Interciencia ISSN: 0378-1844 [email protected] Asociación Interciencia Venezuela

Olivares, Elizabeth; Peña, Eder Fluoride and metals in crassifolia, a medicinal tree from the neotropical savannahs Interciencia, vol. 29, núm. 3, marzo, 2004, pp. 145-152 Asociación Interciencia Caracas, Venezuela

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative FLUORIDE AND METALS IN Byrsonima crassifolia, A MEDICINAL TREE FROM THE NEOTROPICAL SAVANNAHS

Elizabeth Olivares and Eder Peña

SUMMARY

The concentration of fluoride (F) in relation to ele- the content of F was high compared to the normal range in ments (K, Ca, Mg, Mn, Fe, Cu, Zn, Ni, Co, Cr, Al, Pb and Cd) . The difference in concentrations of F, Fe, Al and Pb was and total phenols was evaluated in leaves of Byrsonima highly significant by leaf age and site. In other species, a positive crassifolia. Young, adult and fallen leaves were sampled in a gar- correlation of F to Al and total phenols, and a negative correla- den located outside an aluminium factory in Bolivar State, Venezu- tion to Ca has been reported, while in the present paper a posi- ela and in two sites in the savannah about 5 and 8km away from tive correlation of F to Al, Ca and Fe, but not with total phenols the Al-smelter in the direction of the wind. A positive linear re- was found in B. crassifolia. The presence of metal-fluorine com- gression of F with Mn, Fe, Pb, Al, Ca, Mg, Cr and Zn was found pounds that can be released during medicinal infusions has not for young and adult leaves. Leaves were sampled in two more dis- been previously reported in this species. Washing the leaves with tant sites (B1, B2) and in a contrasting site (M) at a region with- water showed that a high proportion of F and minerals was out Al mining or factories. In the vicinity of the smelter and in B1 present on the surface of the leaf and/or the trichomes.

Introduction blocked the citrate cycle. from Australia contain up to readily to the soil and plants. Fluoracetate is found at low 137mmol·kg-1 fluoracetate Atmospheric pollution and Fluorine is the most abun- concentrations in a wide vari- (O’Hagan and Harper, 1999). repeated application of fertil- dant halogen in the earth’s ety of plants, however it is According to Arnesen (1997) izers and soil amendments, crust, but only six fluorinated accumulated at very high con- the maximum recommended such as phosphatic fertilizers natural products have been centrations (421, 342 and limit for fluorine content in and phosphogypsum, increase isolated, excluding fatty acid 26mmol·kg-1 on a dry weight hay and pasture grass is the total F concentration in homologues (O’Hagan and basis, respectively) in seeds 1.6mmol·kg-1. soils because they contain up Harper, 1999). The first or- of Dichapetalum braunii from Inorganic fluorine is present to 3.5% of F as impurities ganic fluorine compound Tanzania, Gastrolobium bilo- in plants mainly as metal- (Stevens et al., 1997). A high identified, in 1943, was fluo- bum from Australia and Pali- fluorine compounds (Baroni correlation between F emis- racetate, which may cause courea marcgravii from South Fornasiero, 2001). It is the sion levels from Al-smelters death in grazing animals, be- America, presumably for pur- most electronegative element in Norway and F accumula- cause it is transferred to poses of defense. Not only and binds metals forming tion in pine and spruce fluorocitrate and irreversibly seeds can be toxic, as leaves complexes (fluorides, hereun- needles has been reported bound to aconitase, thereby of Oxylobium parviflorum der F), which are adsorbed (Horntvedt, 1995).

KEYWORDS / Aluminium / Fluoride / Iron / Nutrients / Pollution / Received: 09/24/2003. Modified: 02/16/2004. Accepted: 02/27/2004.

Elizabeth Olivares. Ph.D. in Bio- IVIC. Address: Centro de Eco- Eder Peña. Bachelor in Biology, logy (Ecology), Instituto Ve- logía, IVIC. Apartado 21827. Universidad Central de Venezu- nezolano de Investigaciones Caracas 1020A. Venezuela. ela. Researcher Professional, Científicas (IVIC). Researcher, e-mail: [email protected] IVIC. e-mail: [email protected]

MAR 2004, VOL. 29 Nº 3 0378-1844/04/03/145-08 $ 3.00/0 145 RESUMEN

La concentración de fluoruro (F) en relación a elementos mi- brica y en B1 el contenido de F fue alto en comparación a lo nor- nerales (K, Ca, Mg, Mn, Fe, Cu, Zn, Ni, Co, Cr, Al, Pb y Cd) y mal en plantas. Las diferencias en las concentraciones de F, Fe, Al fenoles totales fue evaluada en hojas de Byrsonima crassifolia. Se y Pb por edad y sitio fueron significativas. En otras especies se ha cosecharon hojas jóvenes, adultas y caídas en un jardín situado en reportado una correlación positiva de F con Al y fenoles totales, y las afueras de una industria de aluminio, en el Estado Bolívar, Ve- negativa con Ca. En este trabajo se encontró, en B. crassifolia, una nezuela y en dos sitios en la sabana a una distancia aproximada de correlación positiva de F con Al, Ca y principalmente Fe, pero no 5 y 8km de la fábrica en la dirección del viento. Se encontró una con fenoles. Previamente no se había reportado la presencia de regresión lineal positiva de F con Mn, Fe, Pb, Al, Ca, Mg, Cr y Zn compuestos metálicos con F en esta especie, los cuales pueden ser para hojas jóvenes y adultas. Se colectaron también hojas en otros liberados en infusiones medicinales. El lavado de hojas con agua dos sitios más distantes (B1 y B2) y en un sitio contrastante (M) en mostró que una proporción alta de F y minerales estaba presente una zona sin minería o industrias de Al. En las cercanías a la fá- en la superficie foliar y/o los tricomas.

RESUMO

A concentração de fluoreto (F) em relação a elementos mine- Nas cercanias da fábrica e em B1 o conteúdo de F foi alto em rais (K, Ca, Mg, Mn, Fe, Cu, Zn, Ni, Co, Cr, Al, Pb y Cd) e comparação ao normal em plantas. As diferenças nas concentra- fenóis totais foi avaliada em folhas de Byrsonima crassifolia. Se ções de F, Fe, Al e Pb por idade e lugar foram significativas. Em plantaram folhas jóvens, adultas e caídas em um jardim situado outras espécies se tem reportado uma correlação positiva de F nas aforas de uma industria de alumínio, no Estado Bolívar, Vene- com Al e fenóis totais, e negativa com Ca. Neste trabalho encon- zuela e em dois lugares na savana a uma distância aproximada trou-se, em B. crassifolia, uma correlação positiva de F com Al, de 5 e 8 km da fábrica na direção do vento. Encontrou-se uma Ca e principalmente Fe, mas não com fenóis. Previamente não se regressão lineal positiva de F com Mn, Fe, Pb, Al, Ca, Mg, Cr e havía reportado a presença de compostos metálicos com F nesta Zn para folhas jovens e adultas. Se recolheram também folhas em espécie, os quais podem ser liberados em infusões medicinais. A outros dois lugares mais distantes (B1 e B2) e em um lugar lavagem de folhas com água mostrou que uma proporção alta de contrastante (M) em uma zona sem mineria ou indústrias de Al. F e minerais estava presente na superfície foliar e/ou os tricomas.

Aluminium is the most umes of water by infusion for 1997). The content of total (8º16'N, 62º48'W) located prevalent cation with which F 2h (Berger et al., 1998). It has phenols is positively corre- near Ciudad Guayana, Bolivar complexes in acid soils. been reported that very signifi- lated with F in Pinus nigra State, Venezuela, and two Nagata et al. (1993) found cant reductions in spontaneous (Giertych et al., 1999). sites in the savannah at a dis- evidence that those complexes locomotor activity and explor- The aim of this work was tance of 5km (8º15'N, 62º are taken up and transported atory behavior are caused by a to study the concentration of 50'W) and 8km (8º14'N, in tea plants until they reach dosage of 1.25g dried /kg F and the mineral composi- 62º51'W) west from the fac- the leaf where the complexes aqueous extracts of bark and tion of leaves of B. crassifo- tory. The sampling was done are dissociated. Fung et al. leaves of B. crassifolia lia, and to evaluate the differ- in the prevailing wind direc- (1999) found that soil pH and (Cifuentes et al., 2001). ences in F concentration in tion (North-East) in May extractable Al concentration It is important to know the relation to metals and total 2001. affected the F contents in F content of plants used in phenols, in leaves of different Samples were also col- soils and plant tissues from infusions. Fluorine is phyto- ages, in sites with pollution lected in two other sites in tea plantations. toxic, causing damage in veg- differences due to their prox- Bolívar State in May 2001. The present study was car- etation, wildlife and humans imity to or farness from an Site B1 (8º11'N, 62º52'W) ried out on Byrsonima crassi- (Thomson et al., 1979; Wein- Al-smelter. was located 20km away from folia, a medicinal plant dis- stein and Davison, 2003). the Al-smelter in a savannah tributed in several regions of Fluorosis has been reported in Material and methods close to the intersection Mexico, Central and South inhabitants of Chinese prov- known as km 88 on the road America, used since pre-His- inces and Tibet where tea Plant material from Ciudad Guayana to the panic times. The bark and with a high F content is con- iron mines of El Pao. Site B2 leaves are used to treat sumed (Fung et al., 1999, Young (distal, light green, (8º10'N, 62º47'W) was located coughs, gastrointestinal disor- Cao et al., 2000). A small hairy and soft), adult (basal, in Ciudad Guayana, at a uni- ders, skin infections and amount of F is beneficial in green, leathery) and old versity garden (Universidad snake bites (Martínez-Váz- the prevention of dental caries fallen leaves (red-brownish, Nacional Experimental de quez et al., 1999). In Mexico and is used to treat os- leathery) were sampled Guayana), 15km away from it is the medicinal plant most teoporosis, but high doses from five adult plants of the Al-smelter but in opposite frequently used against diar- may cause damage. Byrsonima crassifolia (L.) direction to the wind and rhea (Leonti et al., 2001). Ad- A positive correlation of F H.B.K. () in smelter. ditionally, ethanol extracts of to Al has been shown in to- each study site. Comparisons were carried leaves showed some trypano- mato, oat, clover and Ti- out with plants from site M, cidal activity (Berger et al., bouchina pulchra (Arnesen, Study sites located at 10º8’N, 67º8'W in 1998). 1997; Stevens et al., 1997; Miranda State, in a tree sa- The dry bark and leaf of B. Domingos et al., 2003), and a Leaf samples were col- vannah without Al industries, crassifolia is extracted in tradi- negative correlation to Ca in lected in a garden in the where leaves were sampled in tional medicine with ten vol- clover and ryegrass (Arnesen, vicinity of an Al-smelter February, 2000.

146 MAR 2004, VOL. 29 Nº 3 Al, 0.5-60ppm Mn, 0.01- Results 2ppm Zn, 0.03-10ppm Cu, 0.1-20ppm Ni, 0.06-15ppm Fluoride, metals and total Cr, 0.1-30ppm Pb, 0.05- phenols in different age leaves 15ppm Co, and 15-800ppm in relation to distance from K. Merck Standards were an Al-smelter used for each element. With a peach leaves standard (ref- In adult leaves the highest erence 1547) from the Na- fluoride (F) concentration tional Institute of Standards (7.0 ±0.1mmol·kg-1) was ob- and Technology, Gaithers- tained close to the factory burg, MD, a 96% Mg, 96% (Figure 1). The concentra- Ca, 99% Al, 97% Mn and tion of Ca, Fe, Mg, Al, Mn 97% Cu recovery was ob- (Figure 1), Zn, Cu, Ni, Cr, tained. Pb and Co (Figure 2) was Total phenols were measured also high in those leaves in colorimetrically in ethanol ex- relation to the other tracts of ground dried leaves, samples. The decrease of F using the Folin-Ciocalteu and metal contents observed method (Amorim et al., 1977). in young and adult leaves in Values were standardized relation to the distance to against chlorogenic acid. the Al-smelter was not ob- served in fallen leaves for F, Washing experiment Ca, Al, Mn, Cu, Ni and Cr. The concentration of K in Young leaves (n=18) were plants close to the factory cut from the plants and was not higher than that of washed with distilled and distant ones. Young leaves deionized water until the tri- showed higher K contents chomes were not visible (1l than adult and fallen leaves. per leaf). The same amount The leaves with the highest of water was used for the not metal concentrations showed hairy adult leaves (n=18). Op- the lowest contents of phe- Figure 1. Mean (±SE) contents of fluoride (F), Ca, Fe, Mg, Al and posite pairs of unwashed nols (Figure 2). Mn in young, adult and fallen leaves of B. crassifolia, collected at young and adult leaves were Table I shows the compari- different distances from an aluminium smelter in May 2001 (n=5). collected from the trees. The son by age, site and their in- sampling was done in B1, B2 teraction, of the differences and in the Al-smelter garden in concentrations found for F, Chemical analyses 0.1M). The supernatant was on October 2002. Minerals metals and phenols in the vi- mixed 1:1 with TISAB-IV and F were measured and the cinity of the Al-smelter. Soluble fluoride (F) was buffer to dissociate F- value obtained in the un- Highly significant differences determined in hot water Complexes and stabilize washed leaves was considered (p<0.001) were found for F, extracts of dried ground pH. The buffer used con- 100%. The difference between Ca, Fe, Al, Mn, Pb and leaves samples. A total of tained 84ml concentrated unwashed and washed was phenols, but no differences 100mg dry mass was in- HCl, 242g TRIZMA base calculated as percent of the were observed for Cu and fused with 5ml of distilled and 230g sodium tartrate total in the leaf content and Ni. and deionized water using (Na2C 4H 4O6.2H2O) diluted considered to be the F or Positive linear regressions a heater block during ap- to 1l, according to the elec- mineral present on the leaf of F with Mn, Fe, Pb, Al, Ca, proximately 1h until boil- trode instruction manual surface and trichomes (when Mg, Cr and Zn were found ing was observed. The ex- (Thermo Orion, 1999). they are present, as in young for young and adult leaves tracts were centrifuged at The leaves were oven-dried leaves). Thus, the distribution (Figure 3, Table II). The 2G for 10min after they in paper sacks at 80ºC for of F and minerals on the sur- power of the regression test were cooled to room tem- 48h and ground. For the face and in the leaf itself was with α= 0.0500 was 1, but perature. The amounts of F measurement of total K, Ca, indicated. for K, Cu, Ni, Co and total in the solutions were de- Mg, Mn, Fe, Cu, Zn, Ni, Co, phenols it was below the de- termined by a F-specific Al, Pb, Cr and Cd, 0.5g Statistics sired power of 0.8 and the ion electrode (Fluoride/ samples were digested in a range of r was only 0.27-0.44. fluoride combination elec- nitric-perchloric acid mixture Two-way ANOVA was A negative slope for K and trode, model 96-09) and (Miller, 1998) and analyzed done to compare the total phenols was found. Cd was registered in an ion ana- by flame atomic absorption concentration of mineral ele- below the detection limit of lyzer (Orion, model 720 spectrophotometry (SpectrAA ments and phenols in B. 0.02ppm. A). The instrument was 55B, Varian Techtron, Austra- crassifolia between three ages When all the study sites and calibrated with standards lia). The detection limits of and three sites with Statistica foliar ages were analyzed to- of known concentrations of the equipment were 2- 6.0 (Statsoft Inc.). Regression gether (data from Figures 1 NaF in distilled and deion- 800ppm Ca, 0.06-15ppm Fe, statistics were estimated with and Table III) a positive corre- ized water (Ionplus-Orion 0.15-20ppm Mg, 0.3-250ppm Sigma plot 2001 (7.0 S). lation between Fe and F for

MAR 2004, VOL. 29 Nº 3 147 Figure 2. Mean (±SE) contents of several metals, K and total Figure 3. Relationship between fluoride concentration and the con- phenols in the same leaves of Figure 1. centrations of Mn, Fe, Pb, Al, Ca, Mg, Cr and Zn, in young ( ) and adult ( ) leaves from Figures 1 and 2. TABLE I VALUES OF THE F-STATISTIC IN TWO-WAY ANOVA FOR young, adult and fallen leaves TABLE II ELEMENTS AND PHENOLS IN B. crassifolia LEAVES from the different sites were REGRESSION SAMPLED IN THE VICINITY OF AN Al SMELTER found, with r= 0.68, n= 121. COEFFICIENTS (r) FOR With Ca, Mn, and Al the r was Parameter Factor 1: Factor 2: Interaction THE RELATIONSHIP 0.49, 0.40 and 0.29 respec- Leaf age Site Factor 1 x 2 BETWEEN Mn, Fe, Pb, Al, tively. The power of the regres- F 475.3054*** 494.8765*** 150.3895*** Ca, Mg, Cr or Zn AND sion test with α= 0.05 was 1, Ca 134.0322*** 25.3782*** 10.5303*** THE CONCENTRATION 0.9970 and 0.9041, respectively, Fe 141.7178*** 245.9785*** 87.7228*** OF F IN YOUNG AND but for Pb, Mg, Zn, Ni and to- Mg 6.0972* 17.8995*** 4.5188* ADULT LEAVES* Al 146.9301*** 278.1162*** 83.7920*** tal phenols it was below the Mn 97.6055*** 111.8843*** 32.2665*** desired power of 0.8. Young Adult Zn 2.9492ns 14.0535** 0.7139ns leaves leaves Cu 1.7466ns 2.2216ns 1.8016ns Fluoride, metals and Ni 2.2811ns 2.9366ns 0.6714ns Mn 0.86 0.88 Cr 2.8891ns 9.7056*** 1.7011ns phenols in leaves from sites Fe 0.83 0.88 Pb 28.8852*** 52.9743*** 19.7021*** with or without Al-industry Pb 0.74 0.90 Co 0.5864ns 7.0127** 2.4805ns Al 0.87 0.88 In old leaves from site B1 the K 4.4817* 0.5517* 0.3404ns Ca 0.55 0.84 Phenols 47.5205*** 208.6566*** 73.4024*** concentration of F reached Mg 0.43 0.80 13.7mmol·kg-1. Lower values The factors considered are 1) age of leaves and 2) the distance from the alu- Cr 0.62 0.76 minium smelter (see Figures 1 and 2), and the interaction between both fac- (Table III) were found at the Zn 0.86 0.69 tors is also shown. Significance levels: * p<0.05, ** p<0.01, *** p<0.001, site in the opposite wind direc- ns: not significant. tion of the smelter (B2) or at *From Figure 3.

148 MAR 2004, VOL. 29 Nº 3 TABLE III CONTENTS OF FLUORIDE, METALS, PHENOLS, SUM OF CATIONS AND F/Al RATIO IN B. Crassifolia LEAVES OF DIFFERENT AGE, IN THREE LOCALITIES Leaf age Young Adult Old(fallen) Element Site B1 B2 M B1 B2 M B1 B2 M F 1.70 ±0.16 0.11 ±0.05 1.09 ±0.15 6.14 ±0.08 0.24 ±0.05 0.94 ±0.01 13.68 ±0.24 0.70 ±0.04 1.29 ±0.16 Ca 78.84 ±3.43 70.49 ±4.44 69.58 ±1.45 146.46±11.74 173.92±17.29 118.33 ±1.59 229.01 ±17.10 242.88±14.98 156.06 ±2.49 Fe 8.71 ±0.96 0.22 ±0.14 4.84 ±0.30 25.26 ±1.91 7.22 ±1.56 4.29 ±0.15 76.12 ±6.10 5.62 ±0.57 6.85 ±0.04 Mg 65.82 ±3.59 96.84 ±6.20 110.41 ±1.20 114.60±13.24 73.77 ±8.36 180.99 ±0.66 65.35 ±5.69 108.33 ±5.93 180.26 ±0.75 Al 5.75 ±0.34 7.84 ±0.82 3.92 ±0.32 10.01 ±0.71 30.18 ±3.82 5.16 ±0.42 20.46 ±2.54 24.86 ±1.71 8.98 ±1.55 Mn 0.21 ±0.01 0.13 ±0.01 0.44 ±0.00 0.43 ±0.05 0.34 ±0.03 0.44 ±0.00 0.56 ±0.05 0.74 ±0.04 0.52 ±0.03 Zn 0.10 ±0.02 0.16 ±0.02 0.15 ±0.02 0.18 ±0.03 0.15 ±0.02 0.12 ±0.03 0.29 ±0.06 0.24 ±0.08 0.46 ±0.08 Cu 0.03 ±0.00 0.04 ±0.00 0.03 ±0.00 0.04 ±0.00 0.03 ±0.00 0.05 ±0.00 0.0 00 0.41 ±0.18 0.18 ±0.01 Ni 0.03 ±0.01 0.09 ±0.01 0.16 ±0.01 0.08 ±0.02 0.11 ±0.00 0.20 ±0.02 0.14 ±0.01 0.10 ±0.01 0.18 ±0.06 Cr 0.18 ±0.06 0.05 ±0.02 0.12 ±0.01 0.08 ±0.02 0.13 ±0.02 0.08 ±0.01 0.08 ±0.00 0.13 ±0.01 0.15 ±0.00 Pb 0.03 ±0.00 0.02 ± 0.00 0.02 ±0.01 0.00 ±0.00 0.06 ±0.01 0.08 ±0.01 0.0 00 0.02 ±0.01 0.01 ±0.01 Co 0.05 ±0.01 0.07 ± 0.01 0.07 ±0.01 0.08 ±0.01 0.13 ±0.01 0.13 ±0.00 0.07 ±0.00 0.05 ±0.01 0.06 ±0.01 K 73.82 ±5.71 104.29±13.99 106.10 ±4.28 82.21±19.86 73.03 ±8.90 59.27 ±7.89 55.81 ±3.37 88.03±12.91 63.10 ±0.98 Total Phenols 816.74±17.89 528.65 ±6.09 933.65 ±8.53 761.82±123.38 944.34 ±1.54 1005.68±13.35 932.57±32.42 1017.19±29.33 541.33 ±4.42 Total Cations 233.18 279.9 295.45 379.05 358.57 368.68 447.45 470.56 415.95 Fe/Al 1.51 0.03 1.23 2.52 0.24 0.83 3.72 0.23 0.76 Values are means ±standard error, N=5. Sampling sites were B1: Savannah along the road from Ciudad Guayana to iron mines at Pao (Bolívar State), located 20km from the Al-smelter, B2: Garden in a University campus in Ciudad Guayana (Bolívar State), and M: Savannah in Miranda State. Sampling was done in May 2001 in B1 and B2, and in February 2000 in M. the site where no Al-industry face and/or trichomes in <0.5mmol·kg-1 according to trated in F than the normal exists (M). As expected, it was young leaves, and was there- Arnesen (1997). Normal F range, but at 8km from the found that Ca and Mg concen- fore washed off with water content of leaves generally factory only fallen leaves tration was higher than K con- (Figure 4) while only in the ranges from 0.1 to 1mmol·kg- were contaminated with F centration in adult and fallen case of Cu no differences 1 (Baroni Fornasiero, 2001). (Figure 1). leaves, but was not in young were observed in washed and Young, adult and fallen leaves Murray (1985) also showed leaves from all sites. In M, but unwashed leaves. Adult leaves of B. crassifolia sampled at high concentrations of F not in B1 nor B2, the concen- showed lower proportions of the garden of the factory and (32mmol·kg-1) in leaves of tration of Mg was higher than Co, Al, Mn, Zn, Cr, Ca, K, 5km away were more concen- Avicennia marina (grey man- that of Ca. The micronutrient Fe Mg, Fe and Ni on the surface was higher in B1, located at the of the leaves than young road to the iron mines. The Al leaves. However, 73% of F Table IV concentration reached high lev- was washed away and was VALUES OF THE F-STATISTIC IN TWO-WAY ANOVA FOR els in B1 and B2. An increase presumably present on the sur- ELEMENTS AND PHENOLS IN B. crassifolia LEAVES of Mn concentration with age face. In adult leaves Zn, Cr, SAMPLED IN THREE LOCALITIES was observed. The concentration Ni and Cu were not washed -1 Parameter Factor 1: Factor 2: Interaction of phenols was >762mmol·kg away, and for Ca, K, Mg and Leaf age Site Factor 1 x 2 and <1017mmol·kg-1, excepting Fe the proportion present on young leaves from site M and the surface was <20%. F 866.1482*** 3039.4121*** 783.2032*** fallen leaves from B2, with A higher proportion of Al Ca 83.5552*** 14.4205*** 3.2200* lower values (529 and was washed when compared to Fe 74.8442*** 179.3934*** 64.3249*** 541mmol·kg-1, respectively). that of the macronutrients K, Ca Mg 3.1029ns 15.8076*** 5.7114** Table IV shows the com- and Mg, and the micronutrients Al 31.7650*** 47.1607*** 11.4263*** parison by age, site and their Fe, Cu and Ni. For other micro- Mn 56.4552*** 0.4676ns 11.8282*** interaction, of the differences nutrients, such as Mn and Zn, Zn 3.8679* 13.8999*** 5.6655** in concentrations found for F, the proportion washed in adult Cu 1.3177ns 2.4961ns 1.1927ns metals and phenols in the leaves was lower than that in three sites studied. Highly sig- young leaves. Ni 3.8243* 13.4723*** 1.7573ns nificant differences (p<0.001) Cr 0.3623ns 0.5651ns 4.2631** Pb 13.4731*** 30.2926*** 16.5412*** were found for F, Fe, Al and Discussion Pb, but no differences were Co 3.8501* 42.9971*** 3.7560* observed for Cu. High fluoride content K 4.9289* 2.3186ns 2.3245ns in samples collected Phenols 19.6610*** 0.0410ns 25.5124*** Washing experiment at an industrial site The factors considered are 1) age of leaves and 2) the distance from the alu- minium smelter (see Table III), and the interaction between both factors is A large proportion of metals Background concentration also shown. Significance levels: * p<0.05, ** p<0.01, *** p<0.001, ns: not and F was located on the sur- of F in plants is usually significant.

MAR 2004, VOL. 29 Nº 3 149 groves), generally without Fluoride in relation to age was higher than in those from leaves of Faramea marginata visible injury, in a study site M (Table III). The concentra- (Rubiaceae) from a swamp located approximately 100m The content of F in young tion of Al was high in adult forest in Brazil (767mmol·kg-1; from the Australian Fluoride leaves was lower than in leaves sampled at the factory Britez et al., 2002). Accord- Chemicals plant. adult or fallen leaves (Figure garden (36mmol·kg-1; Figure ing to Jansen et al. (2002) the In an industrial complex 1 and Table III), as was 1), and in adult and fallen largest recorded content of in Brazil, which includes found in brick tea (Fung et leaves collected in the garden aluminium in any plant is fertilizer industries, steel al., 1999). Leaf age strongly of the University of Guayana 2677mmol·kg-1, in Symplocos works, refineries, chemical affected the F content in (30 and 25mmol·kg-1, respec- spicata (Symplocaceae). and petrochemical plants, a leaves in Salix caprea and tively; Table III). Higher val- The concentration of Al in foliar F concentration of Betula pendula (Vike and ues have been observed in Al Byrsonima crassa, B. coccolo- 36mmol·kg-1 was found in Håbjørg, 1995). accumulators such as Melas- bifolia and B. verbascifolia Lolium multiflorum (Klumpp In Pinus sylvestris (Scots toma malabathicum (Melasto- found by Haridasan (1982) in et al., 1994), a species used pine) an increase in concentra- mataceae) in old leaves the tree savannahs (cerrado as a bioindicator in Europe tion from needle base to tip sampled from greenhouse region) of central Brazil was and North America because was detected for Al, Fe and F grown plants (534mmol·kg-1; between 8 and 13mmol·kg-1, of its accumulation of toxic (Giertych et al., 1997). This Watanabe et al., 1998), Rich- where other species such as elements (F, S, metals). distal accumulation of toxic eria grandis (Euphorbiaceae) Vochysia thyrsoidea reached Domingos et al. (2003) sug- elements led to necroses. from a cloud forest in Ven- 523mmol·kg-1. gested that Al-F complexes Giertych et al. (1999) also ezuela (556mmol·kg-1; Cuen- were taken from the soil in showed an increase of content ca et al., 1991), Craterisper- High Fe contents in leaves the Al-accumulator tree spe- of F with age of needles (5 mum laurinum (Rubiaceae) from Bolivar samples cies Tibouchina pulchra in classes) in Pinus nigra. from a savannah in Liberia this industrial complex in (1352mmol·kg-1; Jansen et al., Fe in adult leaves from the Brazil, where plants are ex- High Al contents in leaves but 2003), Maschalocorymbus co- factory garden (Figure 1) was -1 posed to fluoride-contami- without hyperaccumulation rymbosus sp. (Rubiaceae) from very high (125 ±7mmol·kg ) nated air. a tropical rain forest in Indone- and lower values (<76 Hydrogen fluoride is usu- The concentration of Al in sia (1368mmol·kg-1; Masunaga ±6mmol·kg-1) were found in ally associated with the pro- plants from sites B1 and B2 et al., 1998) and in mature the other study sites (Table duction of aluminium, steel, III). Fe was not measured in brick, tile, phosphate fertil- the genus Byrsonima from the izer, recycling of uranium savannahs by Haridasan fuel and coal combustion. (1982), but in other species Metallic aluminium is pro- from the cerrado region, such duced by the electrolytic re- as Miconia ferruginata it was duction of alumina, which is reported at 4mmol·kg-1. produced from bauxite and Jansen et al. (2003) reported in this process alumina dis- 12mmol·kg-1 in Melastoma ma- solved in cryolite is emitted labathricum and 13mmol·kg-1 together with gas containing in Coccocypselum canescens. F. The vegetation in the vi- In a crop species, such as cinity of aluminium factories Spinacia oleracea, considered accumulated high F contents to contain high Fe levels, in Greece (Malea, 1995). 9mmol·kg-1 has been reported No visible symptoms of (Bhattacharjee et al., 1998). damage were observed in B. However, in Eichhornia cras- crassifolia with high F con- sipes 179mmol·kg-1 Fe have centrations. Therefore, this been found (Larcher, 1995) species is not suitable as a F and Clark and Baligar (2000) bioindicator. Weinstein and indicated that plants normally Davison (2003) described accumulate relatively high Fe visible symptoms of injury before disorders such as in sensitive species exposed bronzing or brown speckling to F-emitting sources, spe- appear, increasing from 5.4 to cially Al-smelters and phos- 12.5mmol·kg-1 in rice and phate fertilizer plants, and from 19.7 to 28.6mmol·kg-1 in listed 24 native species from Rumex. South America suitable as bioindicators for the pres- Total cation and phenols ence of airborne fluorides. contents in trees from The use of bioindicators is Figure 4. Percent distribution of minerals and fluoride on the sur- different sites in relation described as a rapid and in- face (clear part) of young (a) or adult (b) leaves, calculated as the to micronutrients expensive technique that difference of their contents in unwashed and washed leaves. Shad- provides an estimate of the owed part is percentage inside the leaves. Sampling from B1 and The sum of the cation (K, degree of injury at the time B2 sites, and from the Al-smelter garden, in October 2002 (n=18 Ca, Mg, Fe, Mn and Al) con- of survey. for each leaf age). tents was >416mmol·kg-1 in

150 MAR 2004, VOL. 29 Nº 3 fallen leaves from the differ- plant leaves. Baroni Fornasi- centration on phenol synthesis Domingos M, Klumpp A, Rinaldi ent sites (B1, B2, M), but ero (2001, 2003) reported in- in Paul’s Scarlet Rose cells MCS, Modesto IF, Klumpp G, -1 grown in tissue culture. Delitti WBC (2003) Combined <295mmol·kg in young creases in anthocyanin and Physiol. Plant. 39: 91-95. effects of air and soil pollution leaves (Table III). These val- hypothesized that in H. perfo- Arnesen AKM (1997) Availability by fluoride emissions on Ti- ues in B. crassifolia from ratum, where the proantho- of fluoride to plants grown in bouchina pulchra Cogn., at savannahs with oligotrophic cyanidins or condensed tan- contaminated soils. Plant Soil Cubatão, SE Brazil, and their conditions resulted lower than nins are abundant, the red- 191: 13-25. relations with aluminium. Plant Soil 249: 297-308. the total cation content, be- brown pigmentation of the F- Baroni Fornasiero R (2001) Phyto- tween 500 and 700mmol·kg-1, affected areas is related to the toxic effects of fluorides. Plant Fung KF, Zhang ZQ, Wong JWC, Sci. 161: 979-985. Wong M (1999) Fluoride con- for most of the species stud- increased amounts of antho- tents in tea and soil from tea ied by Köhl et al. (1996) in cyanins during the F-induced Baroni Fornasiero B (2003) Fluo- plantations and the release of the Canary Islands. stress. rides effects on Hypericum fluoride into tea liquor during perforatum plants: first field In Bolívar State, Al and Fe infusion. Environ. Pollut. 104: observations. Plant Sci. 165: 197-205. mining, and industrial related 507-513. Washing experiment Giertych MJ, De Temmerman LO, activities, take place. This can Berger I, Barrientos AC, Cáceres Rachwal L (1997) Distribution explain the high levels in fo- A high proportion of F and A, Hernández M, Rastrelli L, of elements along the length of liar Al and Fe concentration metals was found to be on Passreiter CM, Kubelka W Scots pine needles in a heavily reached, in comparison to the surface and/or trichomes (1998) Plants used in Guate- polluted and a control environ- those in M, a site without of the leaves (Figure 4) and mala for the treatment of pro- ment. Tree Physiol. 17: 697- tozoal infections. II. Activity 703. these activities but where Ni could be washed off by rain. of extracts and fractions of mining takes place. Accord- Isermann (1977) showed Pb five Guatemalan plants against Giertych MJ, Karolewski P, De Temmerman LO (1999) Foli- ingly, leaves showed higher deposited by car exhausts on Trypanosoma cruzi. J. Ethnopharmacol. 62: 107-115. age age and pollution alter Ni contents in M (Table III). Lolium perenne was easily content of phenolic compounds The Fe/Al molar ratio in washed (for 15min with dis- Bhattacharjee S, Dasgupta P, Paul and chemical elements in the proximity of the Al fac- tilled water) reducing the AR, Ghosal S, Pahdi KK, Pinus nigra needles. Water Air Pandey LP (1998) Mineral ele- Soil Pollut. 110: 363-377. tory (calculated from Figure lead content by 27%, while ment composition of spinach. 1) was between 1.96 and washing Betula pubescens J. Sci. Food Agricult. 77: 456- Haridasan M (1982) Aluminium 458. accumulation by some cerrado 3.51, and F concentration was leaves with citric acid, pH native species of central Brazil. >3.16mmol·kg-1, except in 2.5, reduced the F content by Blamey FPC, Joyce DC, Edwards Plant Soil 65: 265-273. young leaves (at 0, 5 and 15% (Vike and Håbjørg, DG, Asher CJ (1986) Role of trichomes in sunflower toler- Horntvedt R (1995) Fluoride uptake 8km) or in adult leaves from 1995). ance to manganese toxicity. in conifers related to emissions trees distant 8km from the The young leaves were Plant Soil 91: 171-180. from aluminium smelters in factory. In B1 the Fe/Al ratio washed until trichomes were Norway. The Science of the Britez RM, Watanabe T, Jansen S, Total Environ. 163: 35-37. (Table III) increased with age, not present anymore. Several Reissmann CB, Osaki M to a maximum in fallen examples in the literature (2002) The relationship be- Isermann K (1977) A method to tween aluminium and silicon reduce contamination and up- leaves (Fe/Al= 3.7 and F= showed the role of trichomes take of lead by plants from car 13.68mmol·kg-1). In B2 and in metal tolerance, such as Pb accumulation in leaves of Faramea marginata (Rubi- exhaust gases. Environ. Pollut. M the Fe/Al ratio was <1.23 in Nicotiana tabacum aceae). New Phytol. 156: 437- 12: 199-203. and F <1.29mmol·kg-1. (Martell, 1974), or Mn in 444. Jansen S, Broadley MR, Robbrecht The concentration of Helianthus annuus (Blamey et Cao J, Zhao Y, Liu J, Xirao R, E, Smets E (2002) Aluminum phenols was high in all the al., 1986). Trichome glands Danzeng S (2000) Environ- hyperaccumulation in An- samples (Figure 2 and Table were found in the petioles of mental Fluoride content in Ti- giosperms: a review of its phy- bet. Environ. Res. 83: 333-337. logenetic significance. Bot. III) as compared with the Nymphaea sp., where Rev. 68: 235-269. range reported by Coley polyphenols play a role in Cifuentes C, Gómez-Serranillos MP, Iglesias I, Villar del Fres- Jansen S, Watanabe T, Dessein S, (1983) in tropical plants (48- metal detoxification (Lavid et Smets E, Robbrecht E (2003) -1 no AM (2001) Neuropharma- 1098mmol·kg ). al., 2001 a, b). cological profile of ethno- A comparative study of metal The content of total medicinal plants of . levels in leaves of some Al-ac- J. Ethnopharmacol. 76: 223- cumulating Rubiaceae. Ann. phenols was positively corre- Conclusions Bot. 91: 657-663. lated with F in Pinus nigra 228. (black pine; Giertych et al., B. crassifolia leaves, Clark RB, Baligar VC (2000) Klumpp A, Klumpp G, Domingos sampled in industrial areas Acidic and alkaline soil con- M (1994) Plants as bioin- 1999). However, in the dicators of air pollution at the where Al is processed, straints on plant mineral nutri- present study such a relation tion. In Wilkinson RE (Ed.) Serra do Mar near the indus- was not observed between showed high levels of F asso- Plant-Environment Interactions. trial complex of Cubatão, Bra- ciated to metals. F was not Marcel Dekker. New York, zil. Environ. Pollut. 85: 109- phenols and F in B. crassifo- 116. lia. On the other hand, Baroni only associated to Al, as re- USA. pp. 133-177. Fornasiero (2001) showed by ported previously, but mainly Coley PD (1983) Herbivory and Köhl K, Lösch R, González-Rodrí- to Fe. These mineral elements defensive characteristics of tree guez AM, Jiménez MS, Mo- electron microscopy many rales D (1996) Foliar cation may be present in the medici- species in lowland tropical for- dark stained, vacuolar, elec- est. Ecol. Monographs 53: contents of laurel forest trees tron dense globular inclusions nal infusions, even when 209-233. on the Canary Islands. 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