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INSTRUMENTATION SCIENCE & TECHNOLOGY Vol. 32, No. 3, pp. 321–333, 2004

Determination of Trace Elements in Alternanthera brasiliana and Pfaffia glabrata by SRTXRF: Application in Environmental Pollution Control M. J. Salvador,1, * S. Moreira,3 D. A. Dias,1,2 and O. L. A. D. Zucchi1,2,* 1

Depto. Quı´mica, Faculdade de Filosofia, Cieˆncias e Letras de Ribeiraõ Preto and Depto. Fı´sica e Quı´mica, Faculdade de Cieˆncias Farmaceûticas de Ribeiraõ Preto, Universidade de Saõ Paulo (USP), Ribeiraõ Preto, Brasil 3 Depto. de Recursos Hı´dricos, Faculdade de Engenharia Civil, Universidade Estadual de Campinas (UNICAMP), Campinas, Brasil 2

ABSTRACT Aiming at environmental control, we report the use of synchrotron radiation total reflection x-ray fluorescence (SRTXRF) analysis as a technique for trace elements determination in plants. The analyses were performed in two species (total plant) of Amaranthaceae family: Alternanthera brasiliana from four sites and Pfaffia glabrata from a single site. Some elements, such as P, S, K, Ca, Mn, Fe, Cu, Zn, Sr, and Pb were detected in all samples, and the elements Cl, Ti, Cr, Co, Ni, Br, Rb, Sr, Cd, Sn, Sb, and Ba were detected in some samples. The limit of detection (LOD) varied from 0.315 (Cu) to 121.4 (P) mg g21. Key Words: Synchrotron radiation; X-ray fluorescence technique; Total reflection; SRTXRF; Trace elements; Alternanthera brasiliana; Pfaffia glabrata; Environmental pollution control.

*Correspondence: M. J. Salvador and O. L. A. D. Zucchi, Depto. Fı´sica e Quı´mica, Faculdade de Cieˆncias Farmaceûticas de Ribeiraõ Preto, Universidade de Saõ Paulo (USP), Av. do Cafe´, s/n, CEP 14040-903, Ribeiraõ Preto, SP, Brasil; E-mail: [email protected] or [email protected]. 321 DOI: 10.1081/CI-120030545 Copyright # 2004 by Marcel Dekker, Inc.

1073-9149 (Print); 1525-6030 (Online) www.dekker.com

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INTRODUCTION The anthropogenic introduction of toxic chemical substances into the environment and their transference into different natural compartments (soil, water, and atmosphere), increased difficulty in the purifying self-capacity of the ecosystems. Due to such environmental problems to which postindustrial mankind is exposed, and the allied constant search for new alternatives to restrict the impact on human activities, several technologies have been implemented; for example, bioremediation process[1,2] and the utilization of proper organisms as bioindicators of environmental pollution.[3,4] Bioremediation is a process for which the proper living organisms are used for environment decontamination.[1] Among such techniques, phytoremediation is potentially top-ranked.[3,5,6] It uses plants to degrade, assimilate, and metabolize environmental pollutants (metals, hydrocarbons, pesticides, organic solvents, etc.) in different matrices (soil, sediment, water, and atmosphere). Several methodologies with plants[2,7] (in situ, in vivo, and in vitro) can be used. So, many concepts (phytoaccumulation, phytoextraction, phytoestabilization, phytotransformation, phytovolatilization, and rhizodegradation) are of relevant importance in the remediation of dangerous or undesirable chemical substances. The ability of certain terrestrial plants to absorb and to accumulate metals as Ca, Ni, Zn, Mn, Cu, and Co renders them very attractive for the disinfection of the atmosphere, as well as to indicate possible environmental alterations, using them as bioindicators, for example, in the environmental pollution for metals. In this process, concepts such as phytoaccumulation and phytoextraction are becoming routine. Plants are hyper-accumulators when they store more than 0.01% of Cd, 0.1% of Ni, or 1% of Zn, Fe, or Mn (usual elements found in vegetables) for dry weight of plant matter under natural atmosphere.[2,8] However, mechanisms related to the uptake, translocation, and metal compartment are not well understood despite considerable improvements that have appeared in recent years. In Brazil, Amaranthaceae is an important family with many species employed in folk medicine for the treatment of several diseases and with the ability for adaptation in different natural habitats[9 – 15] towards environmental adversities such as salinity, soil pH, herbicides, altitude, and pollution (edaphic factors). Some authors suggest that species of Amaranthaceae plants, as such Alternanthera philoxeroids, Blutaparon portulacoids, and Gomphrena globosa can accumulate heavy metals.[16 – 20] Indeed, because of such characteristics, some species can be used as bioindicators of environmental pollution and, thence, as an important tool towards bioremediation. Nowadays, environmental contamination can occur frequently. In urban areas, pollution is a serious problem (automotive intense traffic, inadequate discarding of industrial and domestic residues, industrialization, etc.). In rural areas, the environmental pollution also manifests itself mainly in agricultural activities, as a result of the widespread and indiscriminate use of insecticides, herbicides, fungicides, etc. Measures of environmental pollution stand among the main concerns for authorities in urban centers where the increase of the concentration of atmospheric pollutants becomes a risk for the population’s health.[21] Studies on the atmospheric alterations include: global scale modifications (effect stews, destruction of ozone layer), regional problems (aerosol chemistry, acid deposition) and even some restricted problems of daily living (cigarette smoke, dust, volatile solvents, etc.). The aerosols and particulate materials stand among the more abundant pollutants; they are responsible for pollution of the air, water, and soil. Thus, the

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atmospheric pollutants can be either deposited nearby their sources, or they can be transported and affect even distant areas with deleterious effects over the whole ecosystem, i.e., urban and rural areas. Therefore, the effects of the pollution can present local, regional, or global aspects.[4,22] Among the instrumental analytical techniques, energy dispersive x-ray fluorescence (EDXRF) is highly attractive, allowing the identification and quantification of elements present in different matrices.[10,23 – 26] Total reflection x-ray fluorescence (TXRF), one variant of EDXRF, is especially suitable as it permits trace and ultra-trace analysis in different samples. In TXRF, the effects of intensification and absorption of the analytical line by the sample can be considered irrelevant when the samples are deposited on a Perspex disk and total reflection applied. Besides, its advantage as compared with other analytical methods (atomic absorption spectrometry, inductively coupled plasma techniques)[10,27] is higher because it detects various inorganic elements in small sample volumes and presents the possibility to carry out repetitive assays without prior chemical separation of the constituents of the samples. This paper is aimed at the application of the multi-elemental technique of TXRF with synchrotron radiation for determination of trace elements in A. brasiliana and Pfaffia glabrata (Amaranthaceae), total plants collected from various sites. Also, it is aimed at checking the possibility of using some Amaranthaceae species as bioindicators of environmental pollution.

EXPERIMENTAL Instrumentation Measurements were developed in the “Laborato´rio Nacional de Luz Sıńcrotron” (LNLS), Campinas (SP), Brazil.[28] A polychromatic beam, with energy range 4 –22 keV and photon flux 4 109 photons/sec21 at 8 keV, coming from a storage ring (1.37 GeV and 100 mA) with 2 mm width and 1 mm height under total reflection, 0.5 mm of Al absorbed, 1.0 mm of Ta collimator in the detector, distance sample/detector of 5.0 mm and of Si(Li) detector (resolution of 165 eV for energy of 5.9 keV) was employed with an angle of incidence of 1.0 mrad.

Plant Materials Plant samples (total plant) were collected in their natural habitats (Table 1). Specimens are deposited at the herbarium of “Faculdade de Filosofia, Cieˆncias e Letras de Ribeiraõ Preto/Universidade de Saõ Paulo.” The plants were packed properly in plastic bags for transportation and processed at the Laboratory of Organic Chemistry (“Faculdade de Cieˆncias Farmaceuticas de Ribeiraõ Preto/Universidade de Saõ Paulo”). At the laboratory, this material was processed as soon as possible (so that the stabilization was achieved with the minimum contamination possible) and then washed exhaustively in deionized water. Fresh material also went through a drying process in a hot air circulating oven (408C) and was pulverized with a knife mill, resulting in dried, powdered material.

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Salvador et al. Table 1.

Species, data collect, and localization of Amaranthaceae analyzed by SRTXRF.

Species

Register number

Code

Date collected

Collection sitea

Analyzed part

A. brasiliana

NPL 164

A1

02/1999

Total plant

A. brasiliana

NPL 166

A2

02/1999

A. brasiliana

DAD 041

A3

02/2000

A. brasiliana

NPL 282

A4

03/1998

P. glabrata

NPL 224

B4

03/2000

1-Pico das Almas (Bahia) 2-Pico das Almas (Bahia) 3-Furnas (Minas Gerais) 4-Campos de Jordaõ (Saõ Paulo) 5-Campos de Jordaõ (Saõ Paulo)

Total plant Total plant Total plant

Total plant

a

Sampling sites 1 – 3 were collected close to park areas (rural zones) and the samples of sites 4 and 5 were collected near a city (urban zones).

Preparation of the Sample and Standard Solutions The powder from stabilized plants (0.50 g) was submitted to acid digestion (HNO3/ H2O2) in an open system[29] and, after digestion, the volume was brought to 10 mL with deionized water. The mixture from the acid digestion and deionized water were used as a negative experimental control. For 1 mL of each digested sample solution, 10 mL of internal standard (1025 mg Ga mL21) were added and an aliquot of 5 mL was pipetted in the center of a Perspex disk (polished quartz is used as sample carrier, 3 cm diameter), dried by infrared light (obtaining thus the sample in a shaping thin layer, 5 mm diameter) and irradiated in a spectrometer [synchrotron radiation total reflection x-ray fluorescence (SRTXRF) system]. All the samples were analyzed in triplicate. The same procedure was employed to prepare the multielemental standard samples, in five different concentrations, containing the elements Al, Si, K, Ca, Ti, Cr, Fe, Ni, Zn, Se, Sr, and Mo (K-shell lines) and Mo, Cd, Sb, Ba, Pt, Hg, and Pb (L-shell lines) in which the internal standard (element Ga) was also added. Irradiation time was 200 sec for each sample and 100 sec for the standards.

Quantitative Analysis by SRTXRF The samples and standards analyzed, deposited in the Perspex disk, were taken to the spectrometer in order to measure the characteristic x-ray of all elements present in the samples. The x-ray spectra obtained were interpreted with the aid of AXIL software,[10,19,20,25] determining the energy of peaks present and their respective net intensities. For TXRF it is not necessary to correct for the matrix effect, and the fluorescence

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intensity of the element i is directly proportional to its concentration. Ga was used as an internal standard.[19] Ri ¼ S0i Ci

ð1Þ

for S0i ¼

Si SGa

ð2Þ

and Ri ¼

Ii CGa IGa

ð3Þ

where, Si0 represents the relative spectrometer sensitivity for the element i, Ri the product of the relative intensities for the concentration of the internal standard. Ii, IGa are the intensities (cps), Ci, CGa are the concentrations (mg mL21) and Si, SGa are the x-ray spectrometer sensitivity for element i and Ga (cps mg21 mL21), respectively. From the Si0 values of the standard samples, it becomes possible to obtain the Si0 ¼ f(Zi) and to know the concentration of any elements in the samples. The limit of detection (LODi, mg mL21) was determined for the X-shell lines and L-shell lines by Eq. (4)[30,31] and the function LODi ¼ f(Zi) was determined, for the K-shell lines. rffiffiffiffiffiffiffiffiffiffi IBGi CGa ð4Þ LODi ¼ 3 t IGa S0i where IBGi is the background intensity [counts per second (cps)]; IGa the internal standard (Ga) intensity (cps), CGa the internal standard (Ga) concentration (mg mL21), Si0 the relative sensitivity for the element i and t detection time (sec). RESULTS Obtaining, experimentally, the elementary intensities with the help of the standard samples, it was possible to obtain the experimental sensitivities, and the Si ¼ f(Zi) function was determined[19] for K-shell lines [Eq. (5), Fig. 1] and L-shell lines [Eq. (6), Fig. 2]. The x-ray spectrum for A. brasiliana (sample A4), measured 200 sec by SRTXRF, can be seen in the Fig. 3. By Eq. (4), the experimental LOD were obtained (Table 2) and the function for K-shell lines [Eq. (7), Fig. 4] was determined with significant parameters to a level of 95% of probability. Knowing the Ri for the elements present in the samples, the elemental concentrations were calculated for each sample (Table 3). DISCUSSION AND CONCLUSION The elements P, S, K, Ca, Mn, Fe, Cu, Zn, Sr, and Pb are among the principal representatives, as they were found in all samples. The elements Cl, Ti, Cr, Co, Ni, Br, Rb, Cd, Sn, Sb, and Ba were detected only in some samples, their concentrations varying among

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Figure 1. Relative sensitivity (experimental and calculated, Si0 ) for the elements with atomic numbers 13 Zi 42.

the collection sites, indicating different bioaccumulation (Table 3). According to the phytoaccumulation concept,[2,8] it was verified that all studied plants, except sample A4, show over accumulation K, with elemental concentration higher than 1% of dry weight plant tissues. The sample A3 presented a concentration of Br higher than 0.01% on the dry weight. Therefore, these observations indicate possible potential of metal bioaccumulation in these plants. Further studies should be carried out aimed at providing a better understanding for an application of these Amaranthaceae species as bioindicators of environmental pollution or in the phytoremediation processes. The samples of A. brasiliana from four different sites (rural and urban zones), presented considerable variation in the inorganic elements detected. The coefficient of elementary distribution of Mn/Fe, Zn/Fe, Sr/Fe, and Pb/Fe among the samples showed some similarities to each other (Fig. 5). Fe was chosen as normalizing element—this

Figure 2. Relative sensitivity (experimental and calculated, Si0 ) for the elements with atomic numbers 42 Zi 82.

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Figure 3.

327

XRF spectrum of A. brasiliana (sample A4).

Table 2. Experimental LOD (mg g21) for the elements detected in samples of A. brasiliana and P. glabrata (Amaranthaceae) by SRTXRF. Elements (Z) P (15) S (16) Cl (17) K (19) Ca (20) Ti (22) Cr (24) Mn (25) Fe (26) Co (27) Ni (28) Cu (29) Zn (30) Br (35) Rb (37) Sr (38) Cd (48) Sn (50) Sb (51) Ba (56) Pb (82) a

LODi (mean) 121.4 69.18 28.86 9.25 5.02 1.88 0.688 0.421 0.328 0.355 0.324 0.315 0.367 1.68 2.49 3.33 3.40a 3.69a 3.86a 1.81a 2.22a

Not utilized in the function determination.

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Figure 4. LOD (calculate and experimental) for the synchrotron radiation total reflection (SRTXRF) system employed (15 Zi 38).

Table 3. Concentration (mg g21) by the inorganic elements detected in samples of A. brasiliana and P. glabrata (Amaranthaceae) by SRTXRF. Samples analyzed Element P S Cl K Ca Ti Cr Mn Fe Co Ni Cu Zn Br Rb Sr Cd Sn Sb Ba Pb

A1

A2

A3

A4

B4

2,198 2,946 1,022 20,920 9,248 ND 5.62 98.57 679.2 2.60 2.37 4.96 41.53 ND 20.95 47.87 ND ND ND 52.36 5.01

1,706 3,797 ND 28,520 4,705 ND 6.87 95.58 362.8 1.73 ND 5.83 25.6 ND 8.70 ND ND ND 27.26

2,915 1,721 316.3 15,514 2,370 26.61 4.71 96.86 143.4 1.29 3.68 6.41 32.98 101.1 ND 5.96 ND ND ND ND 3.14

1,600 ND 4,720 4,100 5.44 ND 34.10 425.0 ND ND 14.80 ND 31.41 77.20 33.00 10.60 ND 2.41

2,117 3,914 425.1 38,813 6,648 ND 3.52 82.10 235.5 2.19 0.47 3.75 20.00 14.60 ND 44.60 ND ND 36.10 5.28

Note: ND, element not detected; , LODi.

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Figure 5.

329

Distribution coefficients of the elements to samples analyzed.

element was detected in all samples analyzed. The presence of elements Mn, Sr, Pb, Cl, Ti, Ni, Br, Rb, Sr, Cd, Sn, Sb, and Ba in different concentrations, according to the collecting sites, suggesting a possible environmental exposure. The variation in the concentrations of the inorganic elements can also be observed in A. brasiliana (A4) and P. glabrata (B4) collected at the same site. In these samples, the elements Ti, Rb, Sn, and Sb were detected in A4, but Cl, Cr, Co, Ni, and Ba were detected only in B4. On the other hand, P, S, K, Ca, Mn, Fe, Cu, Zn, Rb, Sr, Cd, and Pb were detected in both species. These species were collected (sites 4 and 5) near a city (urban zone) that can present pollutants (automotive traffic, inadequate discard of industrial, and domestic residues) deposited near their sources or that can be transported to other sites and affect distant areas. Statistical analysis was performed and the principal components analysis validates the cluster analysis.[32,33] The results (Figs. 6 and 7) demonstrate that the samples are grouped by similarity of the trace elements composition, according to collection site and presented

Figure 6. Cluster analysis for samples analyzed.

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Figure 7. Principal components analysis for samples analyzed [l1 ¼ 48.71% (Sn), l2 ¼ 28.94% (Br), and l3 ¼ 15.29%].

low similarity to each other, thereby suggesting a different explanation in agreement with the environmental exposition. Based on cluster analysis (Fig. 6), there is a natural variability of grouping. The samples B4 and A2 formed a first group (0.818 ED, Euclidian Distance); B4, A2 and A1 the second group (1.144 ED); B4, A2, A1, and A3 other (1.324 ED) and finally sample A4 (1.678 ED). The components principal analysis (Fig. 7) confirmed the tendency for the grouping of the samples, indicating different composition in terms of inorganic elemental content. In this grouping, the variables were: Sn (l1 ¼ 48.71%, axis X-first component), Br (l2 ¼ 28.94%, axis Y-second component), and Ni (l3 ¼ 15.29%, axis Z-third component). In order to confirm the results given herein, further evaluations should be conducted at other sites, or at the same sites, with other species, focusing other factors such as: determination of the vehicular flow in different collection sites, type of particulate matter in suspension in the atmosphere and its origin, geochemical characterization of soil and water, among other factors, and the reflection of these eco-toxicological problems in the environment. Several elements were detected in A. brasiliana (samples from different sites) and P. glabrata (sample from a single site) by SRTXRF technique, such as Cr, Mn, Ni, Br, Sr, Cd, Sn, and Pb, suggesting the ability for metal bio-accumulation of these plants. The x-ray fluorescence by total reflection with excitation by synchrotron radiation and the method used in this study are appropriate to determinate trace elements in plants—showing that A. brasiliana and P. glabrata (Amaranthaceae) can, thus, be considered as potential

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candidates for further and more detailed studies for application as bioindicators of environmental pollution.

ABBREVIATIONS Cps EDXRF SRTXRF TXRF

Counts per second Energy dispersive x-ray fluorescence Synchrotron radiation total reflection x-ray fluorescence Total reflection x-ray fluorescence

ACKNOWLEDGMENTS The authors are grateful to FAPESP for financial support and fellowship (N. 02/ 01575-2) given to the first author; to CNPq for financial support; Dr. Josafa´ Carlos de Siqueira for the botanical identification and Dr. Norberto Peporine Lopes who helped to collect the samples. Research partially developed at Laborato´rio Nacional de Luz Sıńcrotron—LNLS (project XRF899/01).

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Received November 10, 2003 Accepted December 11, 2003 Manuscript 1420

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