Lead accumulation and location in the shoots of the aquatic liverwort Scapania undulata (L.) Dum. in stream water at greenside mine, England

Aquatic Botany, 33 (1989) 111-122

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Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

L E A D A C C U M U L A T I O N A N D L O C A T I O N IN T H E S H O O T S OF T H E AQUATIC L I V E R W O R T S C A P A N I A U N D U L A T A (L.) D U M . IN S T R E A M W A T E R AT G R E E N S I D E M I N E , E N G L A N D

KENICHI SATAKE', TAKEJIRO TAKAMATSU1, MASAYUKI SOMA 1, KEIKO SHIBATA', MASATAKA NISHIKAWA1, PHILIP J. SAY2 and BRIAN A. WHITTON '3

'National Institute for Environmental Studies, Onogawa 16-2, Tsukuba, Ibaraki 305 (Japan) eNorthern Environmental Consultants, Medomsley Road, Consett, Co. Durham DH8 6SS (Gt. Britain) ~Department of Botany, University of Durham, South Road, Durham DH1 3LE (Gt. Britain) (Accepted for publication 25 October, 1988)

ABSTRACT Satake, K., Takamatsu, T., Soma, M., Shibata, K., Nishikawa, M., Say, P.J. and Whitton, B.A., 1989. Lead accumulation and location in the shoots of the aquatic liverwort Scapania undulata (L.) Dum. in stream water at Greenside Mine, England. Aquat. Bot., 33: 111-122. Lead accumulation in the shoots of the aquatic liverwort Scapania undulata (L.) Dum. collected from a stream at an abandoned lead mine in England was studied with respect to its concentration, localization and chemical forms in the cells. The concentration of Pb in the stream water was 0.02 mg 1-1, and that of Pb in the shoots ranged from 0.7 to 2.4% Pb on a dry weight basis, giving an enrichment ratio of 3.5 X 105-1 '2 >
INTRODUCTION

Scapania undulata (L.) Dum. is an aquatic liverwort of both acidic and nonacidic waters in temperate regions (Inoue, 1962; Iwatsuki and Inoue, 1972; Light, 1975; McLean and Jones, 1975; Hong, 1979, 1980; Satake and Shibata, 1986). Many examples are known of waters where S. undulata is present and which are polluted by heavy metals; recent studies on the chemical composition of S. undulata have shown high concentrations of Zn, Cd, Pb and Hg in the shoots of this liverwort collected from such heavy-metal-rich areas. For instance, concentrations of Pb, Zn and Hg in shoots can be as high as 1.6% Pb, 0.76% Zn and 0.24% Hg on a dry weight basis (Burton and Peterson, 1979a; 0304-3770/89/$03.50

@) 1989 Elsevier Science Publishers B.V.

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Whitton et al., 1982; Satake et al., 1983 ). These results show the tolerance and a marked ability of S. undulata to accumulate heavy metals. A few studies have been reported on the localization of these metals in the cells of bryophytes. Hg accumulation in the cell wall, as fine particles of an HgS compound, has been reported for the aquatic liverwort Jungermannia vulcanicola Steph. (Satake and Miyasaka, 1984a), and Pb accumulation in the nucleus and cell wall as electron-dense particles for the moss Rhytidiadelphus squarrosus (Hedw.) Warnst. (Skaar et al., 1973 ). In addition to these studies, localization of Pb at an extracellular site of Grimmia doniana Sm. has been shown (Brown and Bates, 1972) and that of Zn in the cell wall of Scapania undulata has been suggested (Burton and Peterson, 1979b). Moreover, the importance of ion exchange, complexing or chelation with specific organic groups has been described (Rfihling and Tyler, 1973). These reports suggest possible ways in which the distribution, localization and chemical forms of the heavy metals accumulated in shoots of bryophytes may occur: ( 1 ) inorganic compound (s), which may be sulphide or other insoluble compound (s), in the cell wall and/or nucleus, forming particles; ( 2 ) heavy metal combined with organic matter, occurring in the cell wall or other cell components. The aim of the present study was to clarify these aspects for Pb accumulated by S. undulata. A preliminary survey of the chemical composition of S. undulata, collected from a stream located at a disused lead mine in England, using a wavelengthdispersive X-ray fluorescence spectrometer (WDXRF) showed a high concentration of Pb in the shoots. Other recently developed analytical techniques for observations of cells and determination and characterization of chemical species in their biological matrix, such as transmission electron microscopy with X-ray microanalysis, inductively coupled plasma atomic emission spectrometry, X-ray photoelectron spectrometry and ion chromatography, were also available for this study. The Pb-rich shoots of this S. undulata population were therefore selected for critical study, using a range of different analytical methods. SAMPLING SITE AND MATERIALS

Water and shoots of Scapania undulata were collected from a stream in England, a tributary of Glenridding Beck (pH 6.2-6.5), which passes through Greenside mine, Cumbria. The mine had been closed in 1962. Scaparia undulata occurred at various locations within this stream and the ores of lead were found around the stream. Samples were collected on 3 September 1981 and 12 September 1985; only shoots on submerged stones in the margin of the stream were collected on the first occasion, and water and shoots on the submerged stones at the central part of the stream on the second occasion.

113 SAMPLE PREPARATIONAND ANALYSIS

Water Two sets of stream water, one passed through a membrane filter (Sartorius Minisart NML, pore size 0.45 ttm) at the site and one unfiltered (filtered after 1 week from collection), were collected in Teflon bottles that had been immersed in concentrated nitric acid for more than 2 weeks and washed thoroughly with purified water prepared with the Milli-Q water purification system (consisting of a charcoal fiber column and ion-exchange resin columns for demineralization of water ) (Millipore Co. ). Lead in the water was analyzed by Zeeman atomic absorption spectrophotometry (AAS) (Hitachi 180-80), a method with high sensitivity for Pb.

Shoots of S. undulata Shoots of S. undulata were collected by hand and washed several times in water at the sampling site. The samples were transferred from the field to the laboratory in polyethylene bags (Seinichi unipack E-8). Each shoot was then separated from the colony and washed carefully with purified water to remove attached inorganic and organic particles. The shoots were cut at 0.5- and/or 1-cm intervals from the tip to the base and then dried at 60 °C for at least 24 h. The dried shoots were digested in Teflon bombs (Okamoto and Fuwa, 1984 ) with concentrated nitric acid for 3 h at 140°C. The digested samples were diluted to about one-tenth with purified water and passed through a membrane filter (Sartorius Minisalt NML, pore size 0.45 #m). Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used for the determination of Pb and P in the digested shoots. X-ray photoelectron spectra (XP-spectra) were recorded on a Vacuum Generators ESCALAB 5 apparatus equipped with a 150 ° spherical energy analyzer. The basal parts of the dried shoots were mounted on the sample holder with double-sided adhesive tape, and X-ray photoelectron spectra were recorded on the apparatus. The spectra of powdered whole samples were also recorded. Photoelectron binding energies were determined relative to Au 4f7/2 binding energy (83.8 eV of a gold film vacuum-evaporated onto the sample). The C ls binding energy of S. undulata thus determined was 284.8 eV, which was representative of C in hydrocarbon skeletons and could be used as an internal standard. Uncertainty in the determination of binding energies was within + 0.2 eV. Samples for the characterization of sulphur compounds were stored in a freezer. They were defrosted when required, washed thoroughly with purified water in an ultrasonic washer and dried at 50 °C for 1 day. The sulphur compounds, (1) sulphate, (2) sulphide, (3) elemental S, (4) organic S and (5)

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total S were determined using an ion chromatograph (Dionex Model 10) after conversion to sulphate as shown below: (1) Sulphate in the samples was extracted into 0.1 M Na2CO3 solution. (2) Sulphides: acid-soluble sulphides including PbS were reduced to H2S with 1 M HC1, sulphides other than FeS2 were reduced to H2S with 9 M HCI containing NH2OH, and all sulphides with a mixture of SnC12, HCI and granulated zinc. The evolved H2S gas was trapped and oxidized to sulphate in 0.1 M NaOH solution with H202. The recovery of sulphide using this method was tested for PbS and HgS and found to be about 98%. (3) Elemental S was extracted from the shoots with cyclohexane, then reduced to H2S and finally oxidized to sulphate. (4) Total sulphur in shoots was determined after digestion in Teflon bombs with H202 solution. (5) Organic S, which includes metallothionein-type protein was calculated according to the following relationship: organic S =total S - (sulphate S + sulphide S ), since the analytical values for sulphide S (by Zn-Sn-HC1 reductant) also included those for elemental S, sulphite S and thiosulphate S. Details of the procedure including the recovery test for S-species will be described elsewhere by Takamatsu. The undried shoots of S. undulata were used for observation using a transmission electron microscope (TEM) (JEOL JEM 100C) and analyses using an energy-dispersive X-ray microanalyzer (XMA) (Kevex 7077QJ). The procedure for shoot sample preparation was as follows. Shoots used for observation of cell components were fixed for 2 h at room temperature in 4% glutaraldehyde and washed in 0.1 M phosphate buffer for 30 min. The shoots were then post-fixed in 2% osmic acid for 2 h and washed in 0.1 M phosphate buffer for 1.5 h. Dehydration was done in a graded series of 30, 50, 70, 80, 90, 95 and 100% ethanol. After treatment with a solution of acetone, acetone/resin mixture and resin, the tissues were embedded in Spurr's resin. The unstained 100150-nm thick sections for elemental analyses were mounted on copper grids with a covering of carbon, and analysed using an energy-dispersive X-ray microanalyzer at 80 kV with a probe current of 1-3 × 10- lo A and 10 and/or 20 eV per channel. RESULTS

The concentrations of lead in the shoots of S. undulata from Greenside mine collected in September 1981 and 1985 are summarized in Table 1. The concentration of Pb in the shoots increased from the tip toward the base and attained a maximum of 2.4% in the basal part of the shoot (3-4 cm). On the other hand, the concentration of Pb in water was about 0.02 mg l-1 (1985), so that the enrichment ratio between Pb in the shoot and that in the water was 3.5 × 105 at the shoot tip and 1.2 × 106 at shoot base. The correlation coefficient between the distance from the top of the shoot and the concentration of Pb in the shoot calculated for the samples in 1985

115 TABLE1 The concentrations of Pb and P in the shoots of Scapania undulata sampled from a tributary of Glenridding Beck at Greenside mine on 12 September 1985 and the concentration of Pb in water passed through a membrane filter at the site. The table also shows the concentration of Pb in the shoots of S. undulata sampled from the same site on 3 September 1981 Shoot

Scapania undulata (~g g- 1) 12 September 1985 Pb (/~g g-l, n=3) P (l~g g-l, n=3)

0.0-0.5cm

0.5-1.0cm

1.0-2.0cm

2.0-3.0cm

3.0-4.0cm

7180___ 5 537±17

9990_+515 348_+ 11

14250_+550 400_+ 37

19000±250 447± 10

23500_+550 539_+ 67

13600

15300

17800

3 September 1981 Pb (/~g g-l, n = l )

61401

Water (mg 1-1 ) 12 September 1985 Pb (n=2)

0.02

IValue for 0.0-1.0 c m shoot.

Pb 4f7/2

bSO 4

h,ndulata

'bS

140

135 Binding

Energy

145 (eV)

Fig. 1. X-ray photoelectron spectra of P b compound in the shoots of Scapania undulata from Greenside mine, together with the spectra for the reagents P b S 0 4 and PbS.

116 TABLE 2 Concentrations of S-species in water and shoots of Scapania undulata sampled from a tributary of Glenridding Beck at Greenside mine on 12 September 1985 Total-S

S04-S

S°-S

$2--S

Org-S

Zn-Sn-HCl

Scapaniaundulata 2220_+1901 n Water

4

78_+1 3 1.2 ~

22___7 167_+9 3 6 . .

.

NH2OH-HC1

1 M HCI

39_+8 4

<5 4

1980

.

l#g g-1. 2mg 1 1. n = number of determination.

was 0.996 (P < 0.01 ) and the regression line was: y-- 6080 + 5050x (y, concentration of Pb in the shoot (zg g - l ) ; x, shoot length (cm)). Figure 1 shows the spectra obtained by X-ray photoelectron spectrometry (XPS) analysis, comparing the Pb 4f spectrum of the shoot of S. undulata with those of PbSO4 and PbS. Both the 1981 and 1985 samples gave qualitatively the same spectrum. The binding energy for the Pb 4f7/2 peak of S. undulata was 138.5 eV and those of PbS04 and PbS were 139.2 and 137.5 eV, respectively. Table 2 shows the results of S-speciation in the shoot ofS. undulata. Organic S was the dominant form (90% of total S). Electron micrographs of the cells at the shoot tip (0-1 cm) and the shoot base (3-4 cm) are shown in Figs. 2 and 3, respectively. Figure 2 shows the cell components and Fig. 3 shows the cell components together with bacteria which have invaded the cell wall and formed tunnels. There were no large electron-dense particles in the cell wall or nucleus in the cells at the tip and/or basal part of the shoots. XMA spectra of the cell wall and nucleus at the shoot base are shown in Fig. 4. The spectrum from the cell wall in this region showed peaks for Pb, Fe, Ca and P; XMA spectra of cell components within the cell membrane showed no Pb peak. DISCUSSION

The results obtained by ICP-AES confirmed the marked ability of S. undulata to accumulate Pb in the shoots. The shoot of S. undulata grows from the tip, therefore the shoot base is older than the shoot tip. The linear relationship between the distance from the tip of the shoot and the content of Pb suggests relatively regular growth of this liverwort and a relatively constant concentration of Pb in the water at Glenridding Beck, although seasonal or temporal changes in the concentration of Pb in the water have not been mon-

117

Fig. 2a,b,c. Transmission electron micrographs of the cell components at the shoot tip (0-1 cm) of Scapania undulata. Plate (a) shows cell wall (CW), plasma membrane (pm), nucleus {N), nuclear envelope (ne), nucleolus (no), and mitochondria (M). Plate (b) shows chloroplast (C). Plate (c) shows oil body (O) (unit bar length: 1 #m).

118

Fig. 3a,b,c,d. Transmission electron micrographs of the shoot base (3-4 cm) of Scapania undulata. Plates (a) and (b) show cell wall (CW) with lamina structure, nucleus (N), oil body (O) and chloroplast (C). Plates (c) and (d) show bacteria (B) that have invaded the cell wall (unit bar length: 1/~m).

119

Q

Fe

r

p•bMa,•

Fe

',"1

,,

Ca

..... .,~ t tl I Ii ftlITII,f ::i I;!]l l;!!l I;!!lI::: lii! I.I

(Os) (Cu)

P.b ~ L=

,,,, o,% ,.'"., I=..=,

Pb ~ k/~ ;:::' ........... I:1 ,4 ,,'" I::i Ell.,

b

I

r' tl

.....

(Os) (Cu)

(Os)

., "" E ,:i;

" ..... ~l ,::

4 ,=, " I.:::I..-..=...= .::.

Fig. 4a,b. XMA spectrum and transmission electron micrograph of the cell wall (a) (XI: the analyzed point) and XMA spectrum of the nucleus (b) at shoot base of Scapania undulata. The peaks of elements (Cu, Os) shown in parentheses are not included in the original specimen.

itored at the site. The difference in the concentration of Pb in the shoots between samples obtained in 1981 and those obtained in 1985 seems to be based on differences of microenvironment in the same stream, thus affecting the accumulation rate. However, the high value for Pb at the intercept of the regression line despite this linear relationship indicates that there are two steps involved in Pb accumulation in the shoot.

120 TEM-XMA studies of S. undulata also showed accumulation of Pb in the cells, especially in the thick walls. Taking into account the detection limit for Pb in XMA analysis, the fact that the concentration of Pb in cell walls in the basal part of the shoot was more than 1%, and the fact that some cells in the basal part had lost their cytoplasm, the main Pb compound(s) are probably present in the cell wall. The spectra obtained by XPS allow two conclusions to be made on the basis of comparison with data on the chemical shift in the Pb 4f binding energy for various lead compounds (Morgan and van Wazer, 1973; Nefedov et al., 1979; Pederson, 1982). First, since the Pb 4f spectrum of S. undulata showed a well resolved Pb 4f7/2 and Pb 4f5/2 doublet and the line width of each component was comparable to those of the pure substances, the bonding state of Pb is such that it does not give rise to a large distribution of the photoelectron binding energy. Second, the differences in binding energy among PbS, PbS04 and the Pb compound(s) in the shoot ofS. undulata show that PbS and PbSO4 are not the main compounds in the shoot, although quantification of any contributions from these Pb compounds is difficult. The binding energies of many Pb compounds, such as lead phosphate, lie between those of PbS and PbSO4. However, the concentration of P showed a low correlation with that of Pb (r=0.295), suggesting that the amount of lead phosphate does not exceed 20% of total Pb, even if most of the P in the shoot is combined with Pb. Details of the characterization of Pb in several mosses by XPS have been described elsewhere (Soma et al., 1988). As in the present case, Pohlia ludwigii (Spreng.) Broth. and Pellia endiuiifolia (Dicks.) Dum. were found to exhibit well resolved Pb 4f lines and the Pb 4fl/2 binding energies lay between those of PbSO4 and PbS, although they were slightly higher than that of S. undulata. The speciation of S in the shoot of S. undulata showed a low contribution of PbS as Pb compound because PbS would have been completely dissolved with 1 M HC1 solution owing to its relatively large solubility product of PbS (Kso = 10-26.6- -29.4; Sillen and Martell, 1964). Although 169 and 39 #g g-1 S were vaporized from the sample as H2S in the Zn-SnC12-HC1 and NH2OH-HC1 mixtures, respectively, these amounts may have arisen from organic S decomposed with the acid and reductant mixtures. Since a large portion of the water-soluble sulphate had been removed from the sample by ultrasonic washing, the analyzed sulphate-S level (78/~g g-1) suggests the presence of PbSO4, and is equivalent to 505/~g g-1 Pb. These results show that PbS as well as PbS04 is unlikely to be the major form in which Pb compounds occur in the shoots. This is consistent with the XPS result, provided that the chemical form of Pb existing within the depth of XPS ( < 10 nm) is representative of all Pb species. The chemical form of the main Pb compound(s) in the cell wall is unlikely to be inorganic, but it would be expected to combine with organic matter, including organic S compounds. The findings that (1) there were no large electron-dense particles in the cells and (2) Pb was only detected in the cell wall by XMA analysis, in the shoots

121

of S. undulata from the stream at Greenside mine suggest that Pb is uniformly distributed in the cell wall. There is some possibility that Pb might be distributed in other cell components, although, the content might be less than the detection limit of XMA analysis. These findings are in contrast with those for Rhytidiadelphus squarrosus obtained from roadsides, in which Pb was found to be accumulated in the nucleus as electron-dense particles (Skaar et al., 1973; Gullvfig et al., 1974). One of our results also contrasts with findings for Jungermannia vulcanicola from an acidic stream, which contained a Hg-S compound in the cell wall as electron-dense particles (Satake and Miyasaka, 1984 ), although the other result is not contradictory. The present findings are also different from those of a laboratory experiment on Pb accumulation by the aquatic angiosperm Potamogeton pectinatus L., which contained electron-dense particles in the cell wall (Sharpe and Denny, 1976). The chemical and physical forms of heavy metals accumulated in bryophyte shoots thus appear to differ according to species, the particular heavy metal and the concentrations of metals at the site of bryophyte growth. ACKNOWLEDGEMENTS

We thank Dr. K. Okamoto for AAS analysis of the samples and Mr. R.G. Hardy for help with WDXRF analysis. This research was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Japan, and financial help from the British Council.

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122 Light, J.J., 1975. Clear lakes and aquatic bryophytes in the mountains of Scotland. J. Ecol., 63: 937-944. McLean, R.O. and Jones, A.K., 1975. Studies of tolerance to heavy metals in the flora of the rivers Ystwyth and Clarach, Wales. Freshwater Biol., 5: 431-444. Morgan, W.E. and Van Wazer, J.R., 1973. Binding energy shifts in the X-ray photoelectron spectra of a series of related group IV-a compounds. J. Phys. Chem., 77: 964-969. Nefedov, N.I., SalOn, Ya.V. and Keller, H., 1979. X-ray electron study of lead and mercury compounds. Zh. Neorg. Khim., 24:2564-2566 (Russian J. Inorg. Chem., 24:1425-1426 {1979) ). Okamoto, K. and Fuwa, K. (1984). Low-contamination digestion bomb method using a teflon double vessel for biological materials. Anal. Chem., 56: 1758-1760. Pederson, L.R., 1982. Two dimensional chemical-state plot for lead using XPS. J. Electron Spectrosc. Relat. Phenom., 28: 203-209. R~ihling, A. and Tyler, G., 1973. Heavy metal deposition in Scandinavia. Water, Air, Soil Pollut., 2: 445-455. Satake, K. and Miyasaka, K., 1984a. Evidence of high mercury accumulation in the cell wall of the liverwort Jungermannia vulcanicola Steph. to form particles of a mercury-sulfur compound. J. Bryol., 13: 101-105. Satake, K. and Miyasaka, K., 1984b. Discovery of bacteria in the cell wall of the aquatic liverwort Jungermannia vulcanicola Steph. in an acid stream with pH 4.2-4.6. J. Bryol., 13: 277-279. Satake, K. and Shibata, K., 1986. Bacterial invasion of the cell wall of an aquatic bryophyte Scapania undulata (L.) Dum. in both acidic and near-neutral conditions. Hikobia, 9: 361365. Satake, K., Soma, M., Seyama, H. and Uehiro, T., 1983. Accumulation of mercury in the liverwort Jungermannia vulcanicola Steph. in an acid stream Kashiranashigawa in Japan. Arch. Hydrobiol., 99: 80-92. Sharpe, V. and Denny, P., 1976. Electron microscope studies on the absorption and localization of lead in the leaf tissue ofPotamogetonpectinatus L. J. Exp. Bot., 27: 1156-1162. Sillen, L.G. and Martell, A.E. (Editors), 1964. Stability Constants. The Chemical Society, London, 754 pp. Skaar, H., Ophus, E. and Gullv~g, B.M., 1973. Lead accumulation with nuclei of moss leaf cells. Nature {London), 241: 215-216. Soma, M., Seyama, H. and Satake, K., 1988. X-ray photoelectron spectroscopic analysis of lead accumulated in aquatic bryophytes. Talanta, 35: 68-70. Whitton, B.A., Say, P.J. and Jupp, B.P., 1982. Accumulation of zinc cadmium and lead by the aquatic liverwort Scapania. Environ. Pollut. (Ser. B), 3: 299-316.