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Lichens as a Tool for Biogeochemical Prospecting
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY ARTICLE NO. ES971596
38, 322–335 (1997)
Lichens as a Tool for Biogeochemical Prospecting M. K. Chettri,* T. Sawidis,† and S. Karataglis† *Department of Botany, Amrit Campus, Tribhuvan University, P.O. Box 102, Kathmandu, Nepal; and †Department of Botany, School of Biology, University of Thessaloniki, Gr-54006 Thessaloniki, Greece Received April 11, 1997
The heavy metal content in lichens and vascular plants from abandoned copper mining areas, Gerakario (Kilkis) and Megali Panagia (Chalkidiki), have been compared with metal content in soil in order to assess their efficiency to accumulate five metals (Cu, Mn, Pb, Zn, and Cr). The average metal content in the mineralized soil of Gerakario was, in descending order, Cu, Mn, Pb, Zn, and Cr, and in Chalkidiki it was Cu, Mn, Cr, Pb, and Zn. The epilithic lichens (Neophuscelia pulla) accumulated the highest amount of Cu and Pb, and Xanthoparmelia taractica accumulated the highest amount of Zn. All the lichens revealed significant (P < 0.05) correlation between Cu content in soil and that in thalli. Out of five metals studied, four (Cu, Pb, Mn, and Cr) in the epigeic lichen Cladonia convoluta, two (Cu and Mn) in both epilithic lichen N. pulla and X. taractica, and one (Pb) in vascular plant Minuartia (root) were significantly (P < 0.05) correlated between their metal content in plant tissue and in soil. Further, discoloration of C. convoluta with higher Cu concentrations adds a visible clue for biogeochemical exploration. Thus, lichens along with other symptomatic species will help in locating mining areas. © 1997 Academic Press
INTRODUCTION
Plants growing in heavy-metal-rich soil have been described as excluders, indicators, or accumulators, depending on the basis of metal concentrations in it (Baker, 1981; Legittimo et al., 1995). The basis of the biogeochemical method of prospecting is that the presence of anomalous concentrations of metals in the soil or underlying rocks will be reflected by the presence of those metals in abnormal concentrations in the vegetation (Butcher, 1992; Kabata-Pendias and Pendias, 1992; Badri and Springuel, 1994). As long as the metabolism is nearly not disturbed, i.e., around the optimal range, visible deviation of ‘‘normal’’ plant behavior will not be detectable, even if increased concentration of a heavy metal may be detectable by chemical analysis. As soon as a surplus of the element disturbs the metabolism, reaction will become visible; it may be a chlorosis, discoloration, a necrosis, stunted growth, or teratological performance of plant parts, when symptomatic indication of individual plants is considered (Eleftheriou and Karataglis, 1989; Ernst, 1993). In the case of the presence of open and hidden ore bodies, the first target organ of a plant for heavy metal accumulation is 322 0147-6513/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
the root (Ernst, 1993). Malyuga (1964) was the first to give an account on the importance of the root morphology for mineral prospection. If an ore body is hidden by overburden, but still remains in the root zone, then metal can be brought above ground through plant parts. After the death or fall of plant parts, these metals will accumulate in the upper soil horizon of the overburden (Martin and Coughtrey, 1982; Ernst, 1993; Hobbie, 1995). Bowen (1979) reviewed the average crustal abundance of various metals and their median concentrations in soil. The data suggest that Fe, Ti, Mn, Zn, Ga, Ag, and Hg exhibit a close relationship between parent material and soil concentrations. Ti, Co, Cu, Ni, Cr, and Va appear to be somewhat depleted in soils relative to parent material. In, Bi, Cd, Sn, and As are all somewhat rich in soil relative to parent material. Frequently in mineralized areas, the change in species composition of the plant community relative to neighboring nonmineralized areas causes a dramatic difference in the appearance (physiognomy) of the vegetation (Wild, 1974). Lichens are considered to be an extremist plant (Lange, 1990), because they can grow under extreme environmental conditions and even can stand a high level of heavy metals in mining areas (Lange and Ziegler, 1963; Czehura, 1977; Sawidis et al., 1995). Studies on the capacity of lichens to accumulate heavy metals and other elements were reviewed thoroughly (James, 1973; Nieboer et al., 1977, 1978; Rao et al., 1977; Richardson and Nieboer, 1980; Martin and Coughtrey, 1982; Brown and Beckett, 1985; Puckett, 1988; Tyler, 1989; Brown, 1991; Garty, 1992, 1993). Lichens accumulate and tolerate metals to a high degree because of their relatively large surface area and slow growth rate. Because of the lack of cuticle (or similar) wax covering and their poikilohydric nature, accumulation of metals occurs by passive adsorption and ion exchange (Nieboer and Richardson, 1981). Vascular plants, compared to lichens, have a well-developed root and transport system. Rumex acetosella and Minuartia verna (which was later reported as Minuartia hirsuta; Konstantinou, 1992) have been reported as indicators for ore deposits in northern Greece (Kelepertsis and Andrulakis, 1983; Kelepertsis et al., 1985; Ouzounidou, 1993). Brooks (1972, 1983) concluded that a universal characteristic of indicators is
LICHENS AS A TOOL FOR BIOGEOCHEMICAL PROSPECTING
that they will have a very high elemental content in their ash. In other words, a criterion for new indicators is that they have a higher concentration of the element sought than the surrounding vegetation. Although lichens do not have well organized absorptive systems, they come in contact with metals of upper soil horizon due to wind and rain activity. Due to the poikilohydric nature of lichens, they can accumulate dissolved metals from all the surface of their body. Therefore, it was hypothesized that lichens growing on mineralized soil or rocks would accumulate anomalous concentrations of heavy metals and could be used as a tool for biogeochemical prospecting, especially in remote places where anthropogenic activity is lacking. Therefore, in the present study heavy metal content in lichens and vascular plants collected from abandoned mining areas were compared with metal content in mineralized soil to assess their efficiency to accumulate different metals. MATERIALS AND METHODS
Gerakario and Chalkidiki (M. Panagia) in northern Greece (Fig. 1) have been reported to have varying degrees of porphyry copper mineralization, associated with post-miocene volcanic rocks of rhyolithic composition (Reeves et al., 1986). Vascular plants (VP), such as M. hirsuta subsp. falcata (griseb) Mattf., R. acetosella L., Thymus sibthorpii Bentham, Sanguisorba minor subsp. minor, Dianthus pinifolius Sibth & Sm., were collected from the following mentioned sampling sites. Along with VP, epigeic (EG) lichens like Cladonia convoluta (Lam.) P. Cout. and Cladonia rangiformis Hoffm. (substrate soil), and epilithic (EL) lichens like Neophuscelia pulla and Xanthoparmelia taractica (Kremp.) Hale (substrate stones), were also collected. The sampling sites at Gerakario were five deserted plots (1, 2, 3, 4, and 5) located approximately 100 m
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apart, along the northwest to southeast direction and one undeserted plot (6) in a pasture area (Fig. 2a). At the same time, the same plant species were also collected from rocks in the heart of the mine (1), the sandy field in the center (2), the first contour around the center (3), the pasture area (4), and the oak forest (5) around the mining area of M. Panagia (Chalkidiki) (Fig. 2b). In addition to these lichens, epiphytic (EP) lichens (substrate bark), which appeared only in the forest (5), were also collected for comparison with EG and EL lichens. Along with plant samples, soil (S) samples were also collected from the 6- to 8-cm depth of each plot. The attached foreign particles from collected plant materials were removed carefully with the help of fine plastic forceps. The vascular plant materials were washed thoroughly with running double-deionized water to avoid soil contamination. Lichens were washed for 5 s under running double-deionized water as suggested by Tuba et al. (1994). Plant samples were dried at ambient temperature (25–30°C) for 2–3 days, then oven-dried at 60–80°C for 24–48 h to constant weight, and then pulverized with a mortar and pestle. The pulverized sample was considered a representative sample of that plot as it consisted of many individuals. Soil (S) samples were also dried as mentioned above and sieved through a 0.25-mm mesh. An accurately weighed portion of each sample (1 g dry wt) was placed in an open quartz tube; 8 ml concentrated HNO3 (Merck) was added to each tube and the mixture was left overnight at room temperature. The mixture was then warmed for 2 h at 50°C and subsequently heated at 160°C for 4 h. The extract was filtered through Whatman type 589/2 filters and the filtrate was diluted to 25 ml volume with double-deionized water. The diluted filtrates were analyzed for metal concentration using a Perkin Elmer 2380 atomic absorption spectrometer. The analytical wavelengths were 283.3 nm for Pb, 279.5 for Mn, 357.9 for Cr, 324.7 nm for Cu, and 213.8 nm of Zn (Welz, 1985). The relative standard deviation of the measurements were 8.2% for Pb, 2.5% for Cu, 3.6% for Zn, 4.7% for Mn, and 7.9% for Cr. This procedure provides data for an estimation of the total content of the metal in the thalli (Sawidis et al., 1993). Statistical procedure. To understand the relationship between the different metals studied, correlation coefficient (r) was calculated from linear regression analysis between different metals in soil and different metals in soil and in plant. Bearing in mind the number of pairs of points (n) used in the calculation, the level of significance (P) for a calculated correlation coefficient (r) was obtained by calculating the t value. The calculated value of t was compared with the tabulated value at P < 0.05 using a two-tailed t test and (n − 2) degrees of freedom (Miller and Miller, 1986). RESULTS
FIG. 1. Map of Macedonia, N. Greece, indicating the sampling stations. Gerakario (Kilkis) and Megali Panagia (Chalkidiki) areas with porphyry copper deposits.
The average metal content in the mineralized soil of Gerakario was, in descending order, Cu, Mn, Pb, Zn, and Cr, and in
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FIG. 2. Sampling sites at abandoned porphyry copper deposits (a) Gerakario (Kilkis) and (b) M. Panagia (Chalkidiki).
Chalkidiki it was Cu, Mn, Cr, Pb, and Zn. Total metal load in EL lichens was mostly higher than that in EG lichens or VP (Fig. 3). Epiphytic lichens collected from the oak forest around (5) Chalkidiki mining accumulated higher amounts of Mn than the epigeic lichens or vascular plants from the same plot (Fig. 3).
The accumulation of Cu ranged from 10 to 349 mg/g in leaves, from 10 to 355 mg/g in stems, and from 10.5 to 689 mg/g in roots, respectively. In vascular plants, Cu accumulation was higher in the roots than the leaves or the stems in all species. Accumulation of Mn, Zn, Pb, and Cr varying in different parts of different species indicated free translocation of
FIG. 3. Metal (Cu, Mn, Pb, Zn, and Cr) content in epilithic lichens (EL), epegeic lichens (EG), epiphytic lichens (EP), vascular plants (VP), and soil (S).
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these metals to stem and leaves. Rumex acetosella L. and M. verna (L) Hiern. (which was later identified as M. hirsuta; Konstantinou, 1992) have been reported as biogeochemical indicators for ore deposits in northern Greece (Kelepertsis and Andrulakis, 1983; Kelepertsis et al., 1985). Though, R. acetosella exhibited the highest Cu accumulation in the roots, they were encountered only in Chalkidiki and not in Gerakario. Therefore, M. hirsuta (roots), which exhibited the highest accumulation of Cu (and also most other metals) next to Rumex (Fig. 4) was encountered in all stands, was considered for comparison with epigeic and epilithic lichens in the present study. Cu The amount of Cu measured in the soil and in plant material was mostly higher in Chalkidiki than in Gerakario. The highest
Cu accumulation was observed in N. pulla (Fig. 5) and hyperaccumulation was recorded in it in some plots. All the lichens (EG or EL) considered in the present study demonstrated significant (P < 0.05) correlation between Cu content in thalli and in the soil (Table 1). An elevated amount of Cu was detected in C. convoluta encountered in plots 1–4 of Chalkidiki; at the same time, the ventral surface of the thallus was purplishblack-rimmed instead of the normal greenish-white color. Very low Cu concentrations were detected in epiphytic lichens like Evernia prunastri (9 to 23 mg/g) and Ramalina fraxinea (8.5 mg/g) growing in the oak forest around Chalkidiki mining areas, but in Parmelia sulcata the accumulation of Cu was much higher, ranging from 37 to 87 mg/g. Similarly, low concentrations of Cu were detected in some epigeic lichens like Cladonia ciliata (35 mg/g), Cladonia rangiferina (16 mg/ g), and Cladonia stellaris (16 mg/g) growing in Chalkidiki (4) mining areas.
FIG. 4. Cu content in leaves, stems, and roots of different vascular plants in different plots of abandoned Cu mining areas. (A) Thymus (Gerakario), (B) Minuartia (Gerakario, Chalkidiki), (C) Sanguisorba (Gerakario), (D) Dianthus (Gerakario), (E) Rumex (Chalkidiki).
FIG. 5. Cu accumulation in different EG, EL lichens, Minuartia (roots), and soil.
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TABLE 1 Correlation Coefficient (r) Obtained from Linear Regression between Different Metals in Soil, and between Soil and Plant Tissue S-Pb
S-Mn
S-Cr
S-Zn
S-Cu
1.000 0.545 0.044
1.000 −0.090
1.000
n 4 11 S-Pb S-Mn S-Cr S-Zn S-Cu
1.000 0.356 −0.492 0.234 −0.305
1.000 0.515 0.842** −0.116
CC-Pb CC-Mn CC-Cr CC-Zn CC-Cu
0.624* 0.152 −0.331 0.179 0.488
0.733* 0.773* 0.633* 0.654* 0.134
CR-Pb CR-Mn CR-Cr CR-Zn CR-Cu
0.820** 0.614* −0.298 0.064 −0.383
0.241 0.356 −0.019 0.274 −0.451
N-Pb N-Mn N-Cr N-Zn N-Cu
0.623 0.695* −0.445 0.607 −0.292
0.733 0.801* −0.097 −0.651 0.171
X-Pb X-Mn X-Cr X-Zn X-Cu
0.383 0.811* −0.293 0.493 0.187
0.518 0.824* −0.230 0.327 0.175
M-Pb M-Mn M-Cr M-Zn M-Cu
0.652* 0.873** 0.548 −0.351 −0.067
0.15 0.461 −0.002 0.307 0.191
n 4 11 0.116 0.107 0.822** 0.345 0.267
0.524 0.349 0.444 0.419 0.074
0.030 0.389 0.195 0.356 0.810**
n 4 11 −0.301 −0.376 0.122 0.133 −0.243
0.125 0.125 −0.394 0.102 −0.484
−0.121 −0.118 0.421 0.635* 0.665*
−0.266 −0.357 0.617 −0.150 0.229
0.748* 0.762* 0.108 0.558 −0.051
0.161 0.136 0.221 0.382 0.896*
−0.190 −0.772 0.522 −0.867* −0.274
0.448 0.842* −0.448 0.466 0.131
−0.434 0.375 −0.060 0.698 0.860*
−0.597 −0.310 −0.343 0.625* 0.208
−0.368 0.307 −0.320 0.429 −0.140
0.048 −0.513 −0.335 0.271 0.446
n48
n46
n 4 11
Note. S, soil; CC, C. convoluta; CR, C. rangiformis; N, N. pulla; X, X. taractica, M, Minuartia (root); Pb, lead; Mn, manganese; Cr, chromium; Zn, zinc; Cu, copper. * P < 0.05, **P < 0.01; unmarked, not significant.
Zn The amounts of Zn accumulation in the soil of Gerakario and Chalkidiki do not differ much. High accumulation of Zn was seen in X. taractica and Minuartia (root) in some plots (Fig. 6). Zn and Mn content in soil revealed significant positive correlation (P < 0.05). Significant negative correlation (P < 0.05) was observed in the Zn content of X. taractica thalli and Cr in the soil, whereas significant positive correlation, for the same, was obtained in Minuartia. No significant relation was detected in the soil Zn content and Zn in any plant material encountered in the present study. All EG and EP lichens, except corticolos P. sulcata, found in the oak forest around the Chalkidiki mine, did not indicate
much variation in Zn accumulation. Accumulation of Zn was higher in P. sulcata (85 mg/g) than in the epilithic or epigeic lichens that occurred in the same plot or other plots. Pb Lead content in the soil ranged from 112 to 212 mg/g in Gerakario and 19 to 131 mg/g in Chalkidiki. The highest amount of Pb accumulation was observed in lithophytic lichens (Fig. 7). Cladonia convoluta, which is an epigeic lichen, also accumulated higher Pb amounts than Minuartia. Pb content in plant and in soil was significantly correlated in C. convoluta, C. rangiformis, and Minuartia (root). Pb content in C. convoluta and N. pulla revealed significant correlation with Mn con-
FIG. 6. Zn accumulation in different EG, EL lichens, Minuartia (roots), and soil.
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FIG. 7. Pb accumulation in different EG, EL lichens, Minuartia (roots), and soil.
330 CHETTRI, SAWIDIS, AND KARATAGLIS
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tent in the soil. in the case of N. pulla, Pb content in the thalli was also significantly correlated to Zn content in the soil (Table 1). Low amounts of Pb were detected in some EG lichens like C. ciliata (0.5 mg/g), C. rangiferina (12.5 mg/g), C. stellaris (12.5 mg/g), and Cornicularia aculeata (6 mg/g) collected from the oak forest. Similarly, EP lichens like E. prunastri and R. fraxinea accumulated 13 and 8 mg/g, respectively, whereas 55 mg/g Pb was measured in P. sulcata from the same area. Cr Chromium content in soil ranged from 20 to 45 mg/g in Gerakario and from 29 to 370 mg/g in Chalkidiki area. Cr accumulation was higher in lithophytic lichens than in epigeic lichens or Minuartia root (Fig. 8). Cr content in C. convoluta thalli and in the soil as well demonstrated significant (P < 0.05) correlation. Chromium content in C. convoluta was also significantly correlated to Mn content in soil (Table 1). Low amounts of Cr accumulation were measured in all EP lichens (0.5 to 13 mg/g) and EG lichens (0.5 to 10.9) except C. convoluta (2.24 to 98.72). Mn Manganese concentration in the soil ranged from 100 to 534 mg/g in Gerakario and 157 to 680 mg/g in Chalkidiki area. In all the plots, Mn accumulation was higher in Minuartia (roots) than in lichens (Fig. 9). In some cases manganese was accumulated as much as or more in the stems than in the roots of vascular plants, thus indicating free translocation of this metal in plant tissue. Significant correlation was obtained for Mn content in lichen thalli (C. convoluta, N. pulla, and X. taractica) and in the soil (Table 1). Similarly, Mn content in C. rangiformis, N. pulla, X. taractica, or Minuartia was also significantly correlated to Pb content in the soil, indicating positive relations between Mn accumulation in plant and Pb content in soil. Mn accumulation in both EL lichens was significantly correlated to Zn content in soil. Accumulation of Mn in the epiphytic lichens E. prunastri, P. sulcata, and R. fraxinea encountered in oak forest around M. Panagia (Chalkidiki) mine (plot 5) was measured to be 114, 151, and 115 mg/g, respectively. The amount of Mn accumulation was higher in EP lichens than in EG lichens or Minuartia (root) in the same plot and also higher than EL or EG lichens of most other plots. DISCUSSION
Out of the five metals measured in plant tissues, four metals (Pb, Mn, Cr, and Cu) in C. convoluta thalli, two metals (Pb and Cu) in C. rangiformis, two metals (Mn and Cu) in N. pulla and X. taractica, and one metal (Pb) in Minuartia (root) were significantly (P 4 0.05) correlated with the same metal in soil. Although Kelepertsis and Andrulakis (1983) reported direct relationship between the Cu content of plant ash and of soils in Minuartia, no significant correlation was observed in the pre-
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sent study. Comparing the relations in the soil and plant material, it was observed that significant correlation between Zn and Mn found in soil was also present only in C. convoluta (Table 1). Various edaphic and plant factors responsible for the accumulation of metals in a plant have been mentioned (Chaney, 1973; Martin and Coughtrey, 1982). Metal accumulation in vascular plants is affected by different factors such as: mechanisms of metal movements within the soil, metal distribution within an individual soil profile, root distribution in the soil, root structure and physiology, translocation of metals from roots to shoots in relation to plant growth and development, translocation of metals within above-ground parts of plants in relation to metabolism and development, and loss of metals from plants via senescence, leaf-fall, leaf leaching, exudation, and volatilisation. The differences in metal accumulation in higher plants caused by the mechanism of metal movement within the soil, distribution of metal in each profile, distribution of absorptive organ in the soil, and loss of metal by leaf fall or senescence are completely obscured in lichens. Therefore, metals with higher concentrations in the soil (i.e., Cu, Mn, Pb, and Cr) manifested significant correlation with lichen C. convoluta. The minor changes in trace element contents of the soil substrates did not follow the accumulation pattern of the lichen and moss indicators in any of the cases, except for V in lichen and V and Ni in moss (Tuba et al., 1994). Zinc content in the soil in the present studied mines ranged from 40 to 84 mg/g, which was not so much in comparison to other metals like Cr (20–368 mg/g), Cu (181–819 mg/g), Pb (49–213 mg/g), and Mn (101–679 mg/g). Therefore, the correlation obtained between Zn content in the soil and any plant material was not significant. In addition to this, a general rule concerning cation uptake mechanism involving ion exchange modified by metal complex formation has been formed for third period transition metal cations. The stability of their complexes increases in the series Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+ (Wehrli and Stumm, 1989). As Cu forms the strongest complexes with ligands at binding sites and as both sites of study were with prophyry copper deposits, Cu2+ accumulation in all lichen species tested were significantly correlated. Cladonia convoluta treated with 10−10 to 10−2 M CuCl2 solution demonstrated significant uptake of Cu2+ (Chettri et al., 1996) and total chlorophyll degradation with increasing concentrations (unpublished data). Therefore, it can be speculated that the purplish-black rim on the lower surface of C. convoluta in plots having high concentrations of Cu (Chalkidiki, 1-4) must be due to copper’s toxic effect on chlorophyll. The insignificant correlation between Zn content in the soil and Zn in all lichen thalli (or Minuartia root) strengthens the assumption of Chettri et al. (1996) that the lichens which can accumulate more than one heavy metal would not reflect the actual proportion of those metals in the environment, espe-
FIG. 8. Cr accumulation in different EG, EL lichens, Minuartia (roots), and soil.
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FIG. 9. Mn accumulation in different EG, EL lichens, Minuartia (roots), and soil.
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cially those forming weaker complex in binding sites. They also found that the accumulation of heavy metals, forming weaker complexes, would be affected by an increase in concentrations of other metals which form strong complexes. Although significant correlation coefficient was obtained for Zn accumulation from 10−10 to 10−2 M ZnCl2 solution in C. convoluta and C. rangiformis (Chettri et al., 1996), it was not observed between Zn content in the soil and lichen thalli. Cu formation of a stronger complex than Zn (Wehrli and Stumm, 1989) on one hand, and higher concentrations of Cu in mineralized soil on the other hand, must be the reason for the insignificant correlation. Manganese concentrations in epiphytic lichens were mostly higher than in epilithic and epigeic lichens in most plots. Freezing and cracking of thalli during rainy days with subzero temperatures have been explained as a reason for leakage of Mn and Fe from in situ epilithic R. maciformis thalli (Garty et al., 1995). Manganese is an essential element for plants, and its free translocation to the aerial parts of vascular plants, as mentioned by Bodek et al. (1988), and Taiz and Zeiger (1991), was further supported by the present study. De Bruin (1985) suggested that epiphytic lichens might obtain Ca, Mn, Zn, and Cd from the bark substrate. Another possible factor favoring the accumulation of elements in corticolous species involves the inputs from stemflow and canopy leachates (Pike, 1978). Thus, it can be concluded that low accumulation of Mn in epigeic and epilithic lichens must be due to low temperature (below zero) and rain in winter, which caused leaching of Mn in it. In contrast, epiphytic lichens growing in the oak forest could accumulate Mn from the bark, stemflow, and canopy leachates, on one hand, and, due to the canopy of trees, they are less affected by winter rain, on the other hand. This must also be the reason for the high accumulation of Zn and Cu in P. sulcata growing in the oak forest. Less accumulation of Cr in all epigeic lichens and vascular plants than in epilithic lichens is due to the fact that uptake of this metal occurs as hexavalent chromate (CrO42−) which, however, is rapidly reduced to Cr3+ in all soils. The trivalent form of Cr is absorbed minimally by root and its translocation from root to other parts is low (Streit and Stumm, 1993). As a result, less accumulation of Cr in all epiphytic lichens was obtained in the present study. Even then, elevated accumulation of Cr in C. convoluta was observed in soil with high concentrations of Cr (plot 4 and 5 of Chalkidiki). Though higher amounts of Pb were accumulated in epilithic lichens than in epigeic or higher plants, significant correlation was observed in the epigeic lichens (C. convoluta and C. rangiformis) and the vascular plant M. hirsuta (root). The insignificant correlations in EL lichens are due to small (n) value, less range of Pb content in the soil, and irregularity in Pb accumulation in thalli. In air-polluted areas, the correlation between soil and epigeic lichens (C. convoluta and C. rangiformis) was not achieved due to atmospheric Pb accumulation in thalli (unpublished data).
CONCLUSION
From the present study it can be concluded that epilithic lichens (N. pulla and X. taractica) and the epigeic lichen C. convoluta, which accumulated a higher amount of Cu, Pb, Cr, and Zn, should be included along with other symptomatic plants (like Minuartia or Rumex plants) for geochemical exploration. Inclusion of these lichens along with indicator plants will help to confirm the results. Besides this, high Cu concentrations were detected in mineralized soil where C. convoluta exhibited a blackish-rimmed ventral surface. Thus, the color change in thallus of C. convoluta may be a very useful geobotanical tool in the discovery of copper mineralization. ACKNOWLEDGMENTS M.K.C. thanks the State Scholarships Foundation (IKY), Greece, for awarding scholarships and Euxinos Leschi, Thessaloniki, for providing logistic support.
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Garty, J. (1993). Lichens as biomonitors for heavy metal pollution. In Plants as Biomonitors (B. Markert, Ed.), pp. 193–263. VCH, Weinheim. Garty, J., Harel, Y., and Steinberger, Y. (1995). The role of lichens in the cycling of metals in the Negev Desert. Arch. Environ. Cont. Toxicol. 29, 247–253.
Nieboer, E., and Richardson, D. H. S. (1981). Lichens as monitors of atmospheric deposition. In Atmospheric Pollutants in Natural Waters (S. J. Eisenreich, Ed.), pp. 339–388. American Arbor Science, Ann Arbor, MI.
Hobbie, S. E. (1995). Direct and indirect effects of plant species on biogeochemical processes in arctic ecosystems. Ecol. Stud. (New York) 113, 213– 224. James, P. W. (1973). The effect of air pollutants other than hydrogen fluoride and sulphur dioxide on lichens. In Air Pollution and Lichens (B. W. Ferry, M. S. Baddeley, and D. L. Hawksworth, Eds.), pp. 143–176. The Athlone Press, London. Kabata-Pendias, A., and Pendias, H. (1992). Trace Elements in Soils and Plants. Boca Raton, CRC Press. Kelepertsis, A. E., and Andrulakis, I. (1983). Geobotany-biogeochemistry for mineral exploration of sulphide deposits in northern Greece—Heavy maetal accumulation by Rumex acetosella L and Minuartia verna (L.) Hiern. J. Geochem. Explor. 18, 267–274. Kelepertsis, A. E., Andrulakis, I., and Reeves, R. D. (1985). Rumex acetosella L and Minuartia verna (L.) Hiern. as geobotanical and biogeochemical indicator for ore deposits in northern Greece. J. Geochem. Explor. 23, 203– 212. Konstantinou, M. (1992). Phytosociological Studies of the Vegetation on Metalliferous Soils, in Northern Greece. Ph.D. thesis, Aristotle University of Thessaloniki. Lange, O. L. (1990). Oekophysiologie der Flechten: Extremisten unter den Pflanzen. Abstract in the proceedings ‘‘Botanikertagung in Regensburg,’’ October 1990. Lange, O. L., and Zeigler, H. (1963). The heavy metal content of the lichen Acarospora sinopica from slag dumps in the Harz Mountains: Floristsoziologischen Arbeitsgemeinschaft Mitt. 10, 156. Legittimo, P. C., Ducceschi, L., and Martini, M. (1995). Plant species as indicators of geochemical anomalies: Experiences on Ilex aquifolium (holly). Environ. Geol. (Berlin) 25, 114–118. Malyuga, D. P. (1964). Biogeochemical Methods of Prospecting. Consultants Bureau, New York. Martin, M. H., and Coughtrey, P. J. (1982). Biological Monitoring of Heavy Metal Pollution. Applied Science, London and New York. Miller, J. C., and Miller, J. N. (1986). Statistics for Analytical Chemistry. Ellis Horwood, Chichester. Nieboer, E., Puckett, K. J., Richardson, D. H. S., Tomassini, F. D., and Grace, B. (1977). Ecological and physicochemical aspects of accumulation of heavy metals and sulphur in lichens. In Proceedings, International Conference on Heavy Metals in the Environment (27–31 October, 1975), Toronto, Canada, Vol. II, pp. 331–353.
Ouzounidou, G. (1993). Heavy Metal Effects on Natural Plant Species of Northern Greece’s Metalliferous Soils. Ph.D. Thesis, Aristotle University of Thessaloniki, Greece. Pike, L. H. (1978). The importance of epiphytic lichens in mineral cycling. Bryologist 81, 247–257. Puckett, K. J. (1988). Bryophytes and lichens as monitors of metal deposition. In Lichens, Bryophytes and Air Quality (T. H. Nash III and V. Wirth, Eds.), pp. 231–267. Cramer, Berlin. Rao, D. N., Robitaile, G., and LeBlanc, F. (1977). Influence of heavy metal pollution on lichens and bryophytes. J. Hattori Bot.Lab. 42, 213–239. Reeves, R. D., Kelepertsis, A. E., Andrulakis, I., and Hill, L. F. (1986). Biogeochemical studies of areas of sulphide mineralization in northern Greece. J. Geochem. Explor. 26, 161–175. Richardson, D. H. S., and Nieboer, E. (1980). Surface binding and accumulation of metals in lichens. In Cellular Interactions in Symbiosis and Parasites (C. B. Cook, P. W. Pappas, and E. D. Rudolph, Eds.), pp. 75–94. Ohio State University Press, Columbia. Sawidis, T., Zachariadis, G., Stratis, J., and Ladoukakis, E. (1993). Mosses as biological indicator for monitoring of heavy metal pollution. Fresenius Envir. Bull. 2, 193–199. Sawidis, T., Chettri, M. K., Zachariadis, G., Stratis, J., and Seaward, M. R. D. (1995). Heavy metal bioaccumulation in lichens from Macedonia in North Greece. Toxicol. Environ. Chem. 50, 157–166. Streit, B., and Stumm, W. (1993). Chemical properties of metals and the process of bioaccumulation in terrestrial plants. In Plants as Biomonitors (B. Markert, Ed.), pp. 31–62. VCH, Weinheim/New York/Basel/Cambridge. Taiz, L., and Zeiger, E. (1991). Plant Physiology. Benjamin/Cummings, Redwood City. Tuba, Z., Csintalan, Z., Nagy, Z., Szente, K., and Takacs, Z. (1994). Sampling of terricolous lichen and moss species for trace element analysis, with special reference to bioindication of air pollution. In Environmental Sampling for Trace Analysis (B. Markert, Ed.), pp. 415–434. VCH, Weinheim/New York/Basel/Cambridge/Tokyo. Tyler, G. (1989). Uptake, retention and toxicity of heavy metals in lichens. Water Air Soil Pollut. 47, 321–333. Wehrli, V., and Stumm, W. (1989). Vanadyl in natural waters: Adsorption and hydrolysis promote oxygenation. Geochim. Cosmochim. Acta 53, 69–77. Welz, B. (1985). Atomic Absorption Spectrometry. VCH, Weinheim/Germany. Wild, H. (1974). Geobotanical anomalies in Rhodesia. 4. The vegetation of arsenical soils. Kirkia 9, 123–125.
38, 322–335 (1997)
Lichens as a Tool for Biogeochemical Prospecting M. K. Chettri,* T. Sawidis,† and S. Karataglis† *Department of Botany, Amrit Campus, Tribhuvan University, P.O. Box 102, Kathmandu, Nepal; and †Department of Botany, School of Biology, University of Thessaloniki, Gr-54006 Thessaloniki, Greece Received April 11, 1997
The heavy metal content in lichens and vascular plants from abandoned copper mining areas, Gerakario (Kilkis) and Megali Panagia (Chalkidiki), have been compared with metal content in soil in order to assess their efficiency to accumulate five metals (Cu, Mn, Pb, Zn, and Cr). The average metal content in the mineralized soil of Gerakario was, in descending order, Cu, Mn, Pb, Zn, and Cr, and in Chalkidiki it was Cu, Mn, Cr, Pb, and Zn. The epilithic lichens (Neophuscelia pulla) accumulated the highest amount of Cu and Pb, and Xanthoparmelia taractica accumulated the highest amount of Zn. All the lichens revealed significant (P < 0.05) correlation between Cu content in soil and that in thalli. Out of five metals studied, four (Cu, Pb, Mn, and Cr) in the epigeic lichen Cladonia convoluta, two (Cu and Mn) in both epilithic lichen N. pulla and X. taractica, and one (Pb) in vascular plant Minuartia (root) were significantly (P < 0.05) correlated between their metal content in plant tissue and in soil. Further, discoloration of C. convoluta with higher Cu concentrations adds a visible clue for biogeochemical exploration. Thus, lichens along with other symptomatic species will help in locating mining areas. © 1997 Academic Press
INTRODUCTION
Plants growing in heavy-metal-rich soil have been described as excluders, indicators, or accumulators, depending on the basis of metal concentrations in it (Baker, 1981; Legittimo et al., 1995). The basis of the biogeochemical method of prospecting is that the presence of anomalous concentrations of metals in the soil or underlying rocks will be reflected by the presence of those metals in abnormal concentrations in the vegetation (Butcher, 1992; Kabata-Pendias and Pendias, 1992; Badri and Springuel, 1994). As long as the metabolism is nearly not disturbed, i.e., around the optimal range, visible deviation of ‘‘normal’’ plant behavior will not be detectable, even if increased concentration of a heavy metal may be detectable by chemical analysis. As soon as a surplus of the element disturbs the metabolism, reaction will become visible; it may be a chlorosis, discoloration, a necrosis, stunted growth, or teratological performance of plant parts, when symptomatic indication of individual plants is considered (Eleftheriou and Karataglis, 1989; Ernst, 1993). In the case of the presence of open and hidden ore bodies, the first target organ of a plant for heavy metal accumulation is 322 0147-6513/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
the root (Ernst, 1993). Malyuga (1964) was the first to give an account on the importance of the root morphology for mineral prospection. If an ore body is hidden by overburden, but still remains in the root zone, then metal can be brought above ground through plant parts. After the death or fall of plant parts, these metals will accumulate in the upper soil horizon of the overburden (Martin and Coughtrey, 1982; Ernst, 1993; Hobbie, 1995). Bowen (1979) reviewed the average crustal abundance of various metals and their median concentrations in soil. The data suggest that Fe, Ti, Mn, Zn, Ga, Ag, and Hg exhibit a close relationship between parent material and soil concentrations. Ti, Co, Cu, Ni, Cr, and Va appear to be somewhat depleted in soils relative to parent material. In, Bi, Cd, Sn, and As are all somewhat rich in soil relative to parent material. Frequently in mineralized areas, the change in species composition of the plant community relative to neighboring nonmineralized areas causes a dramatic difference in the appearance (physiognomy) of the vegetation (Wild, 1974). Lichens are considered to be an extremist plant (Lange, 1990), because they can grow under extreme environmental conditions and even can stand a high level of heavy metals in mining areas (Lange and Ziegler, 1963; Czehura, 1977; Sawidis et al., 1995). Studies on the capacity of lichens to accumulate heavy metals and other elements were reviewed thoroughly (James, 1973; Nieboer et al., 1977, 1978; Rao et al., 1977; Richardson and Nieboer, 1980; Martin and Coughtrey, 1982; Brown and Beckett, 1985; Puckett, 1988; Tyler, 1989; Brown, 1991; Garty, 1992, 1993). Lichens accumulate and tolerate metals to a high degree because of their relatively large surface area and slow growth rate. Because of the lack of cuticle (or similar) wax covering and their poikilohydric nature, accumulation of metals occurs by passive adsorption and ion exchange (Nieboer and Richardson, 1981). Vascular plants, compared to lichens, have a well-developed root and transport system. Rumex acetosella and Minuartia verna (which was later reported as Minuartia hirsuta; Konstantinou, 1992) have been reported as indicators for ore deposits in northern Greece (Kelepertsis and Andrulakis, 1983; Kelepertsis et al., 1985; Ouzounidou, 1993). Brooks (1972, 1983) concluded that a universal characteristic of indicators is
LICHENS AS A TOOL FOR BIOGEOCHEMICAL PROSPECTING
that they will have a very high elemental content in their ash. In other words, a criterion for new indicators is that they have a higher concentration of the element sought than the surrounding vegetation. Although lichens do not have well organized absorptive systems, they come in contact with metals of upper soil horizon due to wind and rain activity. Due to the poikilohydric nature of lichens, they can accumulate dissolved metals from all the surface of their body. Therefore, it was hypothesized that lichens growing on mineralized soil or rocks would accumulate anomalous concentrations of heavy metals and could be used as a tool for biogeochemical prospecting, especially in remote places where anthropogenic activity is lacking. Therefore, in the present study heavy metal content in lichens and vascular plants collected from abandoned mining areas were compared with metal content in mineralized soil to assess their efficiency to accumulate different metals. MATERIALS AND METHODS
Gerakario and Chalkidiki (M. Panagia) in northern Greece (Fig. 1) have been reported to have varying degrees of porphyry copper mineralization, associated with post-miocene volcanic rocks of rhyolithic composition (Reeves et al., 1986). Vascular plants (VP), such as M. hirsuta subsp. falcata (griseb) Mattf., R. acetosella L., Thymus sibthorpii Bentham, Sanguisorba minor subsp. minor, Dianthus pinifolius Sibth & Sm., were collected from the following mentioned sampling sites. Along with VP, epigeic (EG) lichens like Cladonia convoluta (Lam.) P. Cout. and Cladonia rangiformis Hoffm. (substrate soil), and epilithic (EL) lichens like Neophuscelia pulla and Xanthoparmelia taractica (Kremp.) Hale (substrate stones), were also collected. The sampling sites at Gerakario were five deserted plots (1, 2, 3, 4, and 5) located approximately 100 m
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apart, along the northwest to southeast direction and one undeserted plot (6) in a pasture area (Fig. 2a). At the same time, the same plant species were also collected from rocks in the heart of the mine (1), the sandy field in the center (2), the first contour around the center (3), the pasture area (4), and the oak forest (5) around the mining area of M. Panagia (Chalkidiki) (Fig. 2b). In addition to these lichens, epiphytic (EP) lichens (substrate bark), which appeared only in the forest (5), were also collected for comparison with EG and EL lichens. Along with plant samples, soil (S) samples were also collected from the 6- to 8-cm depth of each plot. The attached foreign particles from collected plant materials were removed carefully with the help of fine plastic forceps. The vascular plant materials were washed thoroughly with running double-deionized water to avoid soil contamination. Lichens were washed for 5 s under running double-deionized water as suggested by Tuba et al. (1994). Plant samples were dried at ambient temperature (25–30°C) for 2–3 days, then oven-dried at 60–80°C for 24–48 h to constant weight, and then pulverized with a mortar and pestle. The pulverized sample was considered a representative sample of that plot as it consisted of many individuals. Soil (S) samples were also dried as mentioned above and sieved through a 0.25-mm mesh. An accurately weighed portion of each sample (1 g dry wt) was placed in an open quartz tube; 8 ml concentrated HNO3 (Merck) was added to each tube and the mixture was left overnight at room temperature. The mixture was then warmed for 2 h at 50°C and subsequently heated at 160°C for 4 h. The extract was filtered through Whatman type 589/2 filters and the filtrate was diluted to 25 ml volume with double-deionized water. The diluted filtrates were analyzed for metal concentration using a Perkin Elmer 2380 atomic absorption spectrometer. The analytical wavelengths were 283.3 nm for Pb, 279.5 for Mn, 357.9 for Cr, 324.7 nm for Cu, and 213.8 nm of Zn (Welz, 1985). The relative standard deviation of the measurements were 8.2% for Pb, 2.5% for Cu, 3.6% for Zn, 4.7% for Mn, and 7.9% for Cr. This procedure provides data for an estimation of the total content of the metal in the thalli (Sawidis et al., 1993). Statistical procedure. To understand the relationship between the different metals studied, correlation coefficient (r) was calculated from linear regression analysis between different metals in soil and different metals in soil and in plant. Bearing in mind the number of pairs of points (n) used in the calculation, the level of significance (P) for a calculated correlation coefficient (r) was obtained by calculating the t value. The calculated value of t was compared with the tabulated value at P < 0.05 using a two-tailed t test and (n − 2) degrees of freedom (Miller and Miller, 1986). RESULTS
FIG. 1. Map of Macedonia, N. Greece, indicating the sampling stations. Gerakario (Kilkis) and Megali Panagia (Chalkidiki) areas with porphyry copper deposits.
The average metal content in the mineralized soil of Gerakario was, in descending order, Cu, Mn, Pb, Zn, and Cr, and in
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FIG. 2. Sampling sites at abandoned porphyry copper deposits (a) Gerakario (Kilkis) and (b) M. Panagia (Chalkidiki).
Chalkidiki it was Cu, Mn, Cr, Pb, and Zn. Total metal load in EL lichens was mostly higher than that in EG lichens or VP (Fig. 3). Epiphytic lichens collected from the oak forest around (5) Chalkidiki mining accumulated higher amounts of Mn than the epigeic lichens or vascular plants from the same plot (Fig. 3).
The accumulation of Cu ranged from 10 to 349 mg/g in leaves, from 10 to 355 mg/g in stems, and from 10.5 to 689 mg/g in roots, respectively. In vascular plants, Cu accumulation was higher in the roots than the leaves or the stems in all species. Accumulation of Mn, Zn, Pb, and Cr varying in different parts of different species indicated free translocation of
FIG. 3. Metal (Cu, Mn, Pb, Zn, and Cr) content in epilithic lichens (EL), epegeic lichens (EG), epiphytic lichens (EP), vascular plants (VP), and soil (S).
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these metals to stem and leaves. Rumex acetosella L. and M. verna (L) Hiern. (which was later identified as M. hirsuta; Konstantinou, 1992) have been reported as biogeochemical indicators for ore deposits in northern Greece (Kelepertsis and Andrulakis, 1983; Kelepertsis et al., 1985). Though, R. acetosella exhibited the highest Cu accumulation in the roots, they were encountered only in Chalkidiki and not in Gerakario. Therefore, M. hirsuta (roots), which exhibited the highest accumulation of Cu (and also most other metals) next to Rumex (Fig. 4) was encountered in all stands, was considered for comparison with epigeic and epilithic lichens in the present study. Cu The amount of Cu measured in the soil and in plant material was mostly higher in Chalkidiki than in Gerakario. The highest
Cu accumulation was observed in N. pulla (Fig. 5) and hyperaccumulation was recorded in it in some plots. All the lichens (EG or EL) considered in the present study demonstrated significant (P < 0.05) correlation between Cu content in thalli and in the soil (Table 1). An elevated amount of Cu was detected in C. convoluta encountered in plots 1–4 of Chalkidiki; at the same time, the ventral surface of the thallus was purplishblack-rimmed instead of the normal greenish-white color. Very low Cu concentrations were detected in epiphytic lichens like Evernia prunastri (9 to 23 mg/g) and Ramalina fraxinea (8.5 mg/g) growing in the oak forest around Chalkidiki mining areas, but in Parmelia sulcata the accumulation of Cu was much higher, ranging from 37 to 87 mg/g. Similarly, low concentrations of Cu were detected in some epigeic lichens like Cladonia ciliata (35 mg/g), Cladonia rangiferina (16 mg/ g), and Cladonia stellaris (16 mg/g) growing in Chalkidiki (4) mining areas.
FIG. 4. Cu content in leaves, stems, and roots of different vascular plants in different plots of abandoned Cu mining areas. (A) Thymus (Gerakario), (B) Minuartia (Gerakario, Chalkidiki), (C) Sanguisorba (Gerakario), (D) Dianthus (Gerakario), (E) Rumex (Chalkidiki).
FIG. 5. Cu accumulation in different EG, EL lichens, Minuartia (roots), and soil.
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TABLE 1 Correlation Coefficient (r) Obtained from Linear Regression between Different Metals in Soil, and between Soil and Plant Tissue S-Pb
S-Mn
S-Cr
S-Zn
S-Cu
1.000 0.545 0.044
1.000 −0.090
1.000
n 4 11 S-Pb S-Mn S-Cr S-Zn S-Cu
1.000 0.356 −0.492 0.234 −0.305
1.000 0.515 0.842** −0.116
CC-Pb CC-Mn CC-Cr CC-Zn CC-Cu
0.624* 0.152 −0.331 0.179 0.488
0.733* 0.773* 0.633* 0.654* 0.134
CR-Pb CR-Mn CR-Cr CR-Zn CR-Cu
0.820** 0.614* −0.298 0.064 −0.383
0.241 0.356 −0.019 0.274 −0.451
N-Pb N-Mn N-Cr N-Zn N-Cu
0.623 0.695* −0.445 0.607 −0.292
0.733 0.801* −0.097 −0.651 0.171
X-Pb X-Mn X-Cr X-Zn X-Cu
0.383 0.811* −0.293 0.493 0.187
0.518 0.824* −0.230 0.327 0.175
M-Pb M-Mn M-Cr M-Zn M-Cu
0.652* 0.873** 0.548 −0.351 −0.067
0.15 0.461 −0.002 0.307 0.191
n 4 11 0.116 0.107 0.822** 0.345 0.267
0.524 0.349 0.444 0.419 0.074
0.030 0.389 0.195 0.356 0.810**
n 4 11 −0.301 −0.376 0.122 0.133 −0.243
0.125 0.125 −0.394 0.102 −0.484
−0.121 −0.118 0.421 0.635* 0.665*
−0.266 −0.357 0.617 −0.150 0.229
0.748* 0.762* 0.108 0.558 −0.051
0.161 0.136 0.221 0.382 0.896*
−0.190 −0.772 0.522 −0.867* −0.274
0.448 0.842* −0.448 0.466 0.131
−0.434 0.375 −0.060 0.698 0.860*
−0.597 −0.310 −0.343 0.625* 0.208
−0.368 0.307 −0.320 0.429 −0.140
0.048 −0.513 −0.335 0.271 0.446
n48
n46
n 4 11
Note. S, soil; CC, C. convoluta; CR, C. rangiformis; N, N. pulla; X, X. taractica, M, Minuartia (root); Pb, lead; Mn, manganese; Cr, chromium; Zn, zinc; Cu, copper. * P < 0.05, **P < 0.01; unmarked, not significant.
Zn The amounts of Zn accumulation in the soil of Gerakario and Chalkidiki do not differ much. High accumulation of Zn was seen in X. taractica and Minuartia (root) in some plots (Fig. 6). Zn and Mn content in soil revealed significant positive correlation (P < 0.05). Significant negative correlation (P < 0.05) was observed in the Zn content of X. taractica thalli and Cr in the soil, whereas significant positive correlation, for the same, was obtained in Minuartia. No significant relation was detected in the soil Zn content and Zn in any plant material encountered in the present study. All EG and EP lichens, except corticolos P. sulcata, found in the oak forest around the Chalkidiki mine, did not indicate
much variation in Zn accumulation. Accumulation of Zn was higher in P. sulcata (85 mg/g) than in the epilithic or epigeic lichens that occurred in the same plot or other plots. Pb Lead content in the soil ranged from 112 to 212 mg/g in Gerakario and 19 to 131 mg/g in Chalkidiki. The highest amount of Pb accumulation was observed in lithophytic lichens (Fig. 7). Cladonia convoluta, which is an epigeic lichen, also accumulated higher Pb amounts than Minuartia. Pb content in plant and in soil was significantly correlated in C. convoluta, C. rangiformis, and Minuartia (root). Pb content in C. convoluta and N. pulla revealed significant correlation with Mn con-
FIG. 6. Zn accumulation in different EG, EL lichens, Minuartia (roots), and soil.
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FIG. 7. Pb accumulation in different EG, EL lichens, Minuartia (roots), and soil.
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tent in the soil. in the case of N. pulla, Pb content in the thalli was also significantly correlated to Zn content in the soil (Table 1). Low amounts of Pb were detected in some EG lichens like C. ciliata (0.5 mg/g), C. rangiferina (12.5 mg/g), C. stellaris (12.5 mg/g), and Cornicularia aculeata (6 mg/g) collected from the oak forest. Similarly, EP lichens like E. prunastri and R. fraxinea accumulated 13 and 8 mg/g, respectively, whereas 55 mg/g Pb was measured in P. sulcata from the same area. Cr Chromium content in soil ranged from 20 to 45 mg/g in Gerakario and from 29 to 370 mg/g in Chalkidiki area. Cr accumulation was higher in lithophytic lichens than in epigeic lichens or Minuartia root (Fig. 8). Cr content in C. convoluta thalli and in the soil as well demonstrated significant (P < 0.05) correlation. Chromium content in C. convoluta was also significantly correlated to Mn content in soil (Table 1). Low amounts of Cr accumulation were measured in all EP lichens (0.5 to 13 mg/g) and EG lichens (0.5 to 10.9) except C. convoluta (2.24 to 98.72). Mn Manganese concentration in the soil ranged from 100 to 534 mg/g in Gerakario and 157 to 680 mg/g in Chalkidiki area. In all the plots, Mn accumulation was higher in Minuartia (roots) than in lichens (Fig. 9). In some cases manganese was accumulated as much as or more in the stems than in the roots of vascular plants, thus indicating free translocation of this metal in plant tissue. Significant correlation was obtained for Mn content in lichen thalli (C. convoluta, N. pulla, and X. taractica) and in the soil (Table 1). Similarly, Mn content in C. rangiformis, N. pulla, X. taractica, or Minuartia was also significantly correlated to Pb content in the soil, indicating positive relations between Mn accumulation in plant and Pb content in soil. Mn accumulation in both EL lichens was significantly correlated to Zn content in soil. Accumulation of Mn in the epiphytic lichens E. prunastri, P. sulcata, and R. fraxinea encountered in oak forest around M. Panagia (Chalkidiki) mine (plot 5) was measured to be 114, 151, and 115 mg/g, respectively. The amount of Mn accumulation was higher in EP lichens than in EG lichens or Minuartia (root) in the same plot and also higher than EL or EG lichens of most other plots. DISCUSSION
Out of the five metals measured in plant tissues, four metals (Pb, Mn, Cr, and Cu) in C. convoluta thalli, two metals (Pb and Cu) in C. rangiformis, two metals (Mn and Cu) in N. pulla and X. taractica, and one metal (Pb) in Minuartia (root) were significantly (P 4 0.05) correlated with the same metal in soil. Although Kelepertsis and Andrulakis (1983) reported direct relationship between the Cu content of plant ash and of soils in Minuartia, no significant correlation was observed in the pre-
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sent study. Comparing the relations in the soil and plant material, it was observed that significant correlation between Zn and Mn found in soil was also present only in C. convoluta (Table 1). Various edaphic and plant factors responsible for the accumulation of metals in a plant have been mentioned (Chaney, 1973; Martin and Coughtrey, 1982). Metal accumulation in vascular plants is affected by different factors such as: mechanisms of metal movements within the soil, metal distribution within an individual soil profile, root distribution in the soil, root structure and physiology, translocation of metals from roots to shoots in relation to plant growth and development, translocation of metals within above-ground parts of plants in relation to metabolism and development, and loss of metals from plants via senescence, leaf-fall, leaf leaching, exudation, and volatilisation. The differences in metal accumulation in higher plants caused by the mechanism of metal movement within the soil, distribution of metal in each profile, distribution of absorptive organ in the soil, and loss of metal by leaf fall or senescence are completely obscured in lichens. Therefore, metals with higher concentrations in the soil (i.e., Cu, Mn, Pb, and Cr) manifested significant correlation with lichen C. convoluta. The minor changes in trace element contents of the soil substrates did not follow the accumulation pattern of the lichen and moss indicators in any of the cases, except for V in lichen and V and Ni in moss (Tuba et al., 1994). Zinc content in the soil in the present studied mines ranged from 40 to 84 mg/g, which was not so much in comparison to other metals like Cr (20–368 mg/g), Cu (181–819 mg/g), Pb (49–213 mg/g), and Mn (101–679 mg/g). Therefore, the correlation obtained between Zn content in the soil and any plant material was not significant. In addition to this, a general rule concerning cation uptake mechanism involving ion exchange modified by metal complex formation has been formed for third period transition metal cations. The stability of their complexes increases in the series Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+ (Wehrli and Stumm, 1989). As Cu forms the strongest complexes with ligands at binding sites and as both sites of study were with prophyry copper deposits, Cu2+ accumulation in all lichen species tested were significantly correlated. Cladonia convoluta treated with 10−10 to 10−2 M CuCl2 solution demonstrated significant uptake of Cu2+ (Chettri et al., 1996) and total chlorophyll degradation with increasing concentrations (unpublished data). Therefore, it can be speculated that the purplish-black rim on the lower surface of C. convoluta in plots having high concentrations of Cu (Chalkidiki, 1-4) must be due to copper’s toxic effect on chlorophyll. The insignificant correlation between Zn content in the soil and Zn in all lichen thalli (or Minuartia root) strengthens the assumption of Chettri et al. (1996) that the lichens which can accumulate more than one heavy metal would not reflect the actual proportion of those metals in the environment, espe-
FIG. 8. Cr accumulation in different EG, EL lichens, Minuartia (roots), and soil.
332 CHETTRI, SAWIDIS, AND KARATAGLIS
FIG. 9. Mn accumulation in different EG, EL lichens, Minuartia (roots), and soil.
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cially those forming weaker complex in binding sites. They also found that the accumulation of heavy metals, forming weaker complexes, would be affected by an increase in concentrations of other metals which form strong complexes. Although significant correlation coefficient was obtained for Zn accumulation from 10−10 to 10−2 M ZnCl2 solution in C. convoluta and C. rangiformis (Chettri et al., 1996), it was not observed between Zn content in the soil and lichen thalli. Cu formation of a stronger complex than Zn (Wehrli and Stumm, 1989) on one hand, and higher concentrations of Cu in mineralized soil on the other hand, must be the reason for the insignificant correlation. Manganese concentrations in epiphytic lichens were mostly higher than in epilithic and epigeic lichens in most plots. Freezing and cracking of thalli during rainy days with subzero temperatures have been explained as a reason for leakage of Mn and Fe from in situ epilithic R. maciformis thalli (Garty et al., 1995). Manganese is an essential element for plants, and its free translocation to the aerial parts of vascular plants, as mentioned by Bodek et al. (1988), and Taiz and Zeiger (1991), was further supported by the present study. De Bruin (1985) suggested that epiphytic lichens might obtain Ca, Mn, Zn, and Cd from the bark substrate. Another possible factor favoring the accumulation of elements in corticolous species involves the inputs from stemflow and canopy leachates (Pike, 1978). Thus, it can be concluded that low accumulation of Mn in epigeic and epilithic lichens must be due to low temperature (below zero) and rain in winter, which caused leaching of Mn in it. In contrast, epiphytic lichens growing in the oak forest could accumulate Mn from the bark, stemflow, and canopy leachates, on one hand, and, due to the canopy of trees, they are less affected by winter rain, on the other hand. This must also be the reason for the high accumulation of Zn and Cu in P. sulcata growing in the oak forest. Less accumulation of Cr in all epigeic lichens and vascular plants than in epilithic lichens is due to the fact that uptake of this metal occurs as hexavalent chromate (CrO42−) which, however, is rapidly reduced to Cr3+ in all soils. The trivalent form of Cr is absorbed minimally by root and its translocation from root to other parts is low (Streit and Stumm, 1993). As a result, less accumulation of Cr in all epiphytic lichens was obtained in the present study. Even then, elevated accumulation of Cr in C. convoluta was observed in soil with high concentrations of Cr (plot 4 and 5 of Chalkidiki). Though higher amounts of Pb were accumulated in epilithic lichens than in epigeic or higher plants, significant correlation was observed in the epigeic lichens (C. convoluta and C. rangiformis) and the vascular plant M. hirsuta (root). The insignificant correlations in EL lichens are due to small (n) value, less range of Pb content in the soil, and irregularity in Pb accumulation in thalli. In air-polluted areas, the correlation between soil and epigeic lichens (C. convoluta and C. rangiformis) was not achieved due to atmospheric Pb accumulation in thalli (unpublished data).
CONCLUSION
From the present study it can be concluded that epilithic lichens (N. pulla and X. taractica) and the epigeic lichen C. convoluta, which accumulated a higher amount of Cu, Pb, Cr, and Zn, should be included along with other symptomatic plants (like Minuartia or Rumex plants) for geochemical exploration. Inclusion of these lichens along with indicator plants will help to confirm the results. Besides this, high Cu concentrations were detected in mineralized soil where C. convoluta exhibited a blackish-rimmed ventral surface. Thus, the color change in thallus of C. convoluta may be a very useful geobotanical tool in the discovery of copper mineralization. ACKNOWLEDGMENTS M.K.C. thanks the State Scholarships Foundation (IKY), Greece, for awarding scholarships and Euxinos Leschi, Thessaloniki, for providing logistic support.
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