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Hg localisation in Tillandsia usneoides L. (Bromeliaceae), an atmospheric biomonitor
Atmospheric Environment 36 (2002) 881–887
Hg localisation in Tillandsia usneoides L. (Bromeliaceae), an atmospheric biomonitor G.M. Amado Filhoa,*, L.R. Andradeb, M. Farinab, O. Malmc # # Programa Zona Costeira, Instituto de Pesquisas Jardim Botanico do Rio de Janeiro, Rua Pacheco Leao * 915, Jardim Botanico, 22460-030 Rio de Janeiro (RJ), Brazil b ! ! ! # Laboratorio de Biomineralizac-ao, * Departamento de Anatomia, Instituto de Ciencias Biomedicas, CCS/UFRJ, Cidade Universitaria, 21941-590 Rio de Janeiro (RJ), Brazil c ! ! ! Laboratorio de Radioisotopos Eduardo Penna Franca, Instituto de Biof!ısica Carlos Chagas Filho, CCS/UFRJ, Cidade Universitaria, 21941-590 Rio de Janeiro RJ, Brazil a
Received 21 March 2001; received in revised form 4 September 2001; accepted 17 September 2001
Abstract The Spanish moss, Tillandsia usneoides, has been applied as an atmospheric biomonitor of Hg contamination, although the mechanism of metal plant accumulation has not been understood until now. In the present work, analytical scanning electron microscopy (SEM) was used to localize Hg in T. usneoides exposed to a Hg–air-contaminated area during 15 days. After this period, Hg was determined by the flow injection mercury system, and plants were prepared for SEM observation and energy-dispersive X-ray analysis. A concentration of 27027318 mg Hg g 1 was determined in exposed plants. The presented microanalytical results demonstrated that Hg was partly associated with atmospheric particles deposited upon the plant surface, but it was highly absorbed by the scales, stem and leaves surfaces and less absorbed by epidermal cells of T. usneoides. No Hg was detected in mesophyll parenchyma or in vascular system cells. The great surface adsorption area provided by the scales, in addition to the characteristics of T. usneoides morphology, especially of the node region, are suggested to confer the great capability of T. usneoides in Hg holding. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Spanish moss; Atmospheric contamination; Hg; Scanning electron microscopy; EDXA
1. Introduction The increase of mercury transport by the atmosphere has been associated with gold/silver mine activities, burning of fossil fuels, volcanic emissions, geochemical processes, wastes of mercury content of batteries, residues of pesticides, wiring production and chloralkali plant processes (Barghigiani et al., 1991; Bacci et al., 1994; Artaxo et al., 1996; Guimar*aes et al., 1998). Elemental gaseous mercury (Hg0) is the principal form *Corresponding author. Tel./fax: +55-21-2947526. E-mail address: gfi[email protected] (G.M. Amado Filho).
of mercury in the atmosphere. Hg0 is extremely volatile, and can bind to water vapour or airborne particulates. Also it can be oxidized to mercuric ion (Hg+2) photochemically, and methylated by microorganisms (Bacci et al., 1994; Guimar*aes et al., 1998). The atmospheric mercury contamination in a chloralkali plant and surrounding area situated at Rio de Janeiro City (Brazil), has been monitored through transplants of non-contaminated Spanish moss Tillandsia usneoides (Calasans and Malm, 1997). After 15 days of exposition, total Hg concentrations in transplanted T. usneoides reached a maximum of 10,400 mg g 1 dry wt. (Calasans and Malm, 1997).
1352-2310/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 2 - 2 3 1 0 ( 0 1 ) 0 0 4 9 6 - 4
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T. usneoides presents morphological and physiological characteristics that make it suitable for air quality monitoring (Schrimpff, 1984; Benzing and Bermudes, 1991; Padaki et al., 1992; Brighigna et al., 1997; Pyatt et al., 1999). The epiphytic habit determines that the nutrients and moistures are directly uptaken from the atmosphere, without soil contact (Garth, 1964; Brighigna et al., 1988). This species is adapted to hot and dry air conditions, being more appropriate to biomonitor tropical areas than lichens, bryophytes, azalea and pine species, organisms frequently used as atmospheric pollution biomonitors in temperate regions (Steinnes and Krogg, 1977; Pilegaard, 1979; Lodenius and Laaksovirta, 1979; Barghigiani et al., 1991; Bacci et al., 1994; Zhang et al., 1995). The capacity of Tillandsia species to survive in extreme conditions is based on morphological and physiological features. In the Tillandsia genus, the roots are reduced or are absent and act principally for individual fixation in the support substrata, and not to absorb water and nutrients. The stem and leaves are completely covered by scales, that protect the stomatas from desiccation, and are notably hygroscopic, being responsible for the majority of water and aerosols absorption and thus, the dissolved nutrients (Brighigna et al., 1997; Loeschen et al., 1993). The scales increase, significantly, the plant adsorption surface and the protection against desiccation (Brighigna et al., 1988). Tillandsia utilizes the crassulacean acid metabolism (CAM) that reduces water loss by closing their stomatas during the day, when the temperatures and vapour pressure deficit are high, and opening the stomata at night, absorbing the water vapour and the atmospheric CO2. Analytical techniques associated with electron microscopy, like energy-dispersive X-ray analysis (EDXA) have been used to detect, at the ultrastructural level, contaminant elements, like Hg and other heavy metals, helping the understanding of the processes involved in metal accumulation by organisms (Silverberg, 1975; Amado Filho et al., 1996; Haapala, 1998; Lichtenberger and Neumann, 1997; Vesk et al., 1999). The aim of this work is to determine if the available atmospheric Hg was only adsorbed and trapped by the plant external surface structures or if it was absorbed and retained by the plant. The comprehension of the metal accumulation process presented by T. usneoides is important due to its increased utilization as a Hg atmospheric biomonitor. In this way, Hg localization was determined in T. usneoides individuals submitted to high Hg0 atmospheric levels by using analytical scanning electron microscopy (SEM).
2. Material and methods 2.1. Exposure of Tillandsia usneoides to atmospheric Hg An amount of 5 g of live T. usneoides non-contaminated plants, collected at Rio de Janeiro Botanical Garden in October 1999, were introduced in plastic baskets and maintained in the electrolysis room of the chlor-alkali plant, as previously proceeded by Calasans and Malm (1997). Three baskets were positioned at a height of approximately 2 m above the electrolytic cells during 15 days. After this period, the baskets were collected and the plants were prepared for Hg analysis and electron microscopy. 2.2. Hg analysis Plants used for Hg analysis were dried at 401C. Fractions of 3 g dry wt. were digested in a solution of HNO3:H2SO4 (1:1) plus 5.5 ml of KMnO4 5%, in teflon flasks and exposed to microwaves (CEMMDS-2000) for 25 min. After mineralization, 2 ml of H2ONH3Cl and 8 ml of Milli-Q water were added to the sample. Hg concentrations were measured by atomic absorption spectrophotometry with a flow injection system (FIMS-400) and autosampler (AS-90), both from Perkin-Elmer (Bastos et al., 1998). Samples were analysed in triplicates and the results were given in mg g 1 dry wt. 2.3. Conventional and analytical electron microscopy Portions of T. usneoides from individuals of the Hgexposed site and from a non-contaminated area (Rio de Janeiro Botanical Garden) were cut into pieces, approximately 4 mm long, and immediately fixed in glutaraldehyde (2.5%) in cacodilate buffer (0.1 M, pH 7.3) under microwaves (Laboratory Microwave Processor, Pelco mod. RFS59MP, 2.45 gHz) for 10 s at 451C; rinsed twice in buffer, dehydrated in crescent acetone concentrations and dried by the CO2 critical point (Balzers/Union CPD 020) method. The samples were mounted on appropriate stubs and gold-sputtered. The plants were observed and photographed in a Jeol 5310 SEM, operating at 20 kV. For analytical purposes, plants without any chemical fixation were directly mounted on stubs with a conductive carbon cement and coated with carbon (Balzers/Union FL-9496). Samples were analysed by EDXA, performed in JEOL 1200 EX equipped with a scanning attachment system (ASID SSP10). The analysis was done in the spot mode at 40 kV. Qualitative analysis of the elements presented in the samples was obtained by using a Noran-Voyager system (version 4.0). Typical acquisition conditions were: take-off angle=301; livetime=300 s; deadtimeD30 s and beam-
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spot size o50 nm diameter. All the X-ray spectra were acquired in the same operating conditions. As a control for validation of the obtained analytical data, some samples were directly analysed in a low-vacuum (19 Pa) SEM (JEOL 1495), equipped with the same NoranVoyager analytical system, operating at 20 kV.
3. Results and discussion Spanish moss presents a scorpioid dichotomous growth pattern in which stem development alternates with successive nodes, the left branch being dominant at one node, the right branch at the next. The alternate fork at each node opposite to the dominant branch, is a leaf-like branch. The surfaces of all shoots and leaves are completely covered by scales (Figs. 1 and 2). The Hg atmospheric level determined by Calasans and Malm (1997) at the chlor-alkali plant, where the plants were exposed was in a range of 6–62 mg m 3 (mean of 23 mg m 3). The total concentration of Hg in plants, determined (mean and standard deviation of the three baskets samples) after 15 days of air exposure at the chlor-alkali plant, was 27027318 mg g 1, while the concentration in plants of the non-contaminated area was 0.370.01 mg g 1. These results are within the range of concentrations obtained by Calasans and Malm (1997) for both the contaminated (same exposure time) and non-contaminated sites, respectively.
Fig. 2. Enlarged view of the scale observed by SEM; the central disk formed by live cells and the outer shield formed by radially arranged dead cells are clearly seen. Bar=20 mm.
Fig. 3. Typical EDXA spectrum showing Hg peaks (L peaks=9.98 and 11.92 keV; M peak=2.19 keV) obtained from the plant parts where Hg was detected (scales, surface of stem and leaves). C, O, K, Ca and Zn are always detected. The Cu peaks are from the column of the microscope.
Fig. 1. SEM of a transverse section of the stem, showing the scales (S), epidermal cells (arrowhead), mesophyll parenchyma (MP) and the vascular system (VS). Bar=60 mm.
X-ray microanalysis of the exposure plants shows that Hg was detected by using both high- and lowvacuum pressure SEM. The Hg characteristic X-ray emission (L shell peaks=9.98 and 11.92 keV; M shell peak=2.19 keV) confirmed the presence of this element directly in the plant tissues (Figs. 3 and 6) or as a component of irregularly shaped particles adsorbed to the plant surface (Figs. 4 and 5). The same pattern of elemental composition of X-ray spectra,
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Fig. 4. Detailed view (SEM) of the irregularly shaped particles deposited over the scales that contain Hg. Bar=8 mm.
Fig. 6. EDXA spectrum obtained from the epidermal cell wall layer showing the Hg peaks with intensities lower than those shown in Fig. 3. The same acquisition conditions were used in both the spectra.
Fig. 5. EDXA spectrum from the particles shown in Fig. 4. Besides the Hg peaks, Al, Si and Fe were detected.
Fig. 7. EDXA spectrum obtained from the sclerenchymatous fibres, where no Hg was detected.
achieved from the contaminated plants by conventional analytical SEM, was obtained when using lowvacuum analytical SEM (data not shown), indicating that Hg losses caused by electron-beam warming of the sample in the high-vacuum SEM would be unlikely. Chlorine (Ka and Kb peaks) was always associated with the Hg detection and like Hg0, is partially lost during the amalgamation process. Zinc (Ka peak) was systematically associated with Hg and Cl detection. At the chlor-alkali plant, zinc is composed of the coating layer of the amalgamation reservoir into the electrolysis room, and natural abrasion can discharge some micro-particles containing Zn to the surrounding atmosphere. At the irregularly shaped particles adsorbed to the plant surface, Al, Si
and Fe, elements normally present in atmospheric particles from industrial areas (Lee, 1999) were also detected (Fig. 5). Analysis done in the epidermal cell layer (Fig. 1), showed that Hg was present (Fig. 6) in relatively lower amounts than that found in scales or stem surfaces. No Hg was detected (Fig. 7) in the inner parts of the plant, including the mesophyll parenchyma or the sclerenchymatous fibres (Figs. 1 and 8). These fibres are distributed throughout the length of the stem strengthening it, and are composed of the poorly developed vascular system of T. usneoides (Garth, 1964). Although the vascular system plays a weak functional role in Spanish moss, the absence of Hg in vascular system cells indicates that Hg translocation along the plant longitudinal axis has not occurred.
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Fig. 8. Longitudinal section of the stem observed by SEM. Scales (S), epidermal cells (arrowhead), mesophyll parenchyma (MP) and the sclerenchymatous fiber (SF) are seen. Bar=50 mm.
Fig. 9. Transverse section of the node region observed by SEM. Stem (ST), leaf (L) surrounded by external sheaths (ES) are seen. Bar=100 mm.
Experimental data on the accumulation and release kinetics of azalea (Rhododendron sp.) leaves exposed to a constant vapour level of mercury indicates that Hg vapour can concentrate in plant leaves following an irreversible reaction due to Hg0 transformation to a non-volatile ionic form (Hg2+), which presents low mobility within plant tissues (Gaggi et al., 1991). In another experiment, tobacco plants exposed to elemental mercury vapour, accumulates mercury in shoots with no movement to the roots (Suszcynsky and Shann, 1995). These results suggested that Hg did not penetrate through the inner parts of the shoot tissues, where the vascular system is located, and is in agreement with our findings that demonstrated the restriction of Hg distribution to the surface layers of the T. usneoides stem and leaves. Certain higher plants have an armory of metalprotection mechanisms, including metal sequestration by specially produced organic compounds, sequestration in certain cell compartments, metal ion efflux, and organic ligand exudation (Patra and Sharma, 2000). The outer shield of the scales comprise numerous dead cells, each with relatively thick walls and considerable amounts of pectic materials, both of which are hygroscopic in nature (Martin, 1994). The scale capability of water vapour absorption allows all the plant surfaces to be sites of Hg accumulation. In addition, although the morphology of T. usneoides is not characterized as a ‘‘tank’’-type bromeliad (Brighigna et al., 1988),
the node region (Fig. 9) acts as ‘‘micro-tanks’’, providing a reservoir chamber for deposition of particles and water vapour. In a previous work, T. usneoides was used as an atmospheric biomonitor at Alta Floresta (South Amazon region), a city that was a centre for gold commercialization during the gold-rush period in Amazon (1975–1990) and where the re-burning process of the amalgam takes place in gold-dealing shops. Despite the reduction in gold-mining activities, the city still presents high levels of Hg in the environment mainly in soils and buildings but still, some routine emissions are contaminating air, occupational places and surroundings (Malm et al., 1998). In the above cited work, it was verified that part of the T. usneoides Hg concentration was related to the dust adsorbed on the plants surfaces. The presented microanalytical results confirm that the elemental mercury was partly associated with atmospheric particles deposited upon the plant surface, but it was highly absorbed by the scales, by stem and leaves surfaces and less absorbed by epidermal cells of T. usneoides. On the other hand, it was not detected at the inner parts of plant tissues. From these results, it is concluded that the extended surface area caused by the scale abundance is responsible for the adsorption of particles and absorption of water vapour containing Hg, Cl and probably zinc.
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In relation to the traditional Hg determination with instruments, biological monitoring using bromeliads presents some clear advantages, as was previously pointed out by Malm et al. (1998): it allows assessment of hundreds of sites at the same time; it integrates exposure over longer periods of time, and to a certain extent, solves typical problems of atmospheric studies, involving more representative samples, with long-term sampling; very low cost, and giving an idea of bioavailability. However, in the case of T. usneoides, the importance of the Hg deposition process associated with particles adsorption should not be underestimated, as was pointed out by Calasans and Malm (1997), and described by our microanalytical results.
Acknowledgements The authors thank to Fernando Neves Pinto (Lab ! de Radioisotopos/IBCCF, UFRJ) for the Hg quantitative analysis. This work was supported by PRONEX/MCT, JBRJ/MMA, FAPERJ, CNPq & CAPES.
References Amado Filho, G.M., Karez, C.S., Pfeiffer, W.C., YoneshigueValentin, Y., Farina, M., 1996. Accumulation, effects on growth and ultrastuctural localization of Zn in the brown alga Padina gymnospora. Hydrobiologia 326 (7), 451–456. Artaxo, P., Fernandes, E.T., Martins, J.V., Yamasoa, M.A., Xiao, Z., Lindqvist, O., Maenhaut, W., Hacon, S., Campos, R.C., 1996. Large scale atmospheric mercury and trace elements measurements in Amazonia. Proceedings of the Fourth International Conference on Mercury as a Global Pollutant, Hamburg, Germany, 4–8 August. Bacci, E., Gaggi, C., Duccini, M., Barbagli, R., Reanzoni, A., 1994. Mapping mercury in an abandoned Cinnabar mining area by Azalea (Azalea indica) leaf trapping. Chemosphere 29 (4), 641–656. Barghigiani, C., Ristori, T., Bauleo, R., 1991. Pinus as an atmospheric Hg biomonitor. Environmental Technology 12, 1175–1181. Bastos, W.R., Malm, O., Pfeiffer, W.C., Cleary, D., 1998. Establishment and analytical quality control of laboratories for Hg determination in biological and geological samples in the Amazon-Brazil. Ci#encia e Cultura 50 (4), 255–260. Benzing, D.H., Bermudes, D., 1991. Epiphytic bromeliads as air quality monitors in south Florida. Selbyana 21, 162–165. Brighigna, L., Palandri, M.R., Giuffrida, M., Macchi, C., Tani, G., 1988. Ultrastructural features of Tillandsia usneoides L. absorbing trichome during conditions moisture and aridity. Caryologia 41 (2), 111–129. Brighigna, L., Ravanelli, M., Minelli, A., Ercoli, L., 1997. The use of an epiphyte (Tillandsia caput-medusae morren) as
bioindicator of air pollution in Costa Rica. Science of the Total Environment 198, 175–180. Calasans, C.F., Malm, O., 1997. Elemental mercury contamination survey in a chlor-alkali plant by the use of transplanted Spanish moss, Tillandsia usneoides (L.). Science of the Total Environment 208, 165–177. Gaggi, C., Chemello, G., Bacci, E., 1991. Mercury vapor accumulation in azalea leaves. Chemosphere 22, 869–872. Garth, R.E., 1964. The Ecology of Spanish moss (Tillandsia usneoides) its growth and distribution. Ecology 45 (3), 470–481. Guimaraes, J.R.D., Meili, M., Malm, O., Brito, E.M.D., 1998. Hg methylation in sediments and floating meadows of a tropical lake in the Pantanal floodplain, Brazil. Science of the Total Environment 213 (1–3), 165–175. Haapala, H., 1998. The use of SEM/EDX for studying the distribution of air pollutants in the surrounding of the emission source. Environmental Pollution 9, 361–363. Lee, M.R., 1999. Organic–mineral interaction studied by controlled pressure SEM. Microscopy and Microanalysis 35, 19–21. Lichtenberger, O., Neumann, D., 1997. Analytical electron microscopy as a powerful tool in plant cell biology: examples using electron energy loss spectroscopy and Xray microanalysis. European Journal of Cell Biology 73 (4), 378–386. Lodenius, M., Laaksovirta, M., 1979. Mercury content of Hypgymnia physodes and pine needles affected by a chloralkali works at Kuusankoski, SE Finland. Annales Botanici Fennici 16, 7–10. Loeschen, V.S., Martin, C.E., Smith, M., Eder, S.L., 1993. Leaf anatomy and CO2 recycling during crassulacean acid metabolism in twelve epiphytic species of Tillandsia (Bromeliaceae). International Journal of Plant Science 154 (1), 100–106. Malm, O., Fonseca, M.F., Bastos, W.R., Pinto, F.N., 1998. Use of epiphyte plants as biomonitors to map atmospheric mercury in a gold trade center city, Amazonas, Brazil. Science of Total Environment 213 (1–3), 57–64. Martin, C.E., 1994. Physiological ecology of the bromeliaceae. Botanical Review 1 (60), 1–35. Padaki, P.M., McWilliams, E.L., James, W.D., 1992. Use of Spanish moss as an atmospheric monitor for trace elements. Journal of Radioanalytical and Nuclear Chemistry 161 (1), 147–157. Patra, M., Sharma, A., 2000. Mercury toxicity in plants. Botanical Review 66 (3), 379–421. Pilegaard, K., 1979. Heavy metals in bulk precipitation and transplanted Hypgymnia physodes and Dicranoweisia cirrata in the vicinity of Danish steelworks. Water Air and Soil Pollution 11, 77–91. Pyatt, F.B., Grattan, J.P., Lacy, D., Pyatt, A.J., Seaward, R.D., 1999. Comparative effectiveness of Tillandsia usneoides L. and Parmotrema praesorediousum (Nyl.) Hale as bioindicators of atmospheric pollution in Louisiana (USA). Water, Air and Soil Pollution 111, 317–326. Schrimpff, E., 1984. Air pollution patterns in two cities of Colombia, S. A. according to trace substances content of an epiphyte (Tillandsia recurvata L.). Water, Air and Soil Pollution 21, 279–315.
G.M. Amado Filho et al. / Atmospheric Environment 36 (2002) 881–887 Silverberg, B.A., 1975. Ultrastructural localization of lead in Stigeoclonium tenue (Chlorophyceae, Ulotrichales) as demonstrated by cytochemical and X-ray microanalysis. Phycologia 14, 265–274. Steinnes, E., Krogg, H., 1977. Mercury, arsenic and selenium fall-out from and industrial complex studied by means of lichen transplants. Oikos 28, 160–164. Suszcynsky, E.M., Shann, J.R., 1995. Phytotoxicity and accumulation of mercury in tobacco subjected to different
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exposure routes. Environmental Toxicology and Chemistry 14, 61–67. Vesk, P.A., Nockolds, C.E., Allaway, W.G., 1999. Metal localization in water hyacinth roots from an urban wetland. Plant Cell and Environment 22 (2), 149–158. Zhang, L., Planas, D., Qian, J.L., 1995. Mercury concentrations in black spruce (Picea-Marian mill BSP) and lichens in boreal Quebec, Canada. Water, Air and Soil Pollution 85 (1–2), 153–161.
Hg localisation in Tillandsia usneoides L. (Bromeliaceae), an atmospheric biomonitor G.M. Amado Filhoa,*, L.R. Andradeb, M. Farinab, O. Malmc # # Programa Zona Costeira, Instituto de Pesquisas Jardim Botanico do Rio de Janeiro, Rua Pacheco Leao * 915, Jardim Botanico, 22460-030 Rio de Janeiro (RJ), Brazil b ! ! ! # Laboratorio de Biomineralizac-ao, * Departamento de Anatomia, Instituto de Ciencias Biomedicas, CCS/UFRJ, Cidade Universitaria, 21941-590 Rio de Janeiro (RJ), Brazil c ! ! ! Laboratorio de Radioisotopos Eduardo Penna Franca, Instituto de Biof!ısica Carlos Chagas Filho, CCS/UFRJ, Cidade Universitaria, 21941-590 Rio de Janeiro RJ, Brazil a
Received 21 March 2001; received in revised form 4 September 2001; accepted 17 September 2001
Abstract The Spanish moss, Tillandsia usneoides, has been applied as an atmospheric biomonitor of Hg contamination, although the mechanism of metal plant accumulation has not been understood until now. In the present work, analytical scanning electron microscopy (SEM) was used to localize Hg in T. usneoides exposed to a Hg–air-contaminated area during 15 days. After this period, Hg was determined by the flow injection mercury system, and plants were prepared for SEM observation and energy-dispersive X-ray analysis. A concentration of 27027318 mg Hg g 1 was determined in exposed plants. The presented microanalytical results demonstrated that Hg was partly associated with atmospheric particles deposited upon the plant surface, but it was highly absorbed by the scales, stem and leaves surfaces and less absorbed by epidermal cells of T. usneoides. No Hg was detected in mesophyll parenchyma or in vascular system cells. The great surface adsorption area provided by the scales, in addition to the characteristics of T. usneoides morphology, especially of the node region, are suggested to confer the great capability of T. usneoides in Hg holding. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Spanish moss; Atmospheric contamination; Hg; Scanning electron microscopy; EDXA
1. Introduction The increase of mercury transport by the atmosphere has been associated with gold/silver mine activities, burning of fossil fuels, volcanic emissions, geochemical processes, wastes of mercury content of batteries, residues of pesticides, wiring production and chloralkali plant processes (Barghigiani et al., 1991; Bacci et al., 1994; Artaxo et al., 1996; Guimar*aes et al., 1998). Elemental gaseous mercury (Hg0) is the principal form *Corresponding author. Tel./fax: +55-21-2947526. E-mail address: gfi[email protected] (G.M. Amado Filho).
of mercury in the atmosphere. Hg0 is extremely volatile, and can bind to water vapour or airborne particulates. Also it can be oxidized to mercuric ion (Hg+2) photochemically, and methylated by microorganisms (Bacci et al., 1994; Guimar*aes et al., 1998). The atmospheric mercury contamination in a chloralkali plant and surrounding area situated at Rio de Janeiro City (Brazil), has been monitored through transplants of non-contaminated Spanish moss Tillandsia usneoides (Calasans and Malm, 1997). After 15 days of exposition, total Hg concentrations in transplanted T. usneoides reached a maximum of 10,400 mg g 1 dry wt. (Calasans and Malm, 1997).
1352-2310/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 2 - 2 3 1 0 ( 0 1 ) 0 0 4 9 6 - 4
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T. usneoides presents morphological and physiological characteristics that make it suitable for air quality monitoring (Schrimpff, 1984; Benzing and Bermudes, 1991; Padaki et al., 1992; Brighigna et al., 1997; Pyatt et al., 1999). The epiphytic habit determines that the nutrients and moistures are directly uptaken from the atmosphere, without soil contact (Garth, 1964; Brighigna et al., 1988). This species is adapted to hot and dry air conditions, being more appropriate to biomonitor tropical areas than lichens, bryophytes, azalea and pine species, organisms frequently used as atmospheric pollution biomonitors in temperate regions (Steinnes and Krogg, 1977; Pilegaard, 1979; Lodenius and Laaksovirta, 1979; Barghigiani et al., 1991; Bacci et al., 1994; Zhang et al., 1995). The capacity of Tillandsia species to survive in extreme conditions is based on morphological and physiological features. In the Tillandsia genus, the roots are reduced or are absent and act principally for individual fixation in the support substrata, and not to absorb water and nutrients. The stem and leaves are completely covered by scales, that protect the stomatas from desiccation, and are notably hygroscopic, being responsible for the majority of water and aerosols absorption and thus, the dissolved nutrients (Brighigna et al., 1997; Loeschen et al., 1993). The scales increase, significantly, the plant adsorption surface and the protection against desiccation (Brighigna et al., 1988). Tillandsia utilizes the crassulacean acid metabolism (CAM) that reduces water loss by closing their stomatas during the day, when the temperatures and vapour pressure deficit are high, and opening the stomata at night, absorbing the water vapour and the atmospheric CO2. Analytical techniques associated with electron microscopy, like energy-dispersive X-ray analysis (EDXA) have been used to detect, at the ultrastructural level, contaminant elements, like Hg and other heavy metals, helping the understanding of the processes involved in metal accumulation by organisms (Silverberg, 1975; Amado Filho et al., 1996; Haapala, 1998; Lichtenberger and Neumann, 1997; Vesk et al., 1999). The aim of this work is to determine if the available atmospheric Hg was only adsorbed and trapped by the plant external surface structures or if it was absorbed and retained by the plant. The comprehension of the metal accumulation process presented by T. usneoides is important due to its increased utilization as a Hg atmospheric biomonitor. In this way, Hg localization was determined in T. usneoides individuals submitted to high Hg0 atmospheric levels by using analytical scanning electron microscopy (SEM).
2. Material and methods 2.1. Exposure of Tillandsia usneoides to atmospheric Hg An amount of 5 g of live T. usneoides non-contaminated plants, collected at Rio de Janeiro Botanical Garden in October 1999, were introduced in plastic baskets and maintained in the electrolysis room of the chlor-alkali plant, as previously proceeded by Calasans and Malm (1997). Three baskets were positioned at a height of approximately 2 m above the electrolytic cells during 15 days. After this period, the baskets were collected and the plants were prepared for Hg analysis and electron microscopy. 2.2. Hg analysis Plants used for Hg analysis were dried at 401C. Fractions of 3 g dry wt. were digested in a solution of HNO3:H2SO4 (1:1) plus 5.5 ml of KMnO4 5%, in teflon flasks and exposed to microwaves (CEMMDS-2000) for 25 min. After mineralization, 2 ml of H2ONH3Cl and 8 ml of Milli-Q water were added to the sample. Hg concentrations were measured by atomic absorption spectrophotometry with a flow injection system (FIMS-400) and autosampler (AS-90), both from Perkin-Elmer (Bastos et al., 1998). Samples were analysed in triplicates and the results were given in mg g 1 dry wt. 2.3. Conventional and analytical electron microscopy Portions of T. usneoides from individuals of the Hgexposed site and from a non-contaminated area (Rio de Janeiro Botanical Garden) were cut into pieces, approximately 4 mm long, and immediately fixed in glutaraldehyde (2.5%) in cacodilate buffer (0.1 M, pH 7.3) under microwaves (Laboratory Microwave Processor, Pelco mod. RFS59MP, 2.45 gHz) for 10 s at 451C; rinsed twice in buffer, dehydrated in crescent acetone concentrations and dried by the CO2 critical point (Balzers/Union CPD 020) method. The samples were mounted on appropriate stubs and gold-sputtered. The plants were observed and photographed in a Jeol 5310 SEM, operating at 20 kV. For analytical purposes, plants without any chemical fixation were directly mounted on stubs with a conductive carbon cement and coated with carbon (Balzers/Union FL-9496). Samples were analysed by EDXA, performed in JEOL 1200 EX equipped with a scanning attachment system (ASID SSP10). The analysis was done in the spot mode at 40 kV. Qualitative analysis of the elements presented in the samples was obtained by using a Noran-Voyager system (version 4.0). Typical acquisition conditions were: take-off angle=301; livetime=300 s; deadtimeD30 s and beam-
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spot size o50 nm diameter. All the X-ray spectra were acquired in the same operating conditions. As a control for validation of the obtained analytical data, some samples were directly analysed in a low-vacuum (19 Pa) SEM (JEOL 1495), equipped with the same NoranVoyager analytical system, operating at 20 kV.
3. Results and discussion Spanish moss presents a scorpioid dichotomous growth pattern in which stem development alternates with successive nodes, the left branch being dominant at one node, the right branch at the next. The alternate fork at each node opposite to the dominant branch, is a leaf-like branch. The surfaces of all shoots and leaves are completely covered by scales (Figs. 1 and 2). The Hg atmospheric level determined by Calasans and Malm (1997) at the chlor-alkali plant, where the plants were exposed was in a range of 6–62 mg m 3 (mean of 23 mg m 3). The total concentration of Hg in plants, determined (mean and standard deviation of the three baskets samples) after 15 days of air exposure at the chlor-alkali plant, was 27027318 mg g 1, while the concentration in plants of the non-contaminated area was 0.370.01 mg g 1. These results are within the range of concentrations obtained by Calasans and Malm (1997) for both the contaminated (same exposure time) and non-contaminated sites, respectively.
Fig. 2. Enlarged view of the scale observed by SEM; the central disk formed by live cells and the outer shield formed by radially arranged dead cells are clearly seen. Bar=20 mm.
Fig. 3. Typical EDXA spectrum showing Hg peaks (L peaks=9.98 and 11.92 keV; M peak=2.19 keV) obtained from the plant parts where Hg was detected (scales, surface of stem and leaves). C, O, K, Ca and Zn are always detected. The Cu peaks are from the column of the microscope.
Fig. 1. SEM of a transverse section of the stem, showing the scales (S), epidermal cells (arrowhead), mesophyll parenchyma (MP) and the vascular system (VS). Bar=60 mm.
X-ray microanalysis of the exposure plants shows that Hg was detected by using both high- and lowvacuum pressure SEM. The Hg characteristic X-ray emission (L shell peaks=9.98 and 11.92 keV; M shell peak=2.19 keV) confirmed the presence of this element directly in the plant tissues (Figs. 3 and 6) or as a component of irregularly shaped particles adsorbed to the plant surface (Figs. 4 and 5). The same pattern of elemental composition of X-ray spectra,
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Fig. 4. Detailed view (SEM) of the irregularly shaped particles deposited over the scales that contain Hg. Bar=8 mm.
Fig. 6. EDXA spectrum obtained from the epidermal cell wall layer showing the Hg peaks with intensities lower than those shown in Fig. 3. The same acquisition conditions were used in both the spectra.
Fig. 5. EDXA spectrum from the particles shown in Fig. 4. Besides the Hg peaks, Al, Si and Fe were detected.
Fig. 7. EDXA spectrum obtained from the sclerenchymatous fibres, where no Hg was detected.
achieved from the contaminated plants by conventional analytical SEM, was obtained when using lowvacuum analytical SEM (data not shown), indicating that Hg losses caused by electron-beam warming of the sample in the high-vacuum SEM would be unlikely. Chlorine (Ka and Kb peaks) was always associated with the Hg detection and like Hg0, is partially lost during the amalgamation process. Zinc (Ka peak) was systematically associated with Hg and Cl detection. At the chlor-alkali plant, zinc is composed of the coating layer of the amalgamation reservoir into the electrolysis room, and natural abrasion can discharge some micro-particles containing Zn to the surrounding atmosphere. At the irregularly shaped particles adsorbed to the plant surface, Al, Si
and Fe, elements normally present in atmospheric particles from industrial areas (Lee, 1999) were also detected (Fig. 5). Analysis done in the epidermal cell layer (Fig. 1), showed that Hg was present (Fig. 6) in relatively lower amounts than that found in scales or stem surfaces. No Hg was detected (Fig. 7) in the inner parts of the plant, including the mesophyll parenchyma or the sclerenchymatous fibres (Figs. 1 and 8). These fibres are distributed throughout the length of the stem strengthening it, and are composed of the poorly developed vascular system of T. usneoides (Garth, 1964). Although the vascular system plays a weak functional role in Spanish moss, the absence of Hg in vascular system cells indicates that Hg translocation along the plant longitudinal axis has not occurred.
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Fig. 8. Longitudinal section of the stem observed by SEM. Scales (S), epidermal cells (arrowhead), mesophyll parenchyma (MP) and the sclerenchymatous fiber (SF) are seen. Bar=50 mm.
Fig. 9. Transverse section of the node region observed by SEM. Stem (ST), leaf (L) surrounded by external sheaths (ES) are seen. Bar=100 mm.
Experimental data on the accumulation and release kinetics of azalea (Rhododendron sp.) leaves exposed to a constant vapour level of mercury indicates that Hg vapour can concentrate in plant leaves following an irreversible reaction due to Hg0 transformation to a non-volatile ionic form (Hg2+), which presents low mobility within plant tissues (Gaggi et al., 1991). In another experiment, tobacco plants exposed to elemental mercury vapour, accumulates mercury in shoots with no movement to the roots (Suszcynsky and Shann, 1995). These results suggested that Hg did not penetrate through the inner parts of the shoot tissues, where the vascular system is located, and is in agreement with our findings that demonstrated the restriction of Hg distribution to the surface layers of the T. usneoides stem and leaves. Certain higher plants have an armory of metalprotection mechanisms, including metal sequestration by specially produced organic compounds, sequestration in certain cell compartments, metal ion efflux, and organic ligand exudation (Patra and Sharma, 2000). The outer shield of the scales comprise numerous dead cells, each with relatively thick walls and considerable amounts of pectic materials, both of which are hygroscopic in nature (Martin, 1994). The scale capability of water vapour absorption allows all the plant surfaces to be sites of Hg accumulation. In addition, although the morphology of T. usneoides is not characterized as a ‘‘tank’’-type bromeliad (Brighigna et al., 1988),
the node region (Fig. 9) acts as ‘‘micro-tanks’’, providing a reservoir chamber for deposition of particles and water vapour. In a previous work, T. usneoides was used as an atmospheric biomonitor at Alta Floresta (South Amazon region), a city that was a centre for gold commercialization during the gold-rush period in Amazon (1975–1990) and where the re-burning process of the amalgam takes place in gold-dealing shops. Despite the reduction in gold-mining activities, the city still presents high levels of Hg in the environment mainly in soils and buildings but still, some routine emissions are contaminating air, occupational places and surroundings (Malm et al., 1998). In the above cited work, it was verified that part of the T. usneoides Hg concentration was related to the dust adsorbed on the plants surfaces. The presented microanalytical results confirm that the elemental mercury was partly associated with atmospheric particles deposited upon the plant surface, but it was highly absorbed by the scales, by stem and leaves surfaces and less absorbed by epidermal cells of T. usneoides. On the other hand, it was not detected at the inner parts of plant tissues. From these results, it is concluded that the extended surface area caused by the scale abundance is responsible for the adsorption of particles and absorption of water vapour containing Hg, Cl and probably zinc.
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In relation to the traditional Hg determination with instruments, biological monitoring using bromeliads presents some clear advantages, as was previously pointed out by Malm et al. (1998): it allows assessment of hundreds of sites at the same time; it integrates exposure over longer periods of time, and to a certain extent, solves typical problems of atmospheric studies, involving more representative samples, with long-term sampling; very low cost, and giving an idea of bioavailability. However, in the case of T. usneoides, the importance of the Hg deposition process associated with particles adsorption should not be underestimated, as was pointed out by Calasans and Malm (1997), and described by our microanalytical results.
Acknowledgements The authors thank to Fernando Neves Pinto (Lab ! de Radioisotopos/IBCCF, UFRJ) for the Hg quantitative analysis. This work was supported by PRONEX/MCT, JBRJ/MMA, FAPERJ, CNPq & CAPES.
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