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Modification of the sequential elution technique for the extraction of heavy metals from bryophytes
The Science of the Total Environment 241 Ž1999. 53]62
Modification of the sequential elution technique for the extraction of heavy metals from bryophytes a,U M.D. Vazquez , J. Lopez ´ ´ b, A. Carballeira c a
Ecoloxıa, ´ E.P.S., Uni¨ ersidade de Santiago de Compostela, Campus Uni¨ ersitario, 27002 Lugo, Spain b Ecoloxıa, ´ Facultade de Ciencias, Uni¨ ersidade de Vigo, 36200 Vigo, Spain c Ecoloxıa, ´ Facultade de Bioloxıa, ´ Uni¨ ersidade de Santiago de Compostela, 15706, Santiago de Compostela, Spain Received 6 June 1999; accepted 3 July 1999
Abstract The sequential elution technique ŽS.E.T.. has frequently been used to determine the distribution of metals at different cellular sites in contaminated lichens and, to a lesser extent, in mosses. Here we evaluate certain aspects of the technique in order to improve the reliability of the results obtained when it is used on bryophytes. Various experiments were carried out using the aquatic moss Fontinalis antipyretica Hedw. Results confirmed that two consecutive washings are sufficient to ensure total extraction of the extracellular metal. In addition, the possibility of using new extractants to remove the metal that is accumulated at extracellular sites was investigated. Although NiCl 2 Žthe extractant originally used by the authors of the technique wBrown DH, Buck GW. J Briol 1978a;10:199]209; Brown DH, Buck GW. Ann Bot 1978b;42:923]929x. was adequate for metals with low affinity for the binding sites, EDTA and PbŽNO 3 . 2 were more efficient for the extraction for metals with a medium-high affinity for extracellular binding sites. The optimum concentrations of extractant were found to be 10 or 20 mM for NiCl 2 and EDTA, and 50 mM for PbŽNO. 2 . Q 1999 Elsevier Science B.V. All rights reserved. Keywords: Sequential elution technique; Fontinalis antipyretica Hedw; Bryophytes; Heavy metals; Affinity; Extractant; Extracellular metal; Intracellular metal
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Corresponding author. Tel.: q34-982-252231, ext. 23125; fax: q34-982-241835. . E-mail address: [email protected] ŽM.D. Vazquez ´
0048-9697r99r$ - see front matter Q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 8 - 9 6 9 7 Ž 9 9 . 0 0 3 3 7 - X
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1. Introduction The sequential elution technique ŽS.E.T.. was proposed by Brown and Buck Ž1978a,b, 1979. as a method to extract metals that accumulate at different cellular sites in lichens. Since then, the technique has frequently been applied in studies using lichens ŽBeckett and Brown, 1984; Brown, 1987; Brown and Wells, 1988; Brown and Avalos, 1991. and, less often, in studies using mosses ŽWells and Brown, 1987; Brown and Wells, 1990.. Here we describe a procedure that improves the use of lichens and mosses as biomonitors for heavy metal contamination and which involves the sequential removal of the elements that accumulate at discrete cellular sites. First, soluble, intercellular metal is discarded by rinsing the material with water. The extracellular metal, in the form of cations bound to anionic exchange sites in the cell wall and plasma membrane, is then extracted by displacement with another cation with a higher affinity for the exchange sites andror which is found at a higher concentration than the cation to be extracted ŽBrown, 1987; Brown and Avalos, 1991; Brown and Sidhu, 1992.. The soluble, intracellular metal is then extracted by using an acid that alters the permeability of the cell membranes, thus allowing release of the metals ŽBrown and Wells, 1988.. As the action of the acid alone may not be sufficient, the moss must first be oven dried to ensure that the cell membranes are ruptured and that intracellular material is released ŽBuck and Brown, 1979; Brown and Beckett, 1984; Brown, 1987.. An additional fraction consisting of metal bound to the remaining plant material, and which is extremely difficult to extract, is generically referred to as the particulate metal fraction. In order to release and quantify this metal, the organic material must first be digested. As well as particulate material, this fraction also includes insoluble intracellular elements such as metals incorporated into large polymeric molecules ŽBrown, 1984; Brown and Wells, 1988.. The technique has previously been used in mosses, although it is known that differences in the characteristics of the binding sites of the two types of organisms may influence the uptake of
metals ŽNieboer et al., 1976; Brown, 1982; Richardson and Nieboer, 1983; Wells, 1988.. Thus, it was not clear which type and concentration of displacing agent should be used to remove the extracellular metal, or what the optimum incubation times, etc., were. In order to ensure reliable results when using the technique in biomonitoring studies involving mosses, some of the steps involved were modified.
2. Materials and methods 2.1. Plant material The bryophyte Fontinalis antipyretica Hedw was chosen for this study as it is widely used in biomonitoring studies and is common in the NW Iberian Peninsula ŽLopez and Carballeira, 1990, ´ . 1993; Lopez et al., 1994 . ´ Moss samples were always collected from an uncontaminated stretch of a tributary of the river Sar ŽNW Iberian Peninsula .. Samples were rinsed in the river water to remove adherent particles before transporting them to the laboratory in plastic tanks full of the same river water Ž4 " 28C.. To standardize the experimental material, 2-cm apical segments were used ŽWhitton and Dıaz, ´ 1980; Wehr et al., 1983.. The samples were kept in river water in an incubation chamber Ž168C. with aeration until use. 2.2. Incubation of moss samples. Extraction and analysis of metals (general method) Although each of the experiments was carried out separately, the protocol followed was similar for each. Groups of F. antipyretica apices were firstly incubated for 1 h in the corresponding solutions of metals, depending on the experiment. All solutions were made up using distilled water and the samples were aerated appropriately. This step ensures saturation of extracellular sites in the moss and significant accumulation of metal inside the cells ŽWells and Brown, 1990.. Following incubation, the samples were rinsed to remove excess metal, and the metal accumulated at the different cellular sites was extracted
M.D. Vazquez et al. r The Science of the Total En¨ ironment 241 (1999) 53]62 ´
using the S.E.T. procedure, the modifications proposed being tested in each experiment. The general technique used included the following steps: 2.2.1. First elution Between eight and 25 moss apical segments were added to 10 ml of extractant wEDTA, PbŽNO 3 . 2 or NiCl 2 ŽMerck, analytical grade.x, and then incubated for 45 min with constant shaking. The apical segments were then removed and the extracts stored in the fridge Ž4 " 18C. until measurements were made. 2.2.2. Second elution The moss samples were incubated under the same conditions as before, but only for 30 min. The total metal extracted during these first elutions was the extracellular metal fraction. An additional elution was carried out in one experiment to determine whether or not this step was necessary. 2.2.3. Drying After removing the moss samples from the extractant solutions, they were then dried in an oven at 508C, for 24 h, causing the cell membranes to rupture ŽBrown, 1987.. A temperature of 808C has often been used in previous studies, but as some elements may be volatile at this temperature, a range of 40]508C is considered to be preferable ŽMarkert, 1995.. 2.2.4. Third elution The moss samples were removed from the oven and the total dry weight determined. Samples were then further eluted in 10 ml of 1 M HNO3 ŽMerck, Pure grade., for 30 min with shaking. This step leaves the intracellular metal fraction in solution in the acid. 2.2.5. Digestion of residual material For each group of samples apical segments were placed in a glass tube Ž20 ml. with 4 ml of concentrated HNO3 . The tubes were stoppered with glass spheres, which acted like valves, and placed on a hot plate Ž1008C., until the plant material was completely digested. Distilled water was then added to a volume of 10 ml, and the
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tubes were centrifuged at 3000 rev. miny1 for 5 min. These solutions contained the residual metal fraction. Extractions were carried out in triplicate. To avoid interference during spectrometric measurements, 0.1 ml of a solution of Cs]La Ž100 000 ppm. were added to each extract. Lanthanum prevents the formation of refractive compounds during analysis of Mg and Cs acts as a suppressor of ionization ŽWells and Brown, 1987; Brown and Wells, 1990.. The concentration of metals in the extracts was determined by atomic absorption spectrometry ŽPerkin Elmer 2100 instrument . with an airacetylene flame. The exceptions to this were Al Žwhere N2 O was used as an oxidizing agent. and K Žmeasured by flame emission spectrometry.. Standards were made up in the corresponding extractants. The detection limits Žmg ly1 . were: Al Ž30., Ca and Na Ž1., Cd Ž2., Co Ž5., Cu Ž3., K Ž10., Mg Ž0.1., Ni Ž5.5., Pb Ž14. and Zn Ž0.9.. Blank values were always lower than detection limit. The efficiency of the analysis was tested using registered reference material ŽCommunity bureau of reference } BCR no. 61, Platihypnidium riparioides.. Recovery was higher than 95% for all metals except for Al and Na Ž70%.. 2.3. Incubation of moss samples. Extraction and analysis of metals (differentiated by experiment) Although the methodology used was very similar in the four experiments carried out, the variations due to the different aims of each are outlined as follows: 2.3.1. Experiment 1 A sample set of 30 apical segments of F. antipyretica were incubated in 1 l of a solution of Cu Ž3 mg ly1 ., prepared from CuCl 2 , and another set of 30 apical segments in a 20 mM EDTA disodium salt solution ŽC 10 H 14 N2 Na 2 O 8 ? 2H 2 O.. The accumulated metal ŽCu in the first case and Na in the second. was then extracted using EDTA and NiCl 2 Žboth 20 mM., respectively, followed by three consecutive washes Žof 45, 30 and 30 min. to extract the extracellular metal Žinstead of two as in the general S.E.T. method.. The concentra-
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tions of Cu, Na, K, Mg and Ca in the extracts obtained were determined.
2.3.2. Experiment 2 Solutions of different concentrations Ž0, 1, 10, 50, 100 and 200 mg ly1 . of three metals ŽCo, Cu and Pb. were prepared from their corresponding chlorides, except for Pb when nitrate was used. Sets of 75 apical segments were incubated in 1 l of each solution for 1 h, and then subjected to S.E.T. in order to remove the accumulated metals. With half of the samples, 20 mM NiCl 2 was used as the extractant and with the other half, 20 mM EDTA. In addition to the metal added, the concentrations of K and Mg in each of the extracts were measured.
2.3.3. Experiment 3 The apical segments were incubated with six different metals ŽCd, Co, Cu, Ni, Pb and Zn. at a
single concentration of 100 mg lyl . The metals with low affinity for extracellular sites ŽCd and Zn. were extracted using NiCl 2 , and Co, Cu, Ni and Pb were extracted using EDTA. Both extractants were tested at different concentrations Ž10, 20 and 50 mM.. The cellular concentrations of K and Mg were also determined. 2.3.4. Experiment 4 Groups of 75 apices were firstly incubated for 30, 60, 90 and 120 min in a solution of CuCl 2 Ž3 mg ly1 .. For the S.E.T., NiCl 2 , EDTA and PbŽNO 3 . 2 , were used as extractants } all at a concentration of 20 mM. Another group of 75 F. antipyretica Hedw. apices was then incubated for 1 h in 1 l of a solution of Al Ž100 mg ly1 ., prepared from AlŽNO 3 . 3 ŽMerck, analytical grade.. To extract extracellular Al Žan element with a very high affinity for this cellular site., NiCl 2 Ž20 mM., EDTA Ž20 mM. and PbŽNO 3 . 2 Ž10, 20 and 50 mM. were used.
Fig. 1. Concentrations of extracellular metals Žmmol gy1 dry wt.. obtained after incubation of F. antipyretica in solutions of Cu and Na, as a function of the extractant used, ŽNiCl 2 and EDTA, respectively., and the number of consecutive washes Ž1, 2 or 3..
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3. Results and discussion 3.1. Experiment 1: efficacy of elution of extracellular metals In the original S.E.T. method, the extracellular metal fraction was extracted from lichens by two consecutive elutions with NiCl 2 . To determine if these washes were sufficient when using mosses, an experiment was carried out using NiCl 2 and EDTA to extract Na and Cu, respectively. In addition, the effect of the number of washes on the cellular concentrations of three essential elements ŽK, Mg and Ca. was determined. Potassium is mainly found intracellularly in a soluble form, and when the permeability of the membrane is
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altered it is rapidly released from the cell ŽBuck and Brown, 1979; Brown and Wells, 1990.. Magnesium is found in varying amounts both at extracellular sites Žfrom where it is easily displaced., and at intracellular sites ŽBrown, 1982; Brown and Wells, 1988.. Calcium, however, is found mainly in the cell wall ŽWells and Brown, 1990.. It can be seen from Fig. 1 that for both Cu and for Na, the greatest proportion of the extracellular metal is released during the first wash, a small amount is released during the second wash and a negligible amount during the third wash. However, the amount of extracellular K increased considerably with each wash and was almost certainly released from intracellular sites. Some of the K thus released is subsequently retained in
Fig. 2. Concentrations and cellular locations of Cu, Pb and Co, Žpreviously supplied to F. antipyretica at concentrations ranging from 0 to 200 mg ly1 . found after extraction with ŽA. EDTA and ŽB. NiCl 2 , both 20 mM. Cellular concentrations of K and Mg are also shown.
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the extracellular compartment ŽPuckett, 1976.. Larger amounts of Mg and Ca were also extracted, indicating that cationic exchange took place on renewal of the solution. Thus we conclude that a third wash is not a necessary step in the process. 3.2. Experiment 2: selection of extracellular metal extractant In the original S.E.T. NiCl 2 was used to extract metals accumulated at extracellular sites. However, Ni has a relatively low affinity for the anionic sites in the cell wall, and in order to remove strongly bound cations it must be used at concentrations so high that they would damage the plasma membrane ŽBrown and Wells, 1988.. Thus, a second experiment was carried out to evaluate the use of EDTA as an extractant. Results obtained for different metals, using both extractants, were compared. Each of the three metals chosen for this study ŽCo, Cu, and Pb., has a high affinity for extracellular sites ŽNieboer and Richardson, 1981.. The concentrations of metals released, using 20 mM EDTA and NiCl 2 , from extracellular and intracellular sites and also particulate metal from F. antipyretica, can be compared in Fig. 2. For Co, at each concentration supplied, there was slightly more extracellular metal extracted with EDTA than with NiCl 2 . There was also more extracellular K extracted using EDTA, whereas the amount of Mg extracted increased with the amount supplied, and the extractant used had no effect. In general, heavy metals are accumulated almost entirely at extracellular sites, including those which take part in cellular metabolism ŽSatake et al., 1988, 1989; Wells, 1988.. The results obtained for Co using EDTA appear to be the most realistic, although the differences between the two extractants were slight. As previously mentioned, K is generally found intracellularly and small amounts appear to have been lost from these sites when 20 mM EDTA was used. Part of the K released was transferred to extracellular anionic sites, increasing the concentration there. This indicates that EDTA alters the permeability of the
membrane. High concentrations of Co in the medium appear to produce the same effect. There were clear differences in the efficacy of the two extractants in releasing Cu Žmore so than with Co., with EDTA being the more effective. This compound also caused liberation of intracellular K, although with Cu Žat concentrations of 10 mg ly1 or above. NiCl 2 , had the same effect. This appears to indicate that, above this concentration Ž10 mg ly1 ., Cu causes damage to the cell membrane. The extraction of Pb by both of the compounds used was similar to that observed for Cu, although this metal altered the permeability of the cell membrane to a lesser extent. However, with EDTA, and Pb concentrations greater than 10 mg ly1 , a considerable amount of intracellular K was released. We conclude that in metals with a high affinity for extracellular binding sites, such as Co and in particular Cu and Pb, it is better to use EDTA rather than NiCl 2 as the extractant, as Ni produces an incomplete extraction of the extracellular metal. The only disadvantage of using EDTA is that it appears to alter the membrane permeability, thus causing the release of highly mobile elements such as K. Magnesium, however, does not seem to be affected, which suggests that the loss of intracellular heavy metals is slight. 3.3. Experiment 3: efficacy of extracellular metal extractant In previous experiments carried out in both lichens and mosses, 20 mM NiCl 2 was used in the S.E.T. to extract the extracellular metal fraction ŽBeckett and Brown, 1984; Wells and Brown, 1990.. When EDTA was found to be more suitable than NiCl 2 ŽExperiment 2., it was decided to carry out tests to find out if the procedure could be improved by varying the concentration of the extractant. It can be seen from Fig. 3 that the concentrations of Co, Cu, Ni and Pb extracted from each cellular site varied only slightly, even when the concentration of EDTA was increased. However, with Pb and, to a lesser extent with Cu and Ni,
M.D. Vazquez et al. r The Science of the Total En¨ ironment 241 (1999) 53]62 ´
there was a slight decrease in the total amount of metal recovered. There was also a decrease in the proportion of intracellular K when high concentrations of extractant were used. The distribution of Mg removed from F. antipyretica, was very similar in all cases. The concentrations of Cd, Zn and K extracted did not vary with the concentration of NiCl 2 used ŽFig. 4.. A slightly higher concentration of extracellular Mg was obtained by using 50 mM NiCl 2 . Thus there were no appreciable differences in the efficacy of either of the two extractants at
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different concentrations although the use of low concentrations of EDTA avoided unnecessary release of intracellular K. Concentrations of 20 mM can be used, although 10 mM is sufficient. 3.4. Experiment 4: use of Pb(NO3 )2 as an alternati¨ e extractant In the S.E.T., EDTA is effective in displacing metals that are strongly bound to extracellular sites, although as seen above, it can cause loss of intracellular K, especially during the extraction of
Fig. 3. Concentrations and cellular locations of Cu, Pb, Co and Ni Žeach previously supplied at 100 mg ly1 . extracted from F. antipyretica, as a function of the concentration of EDTA used Ž10, 20 and 50 mM.. Cellular concentrations of K and Mg are also shown.
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Fig. 4. Concentrations of extracellular, intracellular and particulate Zn and Cd Žeach previously supplied at 100 mg ly1 . extracted from F. antipyretica, as a function of the concentration of NiCl 2 used Ž10, 20 and 50 mM.. Cellular concentrations of K and Mg are also shown.
Cu. An experiment that compared the concentrations of Cu recovered when using 20 mM NiCl 2 , EDTA and PbŽNO 3 . 2 as extractants, was carried out. The three extractants were also tested for their ability to remove extracellular Al, an element with a very high affinity for extracellular
exchange sites. In this case the first two extractants were used at a concentration of 20 mM and the third at 10, 20 and 50 mM. The amounts of Cu and K extracted from the three cellular sites are shown in Fig. 5. It can be seen that, at each concentration of Cu supplied to
Fig. 5. Concentrations of extracellular, intracellular and particulate Cu and K obtained from F. antipyretica previously incubated in a solution of Cu Ž100 mg ly1 ., as a function of time of incubation Ž0, 30, 60, 90 and 120 min. and of the extractant used: ŽA. NiCl 2 , ŽB. EDTA and ŽC. PbŽNO 3 . 2 .
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the moss, EDTA was the most effective in extracting the extracellular metal. The differences between the other two extractants were small, although in general a smaller amount of total Cu was obtained using PbŽNO 3 . 2 , possibly because of the increase in plant dry weight due to the high molecular weight of Pb. Thus we conclude that EDTA extracted most extracellular Cu, followed by PbŽNO 3 . 2 and NiCl 2 . The distribution of K differed depending on the extractant used. The most marked differences were between EDTA, where almost all of the K extracted was extracellular, and Pb where almost all was intracellular. This result was interesting as Cu tends to cause large losses of intracellular K ŽPuckett, 1976; Vazquez et al., in press., as seen in Experiment 2. ´ Thus PbŽNO 3 . 2 would appear to have the characteristics of a good extractant, although at a different concentration to that used here. The most effective extraction of extracellular Al was achieved by using 20 mM EDTA or 50 mM PbŽNO 3 . 2 ŽFig. 6.. Nickel was almost totally ineffective at extracting Al and in addition, a smaller amount of total metal was obtained than with other extractants, for reasons that are not clear. Loss of intracellular K occurred with EDTA, but not with NiCl 2 or with PbŽNO 3 . 2 at any of the concentrations tested. Appreciable amounts of extracellular Mg were extracted with EDTA, less with NiCl 2 and less still with PbŽNO 3 . 2 , which suggests that with the latter compounds the extraction was not complete.
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To summarize, 50 mM PbŽNO 3 . 2 and 20 mM EDTA were equally effective in extracting extracellular Al. It appears then, that 50 mM PbŽNO 3 . 2 , is a suitable extractant for Al, and furthermore does not seem to alter the cell membrane. It would be interesting in future studies to test the ability of this compound to extract other metals. One disadvantage found was that it is not effective in extracting Mg from the cell wall, a factor that must be taken into consideration in studies involving the determination of the location of this metal.
4. Conclusions From this series of experiments which investigated different aspects of the S.E.T. applied to aquatic bryophytes, the most important conclusions are: 1. two consecutive washes with the chosen extractant are sufficient for the total removal of metal accumulated at extracellular sites; 2. 50 mM PbŽNO 3 . 2 was the most promising compound for use as an extractant of extracellular metal, as it brought about complete removal of extracellular metal without appearing to alter the cell membrane; 3. use of the other two extractants ŽEDTA and NiCl 2 . is, however, perfectly valid under certain circumstances, taking into consideration
Fig. 6. Concentrations and cellular location of Al, K and Mg extracted from F. antipyretica Žpreviously incubated in a solution of Al, 100 mg ly1 ., as a function of the extractant used and its concentration: ŽNi. 20 mM NiCl 2 ; ŽEDTA. 20 mM EDTA; ŽPb. 10, 20 and 50 mM PbŽNO 3 . 2 .
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their limitations. Good results are obtained with 10 or 20 mM NiCl 2 for metals with a low affinity for extracellular binding sites, and 10 or 20 mM EDTA is capable of extracting metals that are strongly bound to extracellular sites, although it causes release of mobile elements from within the cell.
Acknowledgements This study was carried out with funding from the Comision ´ Interministerial de Ciencia y TecŽ .. nologıa AMB94-0273 ´ References Beckett RP, Brown DH. The control of cadmium uptake in the lichen Genus Peltigera. J Exp Bot 1984;35:1071]1082. Brown DH. Mineral nutrition. In: Smith AJE, editor. Bryophyte ecology. London: Academic Press, 1982:383]443. Brown DH. Uptake of mineral elements and their use in pollution monitoring. In: Dyer FA, Duckett JG, editors. The experimental biology of bryophytes. London: Academic Press, 1984:229]255. Brown DH. The location of mineral elements in lichens; implications for metabolism. In: Cramer J, editor. Progress and problems in lichenology in the eighties. Berlin-Stuttgard, 1987:361]375. Brown DH, Avalos A. Chemical control of cadmium uptake by Peltigera. Symbiosis 1991;11:299]311. Brown DH, Beckett RP. Uptake and effect of cations on lichen metabolism. Lichenologist 1984;16:173]188. Brown DH, Buck GW. Cation contents of Acrocarpus and Pleurocarpus mosses growing in a strontium rich substratum. J Briol 1978a;10:199]209. Brown DH, Buck GW. Distribution of potassium, calcium and magnesium in the gametophyte and sporophyte generations of Funaria hygrometrica Hedw. Ann Bot 1978b;42:923]929. Brown DH, Buck GW. Desiccation effects and cation distribution in bryophytes. New Phytol 1979;82:115]125. Brown DH, Sidhu M. Heavy metal uptake, cellular location, and inhibition of moss growth. Cryptogamic Bot 1992;3: 82]85. Brown DH, Wells JM. Sequential elution technique for determining the cellular location of cations. In: Glime JM, editor. Proc Bryol Meth Workshop. Methods in Bryology, Hattori Bot Lab, Mainz, 1988:227]233. Brown DH, Wells JM. Physiological effects of heavy metals on the moss Rhytidiadelphus squarrosus. Ann Bot 1990;66: 641]647.
Buck GW, Brown DH. The effect of desiccation on cation location in lichens. Ann Bot 1979;44:265]277. Lopez J, Carballeira A. A comparative study of pigment ´ contents and response to stress in five species of aquatic bryophytes. Lindbergia 1990;15:188]193. Lopez J, Carballeira A. Interspecific differences in metal ´ bioaccumulation and plant]water concentration ratios in five aquatic bryophytes. Hydrobiologia 1993;263:95]107. Lopez J, Vazquez MD, Carballeira A. Stress responses and ´ metal exchange kinetics following transplants of the aquatic moss Fontinalis antipyretica. Freshwater Biol 1994;32: 185]198. Markert B. Sample preparation Žcleaning, drying, homogenisation. for trace element analysis in plant matrices. Sci Tot Environ 1995;176:45]61. Nieboer E, Richardson DHS. Lichens as monitors of atmospheric deposition. In: Eisenreich SJ, editor. Atmospheric pollutants and natural waters. Ann Arbor, Michigan: Ann Arbor Science, 1981:339]388. Nieboer E, Puckett KJ, Grace B. The uptake of nickel by Umbilicaria muhlenbergii: a physicochemical process. Can J Bot 1976;54:724]733. Puckett KJ. The effect of heavy metals on some aspects of lichen physiology. Can J Bot 1976;54:2695]2703. Richardson DHS, Nieboer E. The uptake of nickel ions by lichen thalli of the genera Umbilicaria and Peltigera. Lichenologist 1983;15:81]88. Satake K, Shibata K, Nishikawa M, Fuwa K. Copper accumulation and location in the moss Scopelophila cataractae. J Briol 1988;15:353]376. Satake K, Takamatsu T, Soma M et al. Lead accumulation and location in the shoots of the aquatic liverwort Scapania undulata ŽL.. Dum in stream water at Greenside Mine, England. Aquat Bot 1989;33:111]122. Vazquez MD, Fernandez JA, Lopez, J, Carballeira A. Effects ´ ´ ´ of water acidity and metal concentration on accumulation and within-plants distribution of metals in the aquatic bryophyte Fontinalis antipyretica Hedw. Water Air Soil Pollut, in press. Wehr JD, Empain A, Mouvet C, Say J, Whitton BA. Methods for processing aquatic mosses used as monitors of heavy metals. Water Res 1983;17:985]992. Wells JM. The role of the cell wall in metal uptake, redistribution and tolerance in the moss Rhytidiadelphus squarrosus. Doctor Th, Univ Bristol, 1988. Wells JM, Brown DH. Factors affecting the kinetics of intra and extracellular cadmium uptake by the moss Rhytidiadelphus squarrosus. New Phytol 1987;105:123]137. Wells JM, Brown D. Ionic control of intracellular and extracellular Cd uptake by the moss Rhytidiadelphus squarrosus ŽHedw. Warnst. New Phytol 1990;116:541]553. Whitton BA, Dıaz ´ BM. Chemistry and plants of streams and rivers with elevated zinc. In: Hemphill DD, editor. Proceedings of 14th Annual Conference on Trace Substances in Environmental Health, Missouri, Columbia, 1980:457]463.
Modification of the sequential elution technique for the extraction of heavy metals from bryophytes a,U M.D. Vazquez , J. Lopez ´ ´ b, A. Carballeira c a
Ecoloxıa, ´ E.P.S., Uni¨ ersidade de Santiago de Compostela, Campus Uni¨ ersitario, 27002 Lugo, Spain b Ecoloxıa, ´ Facultade de Ciencias, Uni¨ ersidade de Vigo, 36200 Vigo, Spain c Ecoloxıa, ´ Facultade de Bioloxıa, ´ Uni¨ ersidade de Santiago de Compostela, 15706, Santiago de Compostela, Spain Received 6 June 1999; accepted 3 July 1999
Abstract The sequential elution technique ŽS.E.T.. has frequently been used to determine the distribution of metals at different cellular sites in contaminated lichens and, to a lesser extent, in mosses. Here we evaluate certain aspects of the technique in order to improve the reliability of the results obtained when it is used on bryophytes. Various experiments were carried out using the aquatic moss Fontinalis antipyretica Hedw. Results confirmed that two consecutive washings are sufficient to ensure total extraction of the extracellular metal. In addition, the possibility of using new extractants to remove the metal that is accumulated at extracellular sites was investigated. Although NiCl 2 Žthe extractant originally used by the authors of the technique wBrown DH, Buck GW. J Briol 1978a;10:199]209; Brown DH, Buck GW. Ann Bot 1978b;42:923]929x. was adequate for metals with low affinity for the binding sites, EDTA and PbŽNO 3 . 2 were more efficient for the extraction for metals with a medium-high affinity for extracellular binding sites. The optimum concentrations of extractant were found to be 10 or 20 mM for NiCl 2 and EDTA, and 50 mM for PbŽNO. 2 . Q 1999 Elsevier Science B.V. All rights reserved. Keywords: Sequential elution technique; Fontinalis antipyretica Hedw; Bryophytes; Heavy metals; Affinity; Extractant; Extracellular metal; Intracellular metal
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Corresponding author. Tel.: q34-982-252231, ext. 23125; fax: q34-982-241835. . E-mail address: [email protected] ŽM.D. Vazquez ´
0048-9697r99r$ - see front matter Q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 8 - 9 6 9 7 Ž 9 9 . 0 0 3 3 7 - X
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1. Introduction The sequential elution technique ŽS.E.T.. was proposed by Brown and Buck Ž1978a,b, 1979. as a method to extract metals that accumulate at different cellular sites in lichens. Since then, the technique has frequently been applied in studies using lichens ŽBeckett and Brown, 1984; Brown, 1987; Brown and Wells, 1988; Brown and Avalos, 1991. and, less often, in studies using mosses ŽWells and Brown, 1987; Brown and Wells, 1990.. Here we describe a procedure that improves the use of lichens and mosses as biomonitors for heavy metal contamination and which involves the sequential removal of the elements that accumulate at discrete cellular sites. First, soluble, intercellular metal is discarded by rinsing the material with water. The extracellular metal, in the form of cations bound to anionic exchange sites in the cell wall and plasma membrane, is then extracted by displacement with another cation with a higher affinity for the exchange sites andror which is found at a higher concentration than the cation to be extracted ŽBrown, 1987; Brown and Avalos, 1991; Brown and Sidhu, 1992.. The soluble, intracellular metal is then extracted by using an acid that alters the permeability of the cell membranes, thus allowing release of the metals ŽBrown and Wells, 1988.. As the action of the acid alone may not be sufficient, the moss must first be oven dried to ensure that the cell membranes are ruptured and that intracellular material is released ŽBuck and Brown, 1979; Brown and Beckett, 1984; Brown, 1987.. An additional fraction consisting of metal bound to the remaining plant material, and which is extremely difficult to extract, is generically referred to as the particulate metal fraction. In order to release and quantify this metal, the organic material must first be digested. As well as particulate material, this fraction also includes insoluble intracellular elements such as metals incorporated into large polymeric molecules ŽBrown, 1984; Brown and Wells, 1988.. The technique has previously been used in mosses, although it is known that differences in the characteristics of the binding sites of the two types of organisms may influence the uptake of
metals ŽNieboer et al., 1976; Brown, 1982; Richardson and Nieboer, 1983; Wells, 1988.. Thus, it was not clear which type and concentration of displacing agent should be used to remove the extracellular metal, or what the optimum incubation times, etc., were. In order to ensure reliable results when using the technique in biomonitoring studies involving mosses, some of the steps involved were modified.
2. Materials and methods 2.1. Plant material The bryophyte Fontinalis antipyretica Hedw was chosen for this study as it is widely used in biomonitoring studies and is common in the NW Iberian Peninsula ŽLopez and Carballeira, 1990, ´ . 1993; Lopez et al., 1994 . ´ Moss samples were always collected from an uncontaminated stretch of a tributary of the river Sar ŽNW Iberian Peninsula .. Samples were rinsed in the river water to remove adherent particles before transporting them to the laboratory in plastic tanks full of the same river water Ž4 " 28C.. To standardize the experimental material, 2-cm apical segments were used ŽWhitton and Dıaz, ´ 1980; Wehr et al., 1983.. The samples were kept in river water in an incubation chamber Ž168C. with aeration until use. 2.2. Incubation of moss samples. Extraction and analysis of metals (general method) Although each of the experiments was carried out separately, the protocol followed was similar for each. Groups of F. antipyretica apices were firstly incubated for 1 h in the corresponding solutions of metals, depending on the experiment. All solutions were made up using distilled water and the samples were aerated appropriately. This step ensures saturation of extracellular sites in the moss and significant accumulation of metal inside the cells ŽWells and Brown, 1990.. Following incubation, the samples were rinsed to remove excess metal, and the metal accumulated at the different cellular sites was extracted
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using the S.E.T. procedure, the modifications proposed being tested in each experiment. The general technique used included the following steps: 2.2.1. First elution Between eight and 25 moss apical segments were added to 10 ml of extractant wEDTA, PbŽNO 3 . 2 or NiCl 2 ŽMerck, analytical grade.x, and then incubated for 45 min with constant shaking. The apical segments were then removed and the extracts stored in the fridge Ž4 " 18C. until measurements were made. 2.2.2. Second elution The moss samples were incubated under the same conditions as before, but only for 30 min. The total metal extracted during these first elutions was the extracellular metal fraction. An additional elution was carried out in one experiment to determine whether or not this step was necessary. 2.2.3. Drying After removing the moss samples from the extractant solutions, they were then dried in an oven at 508C, for 24 h, causing the cell membranes to rupture ŽBrown, 1987.. A temperature of 808C has often been used in previous studies, but as some elements may be volatile at this temperature, a range of 40]508C is considered to be preferable ŽMarkert, 1995.. 2.2.4. Third elution The moss samples were removed from the oven and the total dry weight determined. Samples were then further eluted in 10 ml of 1 M HNO3 ŽMerck, Pure grade., for 30 min with shaking. This step leaves the intracellular metal fraction in solution in the acid. 2.2.5. Digestion of residual material For each group of samples apical segments were placed in a glass tube Ž20 ml. with 4 ml of concentrated HNO3 . The tubes were stoppered with glass spheres, which acted like valves, and placed on a hot plate Ž1008C., until the plant material was completely digested. Distilled water was then added to a volume of 10 ml, and the
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tubes were centrifuged at 3000 rev. miny1 for 5 min. These solutions contained the residual metal fraction. Extractions were carried out in triplicate. To avoid interference during spectrometric measurements, 0.1 ml of a solution of Cs]La Ž100 000 ppm. were added to each extract. Lanthanum prevents the formation of refractive compounds during analysis of Mg and Cs acts as a suppressor of ionization ŽWells and Brown, 1987; Brown and Wells, 1990.. The concentration of metals in the extracts was determined by atomic absorption spectrometry ŽPerkin Elmer 2100 instrument . with an airacetylene flame. The exceptions to this were Al Žwhere N2 O was used as an oxidizing agent. and K Žmeasured by flame emission spectrometry.. Standards were made up in the corresponding extractants. The detection limits Žmg ly1 . were: Al Ž30., Ca and Na Ž1., Cd Ž2., Co Ž5., Cu Ž3., K Ž10., Mg Ž0.1., Ni Ž5.5., Pb Ž14. and Zn Ž0.9.. Blank values were always lower than detection limit. The efficiency of the analysis was tested using registered reference material ŽCommunity bureau of reference } BCR no. 61, Platihypnidium riparioides.. Recovery was higher than 95% for all metals except for Al and Na Ž70%.. 2.3. Incubation of moss samples. Extraction and analysis of metals (differentiated by experiment) Although the methodology used was very similar in the four experiments carried out, the variations due to the different aims of each are outlined as follows: 2.3.1. Experiment 1 A sample set of 30 apical segments of F. antipyretica were incubated in 1 l of a solution of Cu Ž3 mg ly1 ., prepared from CuCl 2 , and another set of 30 apical segments in a 20 mM EDTA disodium salt solution ŽC 10 H 14 N2 Na 2 O 8 ? 2H 2 O.. The accumulated metal ŽCu in the first case and Na in the second. was then extracted using EDTA and NiCl 2 Žboth 20 mM., respectively, followed by three consecutive washes Žof 45, 30 and 30 min. to extract the extracellular metal Žinstead of two as in the general S.E.T. method.. The concentra-
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tions of Cu, Na, K, Mg and Ca in the extracts obtained were determined.
2.3.2. Experiment 2 Solutions of different concentrations Ž0, 1, 10, 50, 100 and 200 mg ly1 . of three metals ŽCo, Cu and Pb. were prepared from their corresponding chlorides, except for Pb when nitrate was used. Sets of 75 apical segments were incubated in 1 l of each solution for 1 h, and then subjected to S.E.T. in order to remove the accumulated metals. With half of the samples, 20 mM NiCl 2 was used as the extractant and with the other half, 20 mM EDTA. In addition to the metal added, the concentrations of K and Mg in each of the extracts were measured.
2.3.3. Experiment 3 The apical segments were incubated with six different metals ŽCd, Co, Cu, Ni, Pb and Zn. at a
single concentration of 100 mg lyl . The metals with low affinity for extracellular sites ŽCd and Zn. were extracted using NiCl 2 , and Co, Cu, Ni and Pb were extracted using EDTA. Both extractants were tested at different concentrations Ž10, 20 and 50 mM.. The cellular concentrations of K and Mg were also determined. 2.3.4. Experiment 4 Groups of 75 apices were firstly incubated for 30, 60, 90 and 120 min in a solution of CuCl 2 Ž3 mg ly1 .. For the S.E.T., NiCl 2 , EDTA and PbŽNO 3 . 2 , were used as extractants } all at a concentration of 20 mM. Another group of 75 F. antipyretica Hedw. apices was then incubated for 1 h in 1 l of a solution of Al Ž100 mg ly1 ., prepared from AlŽNO 3 . 3 ŽMerck, analytical grade.. To extract extracellular Al Žan element with a very high affinity for this cellular site., NiCl 2 Ž20 mM., EDTA Ž20 mM. and PbŽNO 3 . 2 Ž10, 20 and 50 mM. were used.
Fig. 1. Concentrations of extracellular metals Žmmol gy1 dry wt.. obtained after incubation of F. antipyretica in solutions of Cu and Na, as a function of the extractant used, ŽNiCl 2 and EDTA, respectively., and the number of consecutive washes Ž1, 2 or 3..
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3. Results and discussion 3.1. Experiment 1: efficacy of elution of extracellular metals In the original S.E.T. method, the extracellular metal fraction was extracted from lichens by two consecutive elutions with NiCl 2 . To determine if these washes were sufficient when using mosses, an experiment was carried out using NiCl 2 and EDTA to extract Na and Cu, respectively. In addition, the effect of the number of washes on the cellular concentrations of three essential elements ŽK, Mg and Ca. was determined. Potassium is mainly found intracellularly in a soluble form, and when the permeability of the membrane is
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altered it is rapidly released from the cell ŽBuck and Brown, 1979; Brown and Wells, 1990.. Magnesium is found in varying amounts both at extracellular sites Žfrom where it is easily displaced., and at intracellular sites ŽBrown, 1982; Brown and Wells, 1988.. Calcium, however, is found mainly in the cell wall ŽWells and Brown, 1990.. It can be seen from Fig. 1 that for both Cu and for Na, the greatest proportion of the extracellular metal is released during the first wash, a small amount is released during the second wash and a negligible amount during the third wash. However, the amount of extracellular K increased considerably with each wash and was almost certainly released from intracellular sites. Some of the K thus released is subsequently retained in
Fig. 2. Concentrations and cellular locations of Cu, Pb and Co, Žpreviously supplied to F. antipyretica at concentrations ranging from 0 to 200 mg ly1 . found after extraction with ŽA. EDTA and ŽB. NiCl 2 , both 20 mM. Cellular concentrations of K and Mg are also shown.
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the extracellular compartment ŽPuckett, 1976.. Larger amounts of Mg and Ca were also extracted, indicating that cationic exchange took place on renewal of the solution. Thus we conclude that a third wash is not a necessary step in the process. 3.2. Experiment 2: selection of extracellular metal extractant In the original S.E.T. NiCl 2 was used to extract metals accumulated at extracellular sites. However, Ni has a relatively low affinity for the anionic sites in the cell wall, and in order to remove strongly bound cations it must be used at concentrations so high that they would damage the plasma membrane ŽBrown and Wells, 1988.. Thus, a second experiment was carried out to evaluate the use of EDTA as an extractant. Results obtained for different metals, using both extractants, were compared. Each of the three metals chosen for this study ŽCo, Cu, and Pb., has a high affinity for extracellular sites ŽNieboer and Richardson, 1981.. The concentrations of metals released, using 20 mM EDTA and NiCl 2 , from extracellular and intracellular sites and also particulate metal from F. antipyretica, can be compared in Fig. 2. For Co, at each concentration supplied, there was slightly more extracellular metal extracted with EDTA than with NiCl 2 . There was also more extracellular K extracted using EDTA, whereas the amount of Mg extracted increased with the amount supplied, and the extractant used had no effect. In general, heavy metals are accumulated almost entirely at extracellular sites, including those which take part in cellular metabolism ŽSatake et al., 1988, 1989; Wells, 1988.. The results obtained for Co using EDTA appear to be the most realistic, although the differences between the two extractants were slight. As previously mentioned, K is generally found intracellularly and small amounts appear to have been lost from these sites when 20 mM EDTA was used. Part of the K released was transferred to extracellular anionic sites, increasing the concentration there. This indicates that EDTA alters the permeability of the
membrane. High concentrations of Co in the medium appear to produce the same effect. There were clear differences in the efficacy of the two extractants in releasing Cu Žmore so than with Co., with EDTA being the more effective. This compound also caused liberation of intracellular K, although with Cu Žat concentrations of 10 mg ly1 or above. NiCl 2 , had the same effect. This appears to indicate that, above this concentration Ž10 mg ly1 ., Cu causes damage to the cell membrane. The extraction of Pb by both of the compounds used was similar to that observed for Cu, although this metal altered the permeability of the cell membrane to a lesser extent. However, with EDTA, and Pb concentrations greater than 10 mg ly1 , a considerable amount of intracellular K was released. We conclude that in metals with a high affinity for extracellular binding sites, such as Co and in particular Cu and Pb, it is better to use EDTA rather than NiCl 2 as the extractant, as Ni produces an incomplete extraction of the extracellular metal. The only disadvantage of using EDTA is that it appears to alter the membrane permeability, thus causing the release of highly mobile elements such as K. Magnesium, however, does not seem to be affected, which suggests that the loss of intracellular heavy metals is slight. 3.3. Experiment 3: efficacy of extracellular metal extractant In previous experiments carried out in both lichens and mosses, 20 mM NiCl 2 was used in the S.E.T. to extract the extracellular metal fraction ŽBeckett and Brown, 1984; Wells and Brown, 1990.. When EDTA was found to be more suitable than NiCl 2 ŽExperiment 2., it was decided to carry out tests to find out if the procedure could be improved by varying the concentration of the extractant. It can be seen from Fig. 3 that the concentrations of Co, Cu, Ni and Pb extracted from each cellular site varied only slightly, even when the concentration of EDTA was increased. However, with Pb and, to a lesser extent with Cu and Ni,
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there was a slight decrease in the total amount of metal recovered. There was also a decrease in the proportion of intracellular K when high concentrations of extractant were used. The distribution of Mg removed from F. antipyretica, was very similar in all cases. The concentrations of Cd, Zn and K extracted did not vary with the concentration of NiCl 2 used ŽFig. 4.. A slightly higher concentration of extracellular Mg was obtained by using 50 mM NiCl 2 . Thus there were no appreciable differences in the efficacy of either of the two extractants at
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different concentrations although the use of low concentrations of EDTA avoided unnecessary release of intracellular K. Concentrations of 20 mM can be used, although 10 mM is sufficient. 3.4. Experiment 4: use of Pb(NO3 )2 as an alternati¨ e extractant In the S.E.T., EDTA is effective in displacing metals that are strongly bound to extracellular sites, although as seen above, it can cause loss of intracellular K, especially during the extraction of
Fig. 3. Concentrations and cellular locations of Cu, Pb, Co and Ni Žeach previously supplied at 100 mg ly1 . extracted from F. antipyretica, as a function of the concentration of EDTA used Ž10, 20 and 50 mM.. Cellular concentrations of K and Mg are also shown.
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Fig. 4. Concentrations of extracellular, intracellular and particulate Zn and Cd Žeach previously supplied at 100 mg ly1 . extracted from F. antipyretica, as a function of the concentration of NiCl 2 used Ž10, 20 and 50 mM.. Cellular concentrations of K and Mg are also shown.
Cu. An experiment that compared the concentrations of Cu recovered when using 20 mM NiCl 2 , EDTA and PbŽNO 3 . 2 as extractants, was carried out. The three extractants were also tested for their ability to remove extracellular Al, an element with a very high affinity for extracellular
exchange sites. In this case the first two extractants were used at a concentration of 20 mM and the third at 10, 20 and 50 mM. The amounts of Cu and K extracted from the three cellular sites are shown in Fig. 5. It can be seen that, at each concentration of Cu supplied to
Fig. 5. Concentrations of extracellular, intracellular and particulate Cu and K obtained from F. antipyretica previously incubated in a solution of Cu Ž100 mg ly1 ., as a function of time of incubation Ž0, 30, 60, 90 and 120 min. and of the extractant used: ŽA. NiCl 2 , ŽB. EDTA and ŽC. PbŽNO 3 . 2 .
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the moss, EDTA was the most effective in extracting the extracellular metal. The differences between the other two extractants were small, although in general a smaller amount of total Cu was obtained using PbŽNO 3 . 2 , possibly because of the increase in plant dry weight due to the high molecular weight of Pb. Thus we conclude that EDTA extracted most extracellular Cu, followed by PbŽNO 3 . 2 and NiCl 2 . The distribution of K differed depending on the extractant used. The most marked differences were between EDTA, where almost all of the K extracted was extracellular, and Pb where almost all was intracellular. This result was interesting as Cu tends to cause large losses of intracellular K ŽPuckett, 1976; Vazquez et al., in press., as seen in Experiment 2. ´ Thus PbŽNO 3 . 2 would appear to have the characteristics of a good extractant, although at a different concentration to that used here. The most effective extraction of extracellular Al was achieved by using 20 mM EDTA or 50 mM PbŽNO 3 . 2 ŽFig. 6.. Nickel was almost totally ineffective at extracting Al and in addition, a smaller amount of total metal was obtained than with other extractants, for reasons that are not clear. Loss of intracellular K occurred with EDTA, but not with NiCl 2 or with PbŽNO 3 . 2 at any of the concentrations tested. Appreciable amounts of extracellular Mg were extracted with EDTA, less with NiCl 2 and less still with PbŽNO 3 . 2 , which suggests that with the latter compounds the extraction was not complete.
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To summarize, 50 mM PbŽNO 3 . 2 and 20 mM EDTA were equally effective in extracting extracellular Al. It appears then, that 50 mM PbŽNO 3 . 2 , is a suitable extractant for Al, and furthermore does not seem to alter the cell membrane. It would be interesting in future studies to test the ability of this compound to extract other metals. One disadvantage found was that it is not effective in extracting Mg from the cell wall, a factor that must be taken into consideration in studies involving the determination of the location of this metal.
4. Conclusions From this series of experiments which investigated different aspects of the S.E.T. applied to aquatic bryophytes, the most important conclusions are: 1. two consecutive washes with the chosen extractant are sufficient for the total removal of metal accumulated at extracellular sites; 2. 50 mM PbŽNO 3 . 2 was the most promising compound for use as an extractant of extracellular metal, as it brought about complete removal of extracellular metal without appearing to alter the cell membrane; 3. use of the other two extractants ŽEDTA and NiCl 2 . is, however, perfectly valid under certain circumstances, taking into consideration
Fig. 6. Concentrations and cellular location of Al, K and Mg extracted from F. antipyretica Žpreviously incubated in a solution of Al, 100 mg ly1 ., as a function of the extractant used and its concentration: ŽNi. 20 mM NiCl 2 ; ŽEDTA. 20 mM EDTA; ŽPb. 10, 20 and 50 mM PbŽNO 3 . 2 .
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their limitations. Good results are obtained with 10 or 20 mM NiCl 2 for metals with a low affinity for extracellular binding sites, and 10 or 20 mM EDTA is capable of extracting metals that are strongly bound to extracellular sites, although it causes release of mobile elements from within the cell.
Acknowledgements This study was carried out with funding from the Comision ´ Interministerial de Ciencia y TecŽ .. nologıa AMB94-0273 ´ References Beckett RP, Brown DH. The control of cadmium uptake in the lichen Genus Peltigera. J Exp Bot 1984;35:1071]1082. Brown DH. Mineral nutrition. In: Smith AJE, editor. Bryophyte ecology. London: Academic Press, 1982:383]443. Brown DH. Uptake of mineral elements and their use in pollution monitoring. In: Dyer FA, Duckett JG, editors. The experimental biology of bryophytes. London: Academic Press, 1984:229]255. Brown DH. The location of mineral elements in lichens; implications for metabolism. In: Cramer J, editor. Progress and problems in lichenology in the eighties. Berlin-Stuttgard, 1987:361]375. Brown DH, Avalos A. Chemical control of cadmium uptake by Peltigera. Symbiosis 1991;11:299]311. Brown DH, Beckett RP. Uptake and effect of cations on lichen metabolism. Lichenologist 1984;16:173]188. Brown DH, Buck GW. Cation contents of Acrocarpus and Pleurocarpus mosses growing in a strontium rich substratum. J Briol 1978a;10:199]209. Brown DH, Buck GW. Distribution of potassium, calcium and magnesium in the gametophyte and sporophyte generations of Funaria hygrometrica Hedw. Ann Bot 1978b;42:923]929. Brown DH, Buck GW. Desiccation effects and cation distribution in bryophytes. New Phytol 1979;82:115]125. Brown DH, Sidhu M. Heavy metal uptake, cellular location, and inhibition of moss growth. Cryptogamic Bot 1992;3: 82]85. Brown DH, Wells JM. Sequential elution technique for determining the cellular location of cations. In: Glime JM, editor. Proc Bryol Meth Workshop. Methods in Bryology, Hattori Bot Lab, Mainz, 1988:227]233. Brown DH, Wells JM. Physiological effects of heavy metals on the moss Rhytidiadelphus squarrosus. Ann Bot 1990;66: 641]647.
Buck GW, Brown DH. The effect of desiccation on cation location in lichens. Ann Bot 1979;44:265]277. Lopez J, Carballeira A. A comparative study of pigment ´ contents and response to stress in five species of aquatic bryophytes. Lindbergia 1990;15:188]193. Lopez J, Carballeira A. Interspecific differences in metal ´ bioaccumulation and plant]water concentration ratios in five aquatic bryophytes. Hydrobiologia 1993;263:95]107. Lopez J, Vazquez MD, Carballeira A. Stress responses and ´ metal exchange kinetics following transplants of the aquatic moss Fontinalis antipyretica. Freshwater Biol 1994;32: 185]198. Markert B. Sample preparation Žcleaning, drying, homogenisation. for trace element analysis in plant matrices. Sci Tot Environ 1995;176:45]61. Nieboer E, Richardson DHS. Lichens as monitors of atmospheric deposition. In: Eisenreich SJ, editor. Atmospheric pollutants and natural waters. Ann Arbor, Michigan: Ann Arbor Science, 1981:339]388. Nieboer E, Puckett KJ, Grace B. The uptake of nickel by Umbilicaria muhlenbergii: a physicochemical process. Can J Bot 1976;54:724]733. Puckett KJ. The effect of heavy metals on some aspects of lichen physiology. Can J Bot 1976;54:2695]2703. Richardson DHS, Nieboer E. The uptake of nickel ions by lichen thalli of the genera Umbilicaria and Peltigera. Lichenologist 1983;15:81]88. Satake K, Shibata K, Nishikawa M, Fuwa K. Copper accumulation and location in the moss Scopelophila cataractae. J Briol 1988;15:353]376. Satake K, Takamatsu T, Soma M et al. Lead accumulation and location in the shoots of the aquatic liverwort Scapania undulata ŽL.. Dum in stream water at Greenside Mine, England. Aquat Bot 1989;33:111]122. Vazquez MD, Fernandez JA, Lopez, J, Carballeira A. Effects ´ ´ ´ of water acidity and metal concentration on accumulation and within-plants distribution of metals in the aquatic bryophyte Fontinalis antipyretica Hedw. Water Air Soil Pollut, in press. Wehr JD, Empain A, Mouvet C, Say J, Whitton BA. Methods for processing aquatic mosses used as monitors of heavy metals. Water Res 1983;17:985]992. Wells JM. The role of the cell wall in metal uptake, redistribution and tolerance in the moss Rhytidiadelphus squarrosus. Doctor Th, Univ Bristol, 1988. Wells JM, Brown DH. Factors affecting the kinetics of intra and extracellular cadmium uptake by the moss Rhytidiadelphus squarrosus. New Phytol 1987;105:123]137. Wells JM, Brown D. Ionic control of intracellular and extracellular Cd uptake by the moss Rhytidiadelphus squarrosus ŽHedw. Warnst. New Phytol 1990;116:541]553. Whitton BA, Dıaz ´ BM. Chemistry and plants of streams and rivers with elevated zinc. In: Hemphill DD, editor. Proceedings of 14th Annual Conference on Trace Substances in Environmental Health, Missouri, Columbia, 1980:457]463.