW power, for 8 hours. The parameters f and α in the IC-7 are (22.32 ± 0.2) and (-0.0022 ± 0.0002), re- spectively. The samples were irradiated simultaneously with neutron flux monitor Al-Au (0.1%) IRMM-530RA foil cut into 5 mm diameter and 0.1 mm thick.

The usual neutron activation analysis includes the gamma spectroscopy that was performed on an HPGe detector with 15% efficiency. For the spectra analysis - peak area evaluation - the Hyperlab program [44] was used and for the calculation of elemental concentrations, a software package called KAYZERO/SOLCOI [45] was applied.

INAC 2009, Rio de Janeiro, RJ, Brazil. 3. RESULTS & DISCUSSION

Leaf area measurements showed no significant difference between leaves from Serra Talhada and BR 232 (Welch Two Sample t-test, p-value = 0.5564), indicating they had approximately the same age. The NAA analysis results are presented in Tables 2 (soil), 3 (leaves, BR 232) and 4 (leaves, Serra Talhada).

Table 2: Elemental composition of soil from Serra Talhada and BR 232 Concentation Ele – (mg. kg -1 ) ments Serra Talhada BR 232 Plot 1 Plot 2 Plot 3 Plot4 Average Plot 1 Plot 2 Plot 3 Plot4 Average 4535 389 4498 4062 3371 3415 3490 1961 1401 2566.75 Ba ±162 ±72 ±158 ±150 ±999.78 ±122 ±136 ±71 ±50 ±524.23 2.6 5.0 3.8 5.9 4.30 3.2 5 8 3.1 4.74 Br ±0.3 ±0.3 ±0.4 ±0.4 ± 0.70 ±0.4 ±1 ±1 ±0.3 ±1.05 19450 20810 27050 22436.67 Ca < 15700 < 15700 < 15700 < 15700 < 15700 < 15700 ±4046 ±3558 ±4575 ±2339.84 153 99 90 202 136.12 127 205 142 170 161.03 Ce ±5 ±4 ±3 ±7 ±26.12 ±6 ±7 ±5 ±6 ±16.99 10.4 11.5 10.4 28 15.07 24 39 11.9 11.0 21.45 Co ±0.4 ±0.4 ±0.4 ±1 ±4.34 ±1 ±1 ±0.4 ±0.4 ±6.60 96 80 84 150 102.60 60 155 41 48 76.02 Cr ±4 ±3 ±3 ±6 ±16.01 ±3 ±6 ±2 ±2 ±26.71 4.1 3.5 4.3 5.4 4.33 2.3 5.3 7.5 3.5 4.64 Cs ±0.2 ±0.2 ±0.2 ±0.2 ±0.39 ±0.2 ±0.3 ±0.3 ±0.1 ±1.13 2.4 2.0 1.7 4.3 2.60 3.3 4.8 2.4 1.5 3.01 Eu ±0.2 ±0.2 ±0.1 ±0.3 ±0.59 ±0.3 ±0.3 ±0.2 ±0.1 ±0.69 33410 26750 27600 49330 34272.50 59340 81090 28330 25370 48532.50 Fe ±1175 ±942 ±970 ±1732 ±5232.77 ±2082 ±2845 ±995 ±891 ±13296.10 17 19 21 19.31 Ga < 17 < 17 < 17 < 17 < 17 < 17 ±4 ±3 ±4 ±1.16 21 11.5 10.6 10.2 13.40 21 15 22 20.53 Hf < 10 ±1 ±0.4 ±0.4 ±0.4 ±2.66 ±1 ±1 ±1 ±1.75 86400 66660 94760 77690 81377.50 60020 45680 39040 32170 44227.50 K ±4776 ±2659 ±3399 ±2823 ±6017.47 ±2490 ±2269 ±2084 ±1808 ±5942.83 5 48 8 15.23 104 81 7 47.91 La < 5 < 5 ±1 ±2 ±2 ±13.69 ±4 ±3 ±1 ±29.44 4540 9636 3424 4757 5589.25 10480 11160 15430 8881 11487.75 Na ±161 ±360 ±121 ±168 ±1380.16 ±368 ±394 ±542 ±312 ±1398.20 86 58 53 141 84.32 68 127 68 78 85.32 Nd ±4 ±3 ±5 ±11 ±20.16 ±3 ±10 ±4 ±5 ±14.04 358 273 426 389 361.20 185 211 171 127 173.35 Rb ±13 ±11 ±15 ±15 ±32.63 ±8 ±10 ±7 ±5 ±17.60 12.7 12.2 9 23 14.26 29 39 9.4 8.3 21.36 Sc ±0.4 ±0.4 ±0.3 ±1 ±3.02 ±1 ±1 ±0.3 ±0.3 ±7.51 10.2 7.1 5.8 15 9.63 10.9 18 9 8.3 11.49 Sm ±0.4 ±0.3 ±0.3 ±1 ±2.14 ±0.5 ±1 ±1 ±0.5 ±2.18 388 397 272 378 358.93 376 548 461 298 420.80 Sr ±53 ±52 ±43 ±70 ±29.11 ±74 ±102 ±41 ±32 ±53.83 0.81 0.64 0.29 0.4 0.53 2.3 1.9 2 1.12 1.82 Ta ±0.05 ±0.04 ±0.03 ±0.1 ±0.12 ±0.2 ±0.1 ±0.1 ±0.05 ±0.25 15 12.5 6.7 7.5 10.51 18 17 20 29 21.02 Th ±1 ±0.4 ±0.2 ±0.3 ±2.08 ±1 ±1 ±1 ±1 ±2.72 3.1 2.4 1 1.5 2.08 3.7 2.8 4.6 5.1 4.03 U ±0.2 ±0.1 ±0.2 ±0.2 ±0.41 ±0.2 ±0.2 ±0.3 ±0.2 ±0.51 4 8 5 4.0 4.97 Yb < 5 < 5 < 5 < 5 < 5 ±1 ±1 ±1 ±0.2 ±0.91 79 44 18 42 45.74 95 144 74 102 103.53 Zn ±4 ±3 ±2 ±4 ±12.47 ±5 ±8 ±4 ±4 ±14.57 988 560 442 403 598.03 1082 744 1000 942.07 Zr < 4400 ±139 ±59 ±40 ±80 ±134.35 ±222 ±101 ±53 ±88.10

INAC 2009, Rio de Janeiro, RJ, Brazil. Table 3: Elemental composition of leaves of Calotropis procera from BR 232 Concentration Ele- (mg.kg -1 ) ments Plot 1 Plot 2 Plot 3 Plot4 plant 1 plant 2 plant 3 plant 1 plant 2 plant 3 plant 1 plant 2 plant 3 plant 1 plant 2 plant 3 78 77 51 43 42 36 Ba < 16 < 16 < 16 < 16 < 16 < 16 ±6 ±10 ±6 ±9 ±10 ±7 168 193 97 228 187 211 83 126 183 126 98 202 Br ± ±9 ±3 ±8 ±7 ±42 ±3 ±4 ±6 ±4 ±3 ±7 18500 9169 21240 16570 19930 10220 18790 9924 7386 13340 Ca < 4000 < 4000 ±1625 ±2015 ±1325 ±2283 ±3363 ±1070 ±960 ±636 ±1490 ±3018 0.5 0.37 0.7 0.5 0.4 0.3 0.26 0.33 Co < 0.3 < 0.3 < 0.3 < LD ±0.1 ±0.05 ±0.1 ±0.1 ±0.1 ±0.0 ±0.03 ±0.04 4 2.3 2.2 1.7 3.3 2.7 Cr < 1.5 < 1.5 < 1.5 < 1.5 < 1.5 < 1.5 ±1 ±0.4 ±0.5 ±0.3 ±0.4 ±0.4 0.44 Cs < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 ±0.04 197 91 136 Fe < 90 < 90 < 90 < 90 < 90 < 90 < 90 < 90 < 90 ±45 ±22 ±30 24380 59550 41300 38770 38830 31700 31460 28340 33130 36440 37090 K < 949 ±2629 ±2246 ±1493 ±1395 ±1573 ±1288 ±1293 ±1454 ±1363 ±1468 ±1702 0.6 0.51 0.35 La < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 ±0.1 ±0.05 ±0.04 446 1495 4709 606 5695 9391 10320 8617 6318 10280 Na < 300 < 300 ±23 ±56 ±168 ±22 ±205 ±330 ±362 ±326 ±233 ±363 57 8 42 41 15 25 Rb < 8 < 8 < 8 < 8 < 8 < 8 ±3 ±1 ±3 ±3 ±2 ±2 0.03 0.02 0.03 0.04 0.03 0.020 0.02 0.03 Sc < 0.02 < 0.02 < 0.02 < 0.02 ±0.01 ±0.01 ±0.01 ±0.01 ±0.01 ±0.003 ±0.01 ±0.01 1.8 3.2 2.1 9.2 14 22 Se < 2 < 2 < 2 < 2 < 2 < 2 ±0.4 ±0.5 ±0.3 ±0.4 ±1 ±1 0.05 Sm < 0.04 < 0.04 < 0.04 < 0.04 < 0.04 < 0.04 < 0.04 < 0.04 < 0.04 < 0.04 < 0.04 ±0.01 331 98 265 153 217 240 152 133 94 140 Sr < 32 < 32 ±28 ±20 ±28 ±26 ±17 ±22 ±14 ±22 ±15 ±15 0.24 Ta < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 ±0.04 0.37 0.12 0.27 Th < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 ±0.05 ±0.03 ±0.02 20 32 50 30 62 18 27 24 29 Zn < 10 < 10 < 10 ±2 ±3 ±4 ±4 ±4 ±2 ±2 ±2 ±2

Table 4: Elemental composition of leaves of Calotropis procera from Serra Talhada Concentration Ele- (mg.kg -1 ) ments Plot 1 Plot 2 Plot 3 Plot 4 plant 1 plant 2 plant 3 plant 1 plant 2 plant 3 plant 1 plant 2 plant 3 plant 1 plant 2 84 92 1095 157 102 110 91 103 Ba < 63 < 63 < 63 ±13 ±7 ±70 ±8 ±8 ±10 ±8 ±11 71 131 84 143 146 154 616 575 451 181 221 Br ±2 ±5 ±3 ±5 ±5 ±5 ±24 ±20 ±16 ±6 ±8 20790 12750 103600 22500 13170 27240 19240 18610 30900 Ca < 10000 < 10000 ±3457 ±2857 ±26670 ±2396 ±2820 ±3694 ±3824 ±1517 ±4173 0.5 6.6 12.8 5 7.6 3.4 0.46 0.7 Co < 0.4 < 0.4 < 0.4 ±0.1 ±0.2 ±0.5 ±1 ±0.4 0.1± ±0.04 ±0.1 5 6 3.8 25 6.7 6 3 3 2.2 4 Cr < 3 ±1 ±1 ±0.5 ±5 ±0.4 ±1 ±1 ±1 ±0.4 ±1 0.63 0.3 0.72 0.38 Cs < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 ±0.04 ±0.1 ±0.04 ±0.05 157 175 163 Fe < 140 < 140 < 140 < 140 < 140 < 140 < 140 < 140 ±23 ±40 ±23 51340 36560 44540 37840 36780 42230 38890 38640 58770 40280 33180 K ±2031 ±1557 ±1722 ±1834 ±1423 ±1767 ±1466 ±1513 ±2231 ±1433 ±1362 1354 1065 3276 1695 1805 4526 559 2044 7063 1165 795 Na ±49 ±39 ±116 ±62 ±64 ±160 ±21 ±73 ±265 ±42 ±31 26 41 128 131 14 23 210 54 105 66 Rb < 3 ±3 ±5 ±5 ±15 ±1 ±4 ±28 ±3 ±5 ±4 0.04 0.06 0.023 0.043 0.4 0.022 0.032 0.05 0.03 Sc < 0.01 < 0.01 ±0.01 ±0.02 ±0.001 ±0.003 ±0.1 ±0.032 ±0.004 ±0.01 ±0.01 5.1 10.8 93 17 2 2.4 5.7 Se < 1 < 1 < 1 < 1 ±0.4 ±0.4 ±6 ±1 ±1 ±0.2 ± 1.4 1.8 1.4 Sm < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 ±0.2 ±0.2 ±0.3 210 459 266 2047 341 136 2009 309 228 372 538 Sr ±34 ±35 ±21 ±194 ±20 ±17 ±387 ±24 ±27 ±21 ±36 1.25 5 2.0 1.6 Ta < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 < 0.2 ±0.05 ±1 ±0.1 ±0.1 22 21 155 21 27 36 33 30 40 Zn < 13 < 13 ±4 ±2 ±20 ±2 ±6 ±3 ±3 ±2 ±3

INAC 2009, Rio de Janeiro, RJ, Brazil. In [29,30], ashes of leaves of C. procera were analysed, a procedure that might cause the loss of As and Hg compounds, among others, and soil where the specimens were growing was not analysed, either. In [31,33], the aim was to verify deposition of pollutants on the leaves of plants growing on polluted sites; the procedures were more adequate to avoid loss of compounds, but no soil analyses were conducted. In [29-31], no atmospheric pollutant estimation was conducted, either.

This situation, plus the ecological relevance of the C. procera in Brazil, led to the investiga- tion here presented, where it was made an attempt to verify the elements this species can absorb from soil, in polluted and non-polluted sites of Pernambuco, applying a non-destructive analytical technique.

From leaf area measurements, it seems that Calotropis procera leaf growth is not disturbed by more polluted environments; however, comparisons of this kind, so far, were not found in the literature reviewed.

In average, as shown in Table 2, soil from BR 232 plots presents higher elemental concentrations than from Serra Talhada, especially Na. This higher concentration is not unexpected, due to the seashore proximity of BR 232 plots: Recife is on the seashore, and Serra Talhada is 415 km from Recife, in the countryside. Another aspect that is of interest, is that both locali- ties, if there is As and Hg in the soil, their concentrations is bellow our limits of detection, without any probable concern about their presence in those areas.

Some elements will always be present in plant samples, in greater amounts, the so-called macronutrients: Ca, K, Mg, N, P and S. Other elements usually are present, but in small, or even tiny amounts: the micronutrients B, Cl, Cu, Fe, Mg, Mo, Ni and Zn. Another category of plant nutrients are the beneficial ones, which might be present or not, depending on the species (Al, Se and V, for example), or whose absence is not detrimental (Co, Si and Na). This classification has suffered changes along the time, and for a review, see [46].

Plant elemental composition might reflect soil composition; therefore, plant samples from different locations usually present different composition. A comparison of samples from different locations enables a list of elements a certain species is capable of absorbing; so besides the nutrients expected to be found, it has already been described that C. procera can absorb [29-33]: As, Br, Cd, Cr, Hg, Pb, Rb, Sc and Se. From the results presented here, Ba, Cs, La, Sc, Sm, Sr, Ta and Th can be added to the list. The presence of rare-earth elements in that list is not surprising, because they are absorbed by plants (for a review, see [47]).

One of the samples (Serra Talhada, plot 4, plant 3) was not analysed, because fungous growth was found on it. It is surprising that, at a first glance, that the leaf samples from Serra Talhada present higher concentration of the elements analysed, than the samples from BR 232; with exception of Na, again. The proximity to the seashore might explain that, and also indicate some kind of salinity stress in the BR 232 area, causing alterations in growth and consequent less absorption of elements present in the soil. Also, the fact that a certain element is present is not synonymous to its availability to a plant; it might be of interest to verify C. procera resistance to salinity stress, under controlled conditions.

If the results presented here, or in the literature, allow to define, for sure, C. procera as an accumulator of a certain element, that is a question that can not, and should not, be answered

INAC 2009, Rio de Janeiro, RJ, Brazil. yet. Although, there is a very strong evidence to qualify this species as an Se accumulator, considering that Se concentration in soil, in both sites, was bellow the limits of detection, and it was found in some leaf samples, as can be seen in Tables 3 and 4. Also according to the results presented here, it remains to be elucidated if C. procera is capable of absorbing As and Hg, as reported in [32,33], because the levels of these elements in soil, if they exist, are bellow the limits of detection.

C. procera is a species that belongs to a group of plants that should be viewed carefully: latex bearing plants. Latex is an aqueous suspension, of extremely variable composition, as already stated, and that is exuded, for a variable span of time, depending on the species. Figure 1c shows an specimen exuding latex from the stem, after a leaf was cut; that also happens to the leaf, and the amount of latex that is lost is variable. And so is the loss of mass from a sample.

That is why the results in Tables 3 and 4 were presented in a raw form: is it significant to treat them and discuss further than the point of the presence of certain elements, if the samples have lost variable and unknown amounts of latex, i.e., their fresh weight?

Up to now, it was not found any hint that this fact is even considered to affect analytical accuracy, in the literature so far reviewed; jeopardised accuracy leads to biased conclusions, especially when discarding a certain species as an absorber, and even as an accumulator. It seems advisable, first of all, to measure if the latex exuded might alter the elemental composition of a sample from a plant with laticifers; if that is true, to define a better way to sample this kind of species.

Nonetheless, though understandable, and justifiable, to define if a certain species is an accumulator, from field data, it is fundamental to grow it under controlled conditions, since the intricacies of plant nutrition per se are capable of misleading conclusions, as can be reviewed in [46].

Last, but not least, even if it is proved that C. procera is adequate for phytoremediation strategies, one must keep in mind its invasiveness as a severe handicap. What is not usually commented is the management of a certain plant species in remediation technologies; for C. procera , it might be suggested that management should be carried out by constant pruning ( Figure 1a shows its shooting capacity), in order to keep specimens in manageable height, which eases the constant control of the blossoms, that should never be able to fructify.

3. CONCLUSIONS

To determine if a certain plant species is an accumulator, it is necessary to ascertain that trough experiments under controlled conditions, and not only by samples collected in the field.

At the present moment, experiments are being designed to verify if Calotropis procera is an accumulator of As, as stated in the literature, under controlled conditions; so far, that has not been carried out. Also, the establishment of a methodology to sample leaves, without loss of material due to latex exudation, is under progress.

INAC 2009, Rio de Janeiro, RJ, Brazil. To verify if a plant is useful as biomonitor, or in phytoremediation strategies, it is advisable to employ non-destructive and multi-elemental analytical techniques, like neutron activation analysis. The use of k 0-method turns this technique more powerful, because it allows deter- mining elemental composition undreamed of.

Last, but not least, proper management of plant species should be part of any kind of remediation proposals; and that, usually, is not remembered.

ACKNOWLEDGMENTS

The authors wish to thank the help from Carla Patrícia Rodrigues Coutinho, Manuela Cama- rotti Gomes dos Santos, Renata Laranjeiras Gouveia and Viviane Carneiro de Almeida, for collecting the soil and plant samples, and the support from CAPES, CNPq and FAPEMIG.

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