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Analysis of metal-containing granules in the barnacle Tetraclita squamosa
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Inorganic Biochemistry Journal of Inorganic Biochemistry 98 (2004) 1095–1102 www.elsevier.com/locate/jinorgbio
Analysis of metal-containing granules in the barnacle Tetraclita squamosa Ombretta Masala a
a,*
, Paul O’Brien
a,*
, Philip S. Rainbow
b
Manchester Materials Science Centre and Department of Chemistry, The University of Manchester, Oxford Road, M13 9PL Manchester, UK b Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK Received 30 May 2003; received in revised form 11 March 2004; accepted 17 March 2004 Available online 22 April 2004
Abstract Metal-containing granules were isolated from specimens of the barnacle Tetraclita squamosa collected from a metal contaminated site in Hong Kong. The chemical composition, crystallographic and morphological properties of the deposits were analysed by X-ray powder diffraction, electron microscopy, energy dispersive analysis by X-rays and infrared spectroscopy. The most abundant metals were iron, calcium and zinc and the major anion was phosphate. Oxalates, carbonates and sulfur derivates were also found in small amounts. The deposits were amorphous to X-ray and electron diffraction and were approximately spherical in shape with diameters between 100 and 500 nm. 2004 Elsevier Inc. All rights reserved. Keywords: Barnacles; Tetraclita; Granule; Bioaccumulation; Trace metals
1. Introduction Hong Kong is situated on the southern coast of China and consists of Hong Kong Island, Kowloon Peninsula, the New Territories and another 235 surrounding islands. The coastal waters of Hong Kong have faced very high pollution in the recent years due essentially to the dense population (ca. 6 million people, for a total land area of 1068 km2 ), the heavy industrialisation (more than 2000 major and 200,000 minor industries [1], many of which are involved in the processing of toxic metals), the lack of control measures on industrial and agricultural discharges and the specific geographical position (the waters around Hong Kong islands are greatly affected by discharges from the mainland of China drained by the Pearl River). * Corresponding authors. Present address: University of California, Materials, Santa Barbara 93106-5050, USA. Tel.: +1-805-893-7509; fax: +1-805-893-8971 (O. Masala), Tel.: +44-161-275-4653; fax: +44161-275-4616 (P. O’Brien). E-mail addresses: [email protected] (O. Masala), [email protected] (P. O’Brien).
0162-0134/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2004.03.004
Chronic water contamination has required the Hong Kong government to monitor water quality very frequently with particular interest towards metal pollution and its effects on the marine ecosystem [2]. Over the last decade, the use of biomonitors in Hong Kong marine waters has become well established in assessing the variation in bioavailability of toxic metals [3]. In particular, three species of barnacles have been used to monitor metal bioavailability in Hong Kong’s coastal waters, i.e., Balanus amphitrite, Capitulum mitella and Tetraclita squamosa [3–5]. Barnacles accumulate trace metals in small intracellular phosphate granules [6]. Many other invertebrates are known to accumulate trace metals in phosphate granules [6,7]. For example, the snail Helix aspersa accumulates metals in the form of mixed ortho- and pyrophosphate granules [8] and the crab Carcinus maenas in the form of orthophosphate deposits [9]. However while extensive investigations have been carried out on the granules of these two invertebrates, very few have been made on barnacles and it is well known that metal accumulation processes vary from species to species and that different invertebrates accumulate different metals to different degrees [7,10].
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Most of the previous work on the granules formed in barnacles has dealt with the metal content and, to the authors’ knowledge, only one study has been carried out to date to identify the nature of the phosphate species [11]. In that study, enzymatic assay analyses showed that the main anion in the granules of the barnacles Elminius modestus and Semibalanus balanoides was pyrophosphate with orthophosphate and higher polyphosphates present in small amounts. We recently showed that in the presence of metals such as calcium, manganese and zinc, pyrophosphate crystallise to form spherical granules very similar in shape and composition to the deposits found in barnacles [12,13]. This similarity may suggest that pyrophosphate ions play a major role in the metal accumulation processes in barnacles. This paper studies metal-containing granules isolated from the barnacle Tetraclita squamosa, collected from the coastal waters of Aberdeen Harbour, Hong Kong, in April 1998. To date, no detailed investigations have been reported in the literature on the nature of the granules in this species. Infrared microscopy and X-ray microanalysis have been employed to investigate the granules and examine the presence of pyrophosphate and other phosphate species. Understanding metal accumulation in the granules of barnacles is a necessary requisite for any reliable biomonitoring program involving these marine invertebrates. Body metal contents for the same batch of barnacles have been reported earlier by Rainbow and Blackmore [14]. They found that the main contaminants were iron and zinc and other metals were present in traces. Here the opportunity is also taken to compare granule metal contents found in the present work and those reported for the whole body to investigate whether a relationship between accumulation in body tissue and granules exists.
2. Materials and methods Specimens of the barnacle Tetraclita squamosa were collected in April 1998 from either piers or rocks in Aberdeen Harbour, Hong Kong. Barnacles were removed from the location together with their substratum, placed in a clean polythene bag and transported in a cool box to the laboratory where they were kept frozen at )20 C. The samples were later defrosted and the substratum, with the barnacles still attached, was scrubbed thoroughly in deionised water to remove any debris. Barnacles were then removed from their substratum with a stainless steel scraper. The bodies were dissected out of the shells and pooled for isolation of granules after removal of the cirri. A hundred bodies of similar sizes were chosen for the analysis and extraction of granules from the bodies was performed according to the method developed by Walker et al. [15] to give a
granule-rich pellet. Smears from the material after each stage of the extraction procedure were analysed by Xray microanalysis to detect any accidental leaching of granules. X-ray microanalysis enables the identification of small areas of metal accumulation and the presence of little cell debris in the final pellet making this technique more suitable and accurate for the recognition of granules and the study of their metal content over other quantitative methods such as atomic absorption spectroscopy or inductively coupled plasma mass spectrometry. The pellet was also analysed by X-ray powder diffraction (XRPD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and infrared microscopy (IR). The pellet was air-dried before each analysis. XRPD analysis on the dried pellet was carried out using a Philips X’ Pert MPD X-ray powder diffrac and tometer with a Cu Ka radiation (k ¼ 1:5046 A) graphite monochromator. Diffraction patterns were scanned over the angular range 5–80 ð2hÞ, with a step length of 0.04 ð2hÞ. For TEM analysis, the pellet was dissected and randomly selected sections were suspended in acetone, deposited on copper grids coated with carbon film and air-dried. When analysis for copper was required, gold grids were used. Unstained samples were viewed in a Philips CM20 transmission electron microscope. X-ray microanalysis (or energy dispersive analysis by X-rays – EDAX) was carried out with a EDAX DX-4 energy dispersive spectrometer attached to the transmission electron microscope and equipped with an ultrathin window detector able to detect X-rays generated by light elements down to boron. All analyses were carried out at an accelerating voltage of 200 kV, a specimen tilt of 20 and a spot size of 30 nm for a counting time of 100 s. Interpretation of the spectra was carried out by using the standard-less software EDAX DX-4. The atomic percentage of each element was based on the intensity of its characteristic Ka peak defined as the number of counts below the deconvolution curve in a region of interest of 1.2 · FWHM of the peak (with FWHM, Full Width at Half Maximum), minus the background intensity in the same area, divided by the number of counting seconds during which the spectrum was acquired. FWHM of a peak is defined as follows: first a line was drawn by linking the maximum of the peak to the centre of the baseline, then a parallel line to the baseline was made through the centre of the resulting line, resulting in two points of intersection between the line and the peak. The difference in energy values between these points is the FWMH of the peak. The background intensity is defined as the number of counts below the background curve in the same 1.2 · FWHM area divided by the number of counting seconds. Background subtraction was performed manually with the linear interpolation method.
O. Masala et al. / Journal of Inorganic Biochemistry 98 (2004) 1095–1102
For SEM analysis, the pellet was dissected and randomly selected sections were suspended in acetone, deposited on aluminium pin stubs, air-dried, coated with a layer of gold and examined with a Philips 525 scanning electron microscope. IR was carried out on non-diluted samples using an Excalibur Bio-Rad Fourier Transform (FT) spectrometer equipped with a golden gate-attenuated total reflectance accessory and linked to an ImageIRTM focal plane array image detector and a UMA 500 microscope with a 400 400 lm diameter field of view. Fe4 (P2 O7 )3 H2 O and FePO4 4H2 O for IR spectroscopy were purchased from Aldrich and used without any further purifications.
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3. Results and discussion 3.1. Morphological analysis When viewed by TEM and SEM, metal-rich granules were observed either singly or in clusters distributed throughout the pellet (Figs. 1 and 2). Individual granules were mostly spherical but clusters were also often present with irregular shapes and sizes, many larger than 2 lm. However, as shown by the size distribution diagram in Fig. 3, the discrete granules mostly had diameters ranging between 200 and 500 nm, with a mean diameter of ca. 400 nm. Granules with diameters up to 800 nm were occasionally observed
Fig. 1. TEM micrographs of metal-rich granules: (a) shows single granules distributed throughout the pellet; (b) shows the internal concentric pattern in a granule fractured during sample preparation; (c) and (d) show clusters of granules. Single granules are usually smaller than 500 nm but agglomerates of granules can reach up to 2 lm in size.
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Fig. 2. SEM micrographs of (a) discrete granules and (b) clusters of granules. The black arrow points out agglomerates of small granules.
Fig. 3. Distribution of the relative frequency of diameter of phosphate granules. The mean value is 380 nm with a standard deviation of 120 nm. The distribution is based upon examination of electron micrographs of 500 granules recorded during TEM analysis of the pellet.
and appeared to be the result of the fusion of smaller deposits. The differences in size probably reflect different stages of mineralisation of the deposits and indicate that granule formation in the barnacle body is asynchronous. However, as shown by the TEM micrographs in Fig. 1, adjacent granules generally appear to be at a similar stage of formation suggesting that their growth in neighbouring cells or in the same cell could be simultaneous as previously proposed [16]. Granules damaged during sample preparation for TEM analysis showed that the material inside the deposits was arranged in the form of concentric rings around a central dense core (Fig. 1(b)), a pattern already observed in most phosphate granules of invertebrates [17]. The deposits were insoluble in water and acetone and amorphous both to electron and X-ray diffraction (Fig. 4). 3.2. X-ray microanalysis Metal contents of the granules were studied by X-ray microanalysis. Two hundred individual granules were chosen randomly during TEM analysis of the pellet and the results are reported in Table 1. The granules showed
Fig. 4. XRPD pattern of the granule-rich pellet.
a constant composition with iron and phosphorus being always the most abundant elements. Table 1 shows only elements common to all granules (calcium, iron, potassium, oxygen, phosphorus, potassium, silicon, and zinc). Aluminium, arsenic, chlorine, magnesium, manganese, nickel and sulfur were also detected but only occasionally in significant amounts and not in all granules (Fig. 5) so these elements were not included in the table (thus the sum of percentages in the table is lower than 100). The EDAX spectra in Fig. 5 show a number of weak peaks in the region around 5 keV. Several elements such as barium, titanium and lanthanum generate signals in that region, so that an unambiguous assignment of the bands was difficult. It was not possible to determine the presence of sodium in the granules since its Ka peak (1.04 keV) overlapped with the La peaks of zinc (1.01 keV) and copper (0.93 keV). Interferences from the carbon-coated grids used as specimen support did not enable an accurate quantification of carbon. However, the percentage of carbon in the granules appeared to be larger than the percentage usually arising from the coating of the support fittings solely, suggesting that the element could be present in appreciable amounts in the granules. The atomic percentages and percentage ratios (relative to P) calculated for FePO4 , Fe3 (PO4 )2 ,
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Table 1 Atomic percentages and percentage ratios of main elements (with standard deviation) measured over 200 granules Granules % P Fe O Zn Ca K Si
12.33 12.28 63.10 4.03 1.81 1.65 2.50
(1.24) (1.00) (3.40) (1.30) (0.52) (0.54) (1.03)
FePO4
Fe2 P2 O7
Fe3 (PO4 )2
Fe4 (P2 O7 )3
% ratio
%
% ratio
%
% ratio
%
% ratio
%
% ratio
1 1 5.12 0.33 0.15 0.13 0.20
16.70 16.70 66.60
1 1 4
18.20 18.20 63.60
1 1 3.5
15.03 23.08 61.53
1 1.5 4
19.36 12.90 67.74
1 0.7 3.5
Calculated percentages are reported for model phosphate compounds. The % ratio is relative to P.
Rainbow and Blackmore [14] analysed body metal concentrations in the same samples of Tetraclita squamosa and some of the data previously published are reported in Table 2 for comparison purposes (metal body concentrations for calcium and potassium were not reported in their original work). Body metal contents were analysed using atomic absorption spectroscopy and reported as lg/g in mean dry weight so that a direct comparison with the microanalysis data of this work is not possible. However, a general evaluation of body and granule metal contents suggests that granules are greatly enriched with iron but depleted in zinc with respect to body concentrations. With the exception of iron and zinc, the metals reported in Table 2 were found in granules only sporadically and in very small amounts. Significant differences between granule and body metal contents were also found by Pullen and Rainbow [11] when studying the occurrence of pyrophosphate deposits in the barnacles Elminius modestus and Semibalanus balanoides. In both species, the deposits contained calcium, iron, phosphorus, sulfur and zinc (with iron and phosphorus being the most abundant elements) and were richer in iron but poorer in zinc than the body. 3.3. Infrared spectroscopy analysis
Fig. 5. X-ray microanalysis of discrete granules. Sulphur is detected occasionally in considerable amounts (a). Au peaks are artifacts from the support fittings.
Fe4 (P2 O7 )3 and Fe2 P2 O7 are reported in Table 1 for comparison purposes. The data indicate that the percentage ratio of Fe, P and O in the granules is very close to the ratio of the elements in FePO4 and Fe2 P2 O7 suggesting that iron could be present either as Fe(II) or Fe(III) and phosphorus as orthophosphate or pyrophosphate. When sulfur was present in appreciable amounts, the X-ray microanalysis showed that the percentage ratio of Fe/P was often greater than one, suggesting that some of the iron may be bound to sulfur.
The nature of anions in granules was investigated by infrared microscopy. IR spectra of individual granules were collected with the aid of a FT-IR spectrometer linked to an optical microscope and a typical spectrum of a granule is shown in Fig. 6 together with those measured for the model compounds FePO4 4H2 O and Fe4 (P2 O7 )3 H2 O. Assignments of the bands are reported in Table 3 (peak values found in the literature for Fe2 P2 O7 are also reported in the table for comparison purposes). The granule spectrum is dominated by the phosphate and water absorption bands. The bands around 3400 and 1640 cm1 , characteristic of water molecule stretching and bending vibrations respectively [18], indicate that the granules were hydrated. The phosphate absorption bands are noticeable in the region around 1000 cm1 . The peaks at 953 and 744 cm1 are
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Table 2 Metal body concentrations in lg/g dry weight (measured by atomic absorption spectroscopy) of the barnacle Tetraclita squamosa (mean dry weight 0.0342 g) taken from [14] Zn
Fe
As
Mn
Cu
Ag
Cd
Pb
Cr
Ni
5460
450
71.6
17.2
16.7
6.35
3.24
2.76
1.40
1.34
Fig. 6. Infrared spectra of (A) barnacle granule, (B) Fe4 (P2 O7 )3 H2 O and (C) FePO4 4H2 O.
diagnostic of the presence of the pyrophosphate ion [19]. A map illustrating infrared absorption at 953 cm1 in the pellet is shown in Fig. 7. The degree of absorption intensity at this wavelength is represented by different colours and decreases in the order red > yellow > green > blue. The red colours represent the areas where there is the most intense absorption at 953 cm1 , i.e., the absorption of pyrophosphate anion occurs mainly in small discrete regions which may well be identified as the metal-containing granules. The bands at 953 and 744 cm1 show a very close correlation with the P–O–P stretching vibrations of Fe2 P2 O7 and
Fe4 (P2 O7 )3 H2 O (Table 3). The differences in frequency values can be probably attributed to the presence of several cations in the granules and their amorphous state. However, the comparison of the P–O stretching and bending band intensities between the spectra of barnacle granules, iron pyrophosphate and orthophosphate salts suggests that other forms of phosphate may be present in small amounts in the granules (Fig. 6). The absorption bands in the region around 3200 and 1500 cm1 suggest that some of the anions in the granules could be present in the form of urates [20,21]. The peaks at 3267, 3113 and 2932 cm1 can be attributed to the
O. Masala et al. / Journal of Inorganic Biochemistry 98 (2004) 1095–1102
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Table 3 Frequencies (cm1 ), relative intensities (% T),a and assignments of absorption bands in barnacle granules and commercial iron phosphate infrared spectra Granules
Fe4 (P2 O7 )3 H2 O
3437 mb
3300 b
3267 ms 3113 w 2932 wb 2365 w 2168 w 1655 sh 1624 s 1555 s 1443 m 1377 s 1315 m 1261 w 1153 m 1072 m 1068 s 1026 s 953 m 898 w 825 w 744 sh 700 sh a
Fe2 P2 O7 ([29])
1638 w
FePO4 4H2 O
Assignment
3200 b
O–H stretch of H2 O, N–H stretch of C5 H3 O3 N4 , C–H stretch of alkenes, alkynes and alkanes
1645 m
1040 s 941 s
1130 sb 1091 s 1040 s 960 s
760 w
714 m
997 s
C–O stretch of free CO2 (air) CBC stretch O–H bend of H2 O C–O stretch of C2 O2 4 /C5 H3 O3 N4 N–H vibrations C–O stretch of C5 H3 O3 N4 /C2 O2 4 C–O stretch of C5 H3 O3 N4 C–O stretch of C2 O2 4 P–O stretch of PO3 4 P–O stretch of PO3 4 P–O stretch of PO3 4 P–O stretch of PO3 4 P–O stretch of PO3 4 P–O–P stretch of P2 O4 7 H2 O vibration modes H2 O vibration modes P–O–P stretch of P2 O4 7 C–O bend of C5 H3 O3 N4 /C2 O2 4
b, broad; m, medium; s, strong; sh, shoulder; w, weak.
Fig. 7. Infrared absorption map at 953 cm1 in the granule-rich pellet. The IR spectrum at the crossing point of the broken lines is given in Fig. 6(A).
stretching vibrations of carbonyl groups and the bending vibrations of the NH groups, respectively. It is likely that oxalate and carbonate may be also present in small amounts in the granules, as suggested by the peaks at 1315 and 1443 cm1 , respectively [22–24]. Carbonate, oxalate and urate have often been detected together with phosphate in metal-containing granules. For example, in the phosphate granules isolated from the kidney of the scallop Pecten maximus, up to 7% (w/w) of the deposits were composed of oxalate [25]. Granules with a similar composition were also isolated from the kidney of the invertebrates Pinna nobilis and Tridacna maxima [26]. Urates have been found together with carbonates, oxalates and phosphates in granular concretions isolated from the marine invertebrate Molgula manhattensis [27]. X-ray microanalysis showed the presence of considerable amounts of sulfur in the granules but there is no evidence from the IR spectrum of the granules that it may be present as sulphate. Organic compounds containing S–C bonds are known to absorb in the region between 700 and 600 cm1 [28] but the poor resolution of the spectrum in this region does not enable an unequivocal determination.
4. Conclusions stretching vibrations of NH and CH bonds of the purinic ring of the urate group. The strong absorption bands at 1624 and 1550 cm1 can be assigned to the
In the present study we have shown that the granules isolated from the barnacle Tetraclita squamosa are rich
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in metals which are mainly bound to phosphates. It is difficult to discern which form of phosphate predominates, however IR spectroscopy analysis showed that a certain amount is in the form of pyrophosphate. Carbonates, oxalates and urates are also present in smaller quantities. Sulfur has been also found occasionally in considerable amounts, mainly bound to carbon. Different trace metals are present in the granules with calcium, iron, potassium and zinc being the most abundant. These features are common to the barnacles Elminius modestus and Semibalanus balanoides [11]. In these species metal-rich granules are mainly composed by phosphates (with pyrophosphate being the major species), along with significant amounts of sulfur in organic form. Such a relationship is not surprising. Barnacles are known to be strong accumulators of metals over time and one of the dominant detoxification processes involves the incorporation of accumulated trace metals into phosphate deposits. The role of pyrophosphate and other forms of phosphates as candidates for metal detoxification has been already discussed [8,30,31]. Since phosphates (and in particular pyrophosphate) form insoluble salts with most metal ions, they act as ideal sequestering agents for potentially toxic trace metals. In this work, we have also shown that variations may occur between granule and body metal accumulation. The granules appeared to be enriched with particular trace metals relative to body concentrations and this is in accordance with the theory that granules act as detoxification sites for toxic metals [7]. Such variations make the choice between studying the fate of metals at body levels or into inorganic granules a delicate issue for any reliable biomonitoring programme. A detailed knowledge of granule metal incorporation by barnacles is essential to understand the process by which these living organisms accumulate trace metals and is the basis of a reliable biomonitoring programme.
Acknowledgements We thank La Regione Autonoma della Sardegna (Italy) for a Ph.D. studentship to Ombretta Masala. We are also grateful to Graham Blackmore for collecting the barnacles.
References [1] B. Morton, Mar. Pollut. Bull. 20 (1989) 310–318. [2] Hong Kong: The Environmental Challenge, Hong Kong Environmental Protection Department, 1996. [3] G. Blackmore, Sci. Total Environ. 214 (1998) 21–48. [4] G. Blackmore, B. Morton, Z.G. Huang, Mar. Pollut. Bull. 36 (1998) 32–40. [5] W. Fialkowski, W.A. Newman, Mar. Pollut. Bull. 36 (1998) 138– 143. [6] P.S. Rainbow, J. Mar. Biol. Assoc. UK 77 (1997) 195–210. [7] S.P. Hopkin, Ecophysiology of Metals in Terrestrial Invertebrates, Elsevier Applied Science, Amsterdam, 1989. [8] B. Howard, P.C.H. Mitchell, A. Ritchie, K. Simkiss, M.G. Taylor, Biochem. J. 194 (1981) 507–511. [9] K. Simkiss, M.G. Taylor, G.N. Greaves, J. Inorg. Biochem. 39 (1990) 17–23. [10] P. Rainbow, Metal Metabolism in Aquatic Environments, Chapman & Hall, London, 1998. [11] J.S.H. Pullen, P.S. Rainbow, J. Exp. Mar. Biol. Ecol. 150 (1991) 249–266. [12] O. Masala, E.J.L. McInnes, P. O’Brien, Inorg. Chim. Acta 339 (2002) 336–372. [13] O. Masala, P. O’Brien, G. Rafeletos, Crys. Growth Des. 3 (2003) 431–434. [14] P.S. Rainbow, G. Blackmore, Mar. Environ. Res. 51 (2001) 441– 463. [15] G. Walker, P.S. Rainbow, P. Foster, D.L. Holland, Mar. Biol. 33 (1975) 161–166. [16] A.Z. Mason, K. Simkiss, Exp. Cell Res. 139 (1982) 383–391. [17] G. Walker, P.S. Rainbow, P. Foster, D.J. Crisp, Mar. Biol. 30 (1975) 57–65. [18] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds, fifth ed., Wiley Interscience, New York, 1997. [19] D.E.C. Corbridge, E.J. Lowe, J. Chem. Soc. (1954) 493–502. [20] N.S. Mandel, G.S. Mandel, J. Am. Chem. Soc. 98 (1976) 2319– 2323. [21] P. Carmona, J. Solid State Chem. 55 (1984) 293–298. [22] I. Petrov, B. Soptrajanov, Spectrochim. Acta Part A 31 (1975) 309–316. [23] H. Effenberger, Monatsh. Chem. 112 (1981) 899–909. [24] F.A. Andersen, L. Brecevic, Acta Chem. Scand. 45 (1991) 1018– 1024. [25] J. Overnell, J. Exp. Mar. Biol. Ecol. 52 (1981) 173–183. [26] N.G. Carmichael, K. Squibb, B.A. Fowler, J. Fish. Res. Board Can. 39 (1979) 1149–1155. [27] M.B. Saffo, H.A. Lowenstam, Science 200 (1978) 1166–1168. [28] R.M. Silverstein, F.X. Webster, Spectrometric Identification of Organic Compounds, sixth ed., Wiley Interscience, New York, 1998. [29] E.J. Baran, I.L. Botto, A.G. Nord, J. Mol. Struct. 143 (1986) 151– 154. [30] K. Simkiss, Trends Biochem. Sci. 6 (1981) 3–5. [31] K. Simkiss, M.G. Taylor, Mar. Environ. Res. 28 (1989) 211–214.
Inorganic Biochemistry Journal of Inorganic Biochemistry 98 (2004) 1095–1102 www.elsevier.com/locate/jinorgbio
Analysis of metal-containing granules in the barnacle Tetraclita squamosa Ombretta Masala a
a,*
, Paul O’Brien
a,*
, Philip S. Rainbow
b
Manchester Materials Science Centre and Department of Chemistry, The University of Manchester, Oxford Road, M13 9PL Manchester, UK b Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK Received 30 May 2003; received in revised form 11 March 2004; accepted 17 March 2004 Available online 22 April 2004
Abstract Metal-containing granules were isolated from specimens of the barnacle Tetraclita squamosa collected from a metal contaminated site in Hong Kong. The chemical composition, crystallographic and morphological properties of the deposits were analysed by X-ray powder diffraction, electron microscopy, energy dispersive analysis by X-rays and infrared spectroscopy. The most abundant metals were iron, calcium and zinc and the major anion was phosphate. Oxalates, carbonates and sulfur derivates were also found in small amounts. The deposits were amorphous to X-ray and electron diffraction and were approximately spherical in shape with diameters between 100 and 500 nm. 2004 Elsevier Inc. All rights reserved. Keywords: Barnacles; Tetraclita; Granule; Bioaccumulation; Trace metals
1. Introduction Hong Kong is situated on the southern coast of China and consists of Hong Kong Island, Kowloon Peninsula, the New Territories and another 235 surrounding islands. The coastal waters of Hong Kong have faced very high pollution in the recent years due essentially to the dense population (ca. 6 million people, for a total land area of 1068 km2 ), the heavy industrialisation (more than 2000 major and 200,000 minor industries [1], many of which are involved in the processing of toxic metals), the lack of control measures on industrial and agricultural discharges and the specific geographical position (the waters around Hong Kong islands are greatly affected by discharges from the mainland of China drained by the Pearl River). * Corresponding authors. Present address: University of California, Materials, Santa Barbara 93106-5050, USA. Tel.: +1-805-893-7509; fax: +1-805-893-8971 (O. Masala), Tel.: +44-161-275-4653; fax: +44161-275-4616 (P. O’Brien). E-mail addresses: [email protected] (O. Masala), [email protected] (P. O’Brien).
0162-0134/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2004.03.004
Chronic water contamination has required the Hong Kong government to monitor water quality very frequently with particular interest towards metal pollution and its effects on the marine ecosystem [2]. Over the last decade, the use of biomonitors in Hong Kong marine waters has become well established in assessing the variation in bioavailability of toxic metals [3]. In particular, three species of barnacles have been used to monitor metal bioavailability in Hong Kong’s coastal waters, i.e., Balanus amphitrite, Capitulum mitella and Tetraclita squamosa [3–5]. Barnacles accumulate trace metals in small intracellular phosphate granules [6]. Many other invertebrates are known to accumulate trace metals in phosphate granules [6,7]. For example, the snail Helix aspersa accumulates metals in the form of mixed ortho- and pyrophosphate granules [8] and the crab Carcinus maenas in the form of orthophosphate deposits [9]. However while extensive investigations have been carried out on the granules of these two invertebrates, very few have been made on barnacles and it is well known that metal accumulation processes vary from species to species and that different invertebrates accumulate different metals to different degrees [7,10].
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O. Masala et al. / Journal of Inorganic Biochemistry 98 (2004) 1095–1102
Most of the previous work on the granules formed in barnacles has dealt with the metal content and, to the authors’ knowledge, only one study has been carried out to date to identify the nature of the phosphate species [11]. In that study, enzymatic assay analyses showed that the main anion in the granules of the barnacles Elminius modestus and Semibalanus balanoides was pyrophosphate with orthophosphate and higher polyphosphates present in small amounts. We recently showed that in the presence of metals such as calcium, manganese and zinc, pyrophosphate crystallise to form spherical granules very similar in shape and composition to the deposits found in barnacles [12,13]. This similarity may suggest that pyrophosphate ions play a major role in the metal accumulation processes in barnacles. This paper studies metal-containing granules isolated from the barnacle Tetraclita squamosa, collected from the coastal waters of Aberdeen Harbour, Hong Kong, in April 1998. To date, no detailed investigations have been reported in the literature on the nature of the granules in this species. Infrared microscopy and X-ray microanalysis have been employed to investigate the granules and examine the presence of pyrophosphate and other phosphate species. Understanding metal accumulation in the granules of barnacles is a necessary requisite for any reliable biomonitoring program involving these marine invertebrates. Body metal contents for the same batch of barnacles have been reported earlier by Rainbow and Blackmore [14]. They found that the main contaminants were iron and zinc and other metals were present in traces. Here the opportunity is also taken to compare granule metal contents found in the present work and those reported for the whole body to investigate whether a relationship between accumulation in body tissue and granules exists.
2. Materials and methods Specimens of the barnacle Tetraclita squamosa were collected in April 1998 from either piers or rocks in Aberdeen Harbour, Hong Kong. Barnacles were removed from the location together with their substratum, placed in a clean polythene bag and transported in a cool box to the laboratory where they were kept frozen at )20 C. The samples were later defrosted and the substratum, with the barnacles still attached, was scrubbed thoroughly in deionised water to remove any debris. Barnacles were then removed from their substratum with a stainless steel scraper. The bodies were dissected out of the shells and pooled for isolation of granules after removal of the cirri. A hundred bodies of similar sizes were chosen for the analysis and extraction of granules from the bodies was performed according to the method developed by Walker et al. [15] to give a
granule-rich pellet. Smears from the material after each stage of the extraction procedure were analysed by Xray microanalysis to detect any accidental leaching of granules. X-ray microanalysis enables the identification of small areas of metal accumulation and the presence of little cell debris in the final pellet making this technique more suitable and accurate for the recognition of granules and the study of their metal content over other quantitative methods such as atomic absorption spectroscopy or inductively coupled plasma mass spectrometry. The pellet was also analysed by X-ray powder diffraction (XRPD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and infrared microscopy (IR). The pellet was air-dried before each analysis. XRPD analysis on the dried pellet was carried out using a Philips X’ Pert MPD X-ray powder diffrac and tometer with a Cu Ka radiation (k ¼ 1:5046 A) graphite monochromator. Diffraction patterns were scanned over the angular range 5–80 ð2hÞ, with a step length of 0.04 ð2hÞ. For TEM analysis, the pellet was dissected and randomly selected sections were suspended in acetone, deposited on copper grids coated with carbon film and air-dried. When analysis for copper was required, gold grids were used. Unstained samples were viewed in a Philips CM20 transmission electron microscope. X-ray microanalysis (or energy dispersive analysis by X-rays – EDAX) was carried out with a EDAX DX-4 energy dispersive spectrometer attached to the transmission electron microscope and equipped with an ultrathin window detector able to detect X-rays generated by light elements down to boron. All analyses were carried out at an accelerating voltage of 200 kV, a specimen tilt of 20 and a spot size of 30 nm for a counting time of 100 s. Interpretation of the spectra was carried out by using the standard-less software EDAX DX-4. The atomic percentage of each element was based on the intensity of its characteristic Ka peak defined as the number of counts below the deconvolution curve in a region of interest of 1.2 · FWHM of the peak (with FWHM, Full Width at Half Maximum), minus the background intensity in the same area, divided by the number of counting seconds during which the spectrum was acquired. FWHM of a peak is defined as follows: first a line was drawn by linking the maximum of the peak to the centre of the baseline, then a parallel line to the baseline was made through the centre of the resulting line, resulting in two points of intersection between the line and the peak. The difference in energy values between these points is the FWMH of the peak. The background intensity is defined as the number of counts below the background curve in the same 1.2 · FWHM area divided by the number of counting seconds. Background subtraction was performed manually with the linear interpolation method.
O. Masala et al. / Journal of Inorganic Biochemistry 98 (2004) 1095–1102
For SEM analysis, the pellet was dissected and randomly selected sections were suspended in acetone, deposited on aluminium pin stubs, air-dried, coated with a layer of gold and examined with a Philips 525 scanning electron microscope. IR was carried out on non-diluted samples using an Excalibur Bio-Rad Fourier Transform (FT) spectrometer equipped with a golden gate-attenuated total reflectance accessory and linked to an ImageIRTM focal plane array image detector and a UMA 500 microscope with a 400 400 lm diameter field of view. Fe4 (P2 O7 )3 H2 O and FePO4 4H2 O for IR spectroscopy were purchased from Aldrich and used without any further purifications.
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3. Results and discussion 3.1. Morphological analysis When viewed by TEM and SEM, metal-rich granules were observed either singly or in clusters distributed throughout the pellet (Figs. 1 and 2). Individual granules were mostly spherical but clusters were also often present with irregular shapes and sizes, many larger than 2 lm. However, as shown by the size distribution diagram in Fig. 3, the discrete granules mostly had diameters ranging between 200 and 500 nm, with a mean diameter of ca. 400 nm. Granules with diameters up to 800 nm were occasionally observed
Fig. 1. TEM micrographs of metal-rich granules: (a) shows single granules distributed throughout the pellet; (b) shows the internal concentric pattern in a granule fractured during sample preparation; (c) and (d) show clusters of granules. Single granules are usually smaller than 500 nm but agglomerates of granules can reach up to 2 lm in size.
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Fig. 2. SEM micrographs of (a) discrete granules and (b) clusters of granules. The black arrow points out agglomerates of small granules.
Fig. 3. Distribution of the relative frequency of diameter of phosphate granules. The mean value is 380 nm with a standard deviation of 120 nm. The distribution is based upon examination of electron micrographs of 500 granules recorded during TEM analysis of the pellet.
and appeared to be the result of the fusion of smaller deposits. The differences in size probably reflect different stages of mineralisation of the deposits and indicate that granule formation in the barnacle body is asynchronous. However, as shown by the TEM micrographs in Fig. 1, adjacent granules generally appear to be at a similar stage of formation suggesting that their growth in neighbouring cells or in the same cell could be simultaneous as previously proposed [16]. Granules damaged during sample preparation for TEM analysis showed that the material inside the deposits was arranged in the form of concentric rings around a central dense core (Fig. 1(b)), a pattern already observed in most phosphate granules of invertebrates [17]. The deposits were insoluble in water and acetone and amorphous both to electron and X-ray diffraction (Fig. 4). 3.2. X-ray microanalysis Metal contents of the granules were studied by X-ray microanalysis. Two hundred individual granules were chosen randomly during TEM analysis of the pellet and the results are reported in Table 1. The granules showed
Fig. 4. XRPD pattern of the granule-rich pellet.
a constant composition with iron and phosphorus being always the most abundant elements. Table 1 shows only elements common to all granules (calcium, iron, potassium, oxygen, phosphorus, potassium, silicon, and zinc). Aluminium, arsenic, chlorine, magnesium, manganese, nickel and sulfur were also detected but only occasionally in significant amounts and not in all granules (Fig. 5) so these elements were not included in the table (thus the sum of percentages in the table is lower than 100). The EDAX spectra in Fig. 5 show a number of weak peaks in the region around 5 keV. Several elements such as barium, titanium and lanthanum generate signals in that region, so that an unambiguous assignment of the bands was difficult. It was not possible to determine the presence of sodium in the granules since its Ka peak (1.04 keV) overlapped with the La peaks of zinc (1.01 keV) and copper (0.93 keV). Interferences from the carbon-coated grids used as specimen support did not enable an accurate quantification of carbon. However, the percentage of carbon in the granules appeared to be larger than the percentage usually arising from the coating of the support fittings solely, suggesting that the element could be present in appreciable amounts in the granules. The atomic percentages and percentage ratios (relative to P) calculated for FePO4 , Fe3 (PO4 )2 ,
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Table 1 Atomic percentages and percentage ratios of main elements (with standard deviation) measured over 200 granules Granules % P Fe O Zn Ca K Si
12.33 12.28 63.10 4.03 1.81 1.65 2.50
(1.24) (1.00) (3.40) (1.30) (0.52) (0.54) (1.03)
FePO4
Fe2 P2 O7
Fe3 (PO4 )2
Fe4 (P2 O7 )3
% ratio
%
% ratio
%
% ratio
%
% ratio
%
% ratio
1 1 5.12 0.33 0.15 0.13 0.20
16.70 16.70 66.60
1 1 4
18.20 18.20 63.60
1 1 3.5
15.03 23.08 61.53
1 1.5 4
19.36 12.90 67.74
1 0.7 3.5
Calculated percentages are reported for model phosphate compounds. The % ratio is relative to P.
Rainbow and Blackmore [14] analysed body metal concentrations in the same samples of Tetraclita squamosa and some of the data previously published are reported in Table 2 for comparison purposes (metal body concentrations for calcium and potassium were not reported in their original work). Body metal contents were analysed using atomic absorption spectroscopy and reported as lg/g in mean dry weight so that a direct comparison with the microanalysis data of this work is not possible. However, a general evaluation of body and granule metal contents suggests that granules are greatly enriched with iron but depleted in zinc with respect to body concentrations. With the exception of iron and zinc, the metals reported in Table 2 were found in granules only sporadically and in very small amounts. Significant differences between granule and body metal contents were also found by Pullen and Rainbow [11] when studying the occurrence of pyrophosphate deposits in the barnacles Elminius modestus and Semibalanus balanoides. In both species, the deposits contained calcium, iron, phosphorus, sulfur and zinc (with iron and phosphorus being the most abundant elements) and were richer in iron but poorer in zinc than the body. 3.3. Infrared spectroscopy analysis
Fig. 5. X-ray microanalysis of discrete granules. Sulphur is detected occasionally in considerable amounts (a). Au peaks are artifacts from the support fittings.
Fe4 (P2 O7 )3 and Fe2 P2 O7 are reported in Table 1 for comparison purposes. The data indicate that the percentage ratio of Fe, P and O in the granules is very close to the ratio of the elements in FePO4 and Fe2 P2 O7 suggesting that iron could be present either as Fe(II) or Fe(III) and phosphorus as orthophosphate or pyrophosphate. When sulfur was present in appreciable amounts, the X-ray microanalysis showed that the percentage ratio of Fe/P was often greater than one, suggesting that some of the iron may be bound to sulfur.
The nature of anions in granules was investigated by infrared microscopy. IR spectra of individual granules were collected with the aid of a FT-IR spectrometer linked to an optical microscope and a typical spectrum of a granule is shown in Fig. 6 together with those measured for the model compounds FePO4 4H2 O and Fe4 (P2 O7 )3 H2 O. Assignments of the bands are reported in Table 3 (peak values found in the literature for Fe2 P2 O7 are also reported in the table for comparison purposes). The granule spectrum is dominated by the phosphate and water absorption bands. The bands around 3400 and 1640 cm1 , characteristic of water molecule stretching and bending vibrations respectively [18], indicate that the granules were hydrated. The phosphate absorption bands are noticeable in the region around 1000 cm1 . The peaks at 953 and 744 cm1 are
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Table 2 Metal body concentrations in lg/g dry weight (measured by atomic absorption spectroscopy) of the barnacle Tetraclita squamosa (mean dry weight 0.0342 g) taken from [14] Zn
Fe
As
Mn
Cu
Ag
Cd
Pb
Cr
Ni
5460
450
71.6
17.2
16.7
6.35
3.24
2.76
1.40
1.34
Fig. 6. Infrared spectra of (A) barnacle granule, (B) Fe4 (P2 O7 )3 H2 O and (C) FePO4 4H2 O.
diagnostic of the presence of the pyrophosphate ion [19]. A map illustrating infrared absorption at 953 cm1 in the pellet is shown in Fig. 7. The degree of absorption intensity at this wavelength is represented by different colours and decreases in the order red > yellow > green > blue. The red colours represent the areas where there is the most intense absorption at 953 cm1 , i.e., the absorption of pyrophosphate anion occurs mainly in small discrete regions which may well be identified as the metal-containing granules. The bands at 953 and 744 cm1 show a very close correlation with the P–O–P stretching vibrations of Fe2 P2 O7 and
Fe4 (P2 O7 )3 H2 O (Table 3). The differences in frequency values can be probably attributed to the presence of several cations in the granules and their amorphous state. However, the comparison of the P–O stretching and bending band intensities between the spectra of barnacle granules, iron pyrophosphate and orthophosphate salts suggests that other forms of phosphate may be present in small amounts in the granules (Fig. 6). The absorption bands in the region around 3200 and 1500 cm1 suggest that some of the anions in the granules could be present in the form of urates [20,21]. The peaks at 3267, 3113 and 2932 cm1 can be attributed to the
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Table 3 Frequencies (cm1 ), relative intensities (% T),a and assignments of absorption bands in barnacle granules and commercial iron phosphate infrared spectra Granules
Fe4 (P2 O7 )3 H2 O
3437 mb
3300 b
3267 ms 3113 w 2932 wb 2365 w 2168 w 1655 sh 1624 s 1555 s 1443 m 1377 s 1315 m 1261 w 1153 m 1072 m 1068 s 1026 s 953 m 898 w 825 w 744 sh 700 sh a
Fe2 P2 O7 ([29])
1638 w
FePO4 4H2 O
Assignment
3200 b
O–H stretch of H2 O, N–H stretch of C5 H3 O3 N4 , C–H stretch of alkenes, alkynes and alkanes
1645 m
1040 s 941 s
1130 sb 1091 s 1040 s 960 s
760 w
714 m
997 s
C–O stretch of free CO2 (air) CBC stretch O–H bend of H2 O C–O stretch of C2 O2 4 /C5 H3 O3 N4 N–H vibrations C–O stretch of C5 H3 O3 N4 /C2 O2 4 C–O stretch of C5 H3 O3 N4 C–O stretch of C2 O2 4 P–O stretch of PO3 4 P–O stretch of PO3 4 P–O stretch of PO3 4 P–O stretch of PO3 4 P–O stretch of PO3 4 P–O–P stretch of P2 O4 7 H2 O vibration modes H2 O vibration modes P–O–P stretch of P2 O4 7 C–O bend of C5 H3 O3 N4 /C2 O2 4
b, broad; m, medium; s, strong; sh, shoulder; w, weak.
Fig. 7. Infrared absorption map at 953 cm1 in the granule-rich pellet. The IR spectrum at the crossing point of the broken lines is given in Fig. 6(A).
stretching vibrations of carbonyl groups and the bending vibrations of the NH groups, respectively. It is likely that oxalate and carbonate may be also present in small amounts in the granules, as suggested by the peaks at 1315 and 1443 cm1 , respectively [22–24]. Carbonate, oxalate and urate have often been detected together with phosphate in metal-containing granules. For example, in the phosphate granules isolated from the kidney of the scallop Pecten maximus, up to 7% (w/w) of the deposits were composed of oxalate [25]. Granules with a similar composition were also isolated from the kidney of the invertebrates Pinna nobilis and Tridacna maxima [26]. Urates have been found together with carbonates, oxalates and phosphates in granular concretions isolated from the marine invertebrate Molgula manhattensis [27]. X-ray microanalysis showed the presence of considerable amounts of sulfur in the granules but there is no evidence from the IR spectrum of the granules that it may be present as sulphate. Organic compounds containing S–C bonds are known to absorb in the region between 700 and 600 cm1 [28] but the poor resolution of the spectrum in this region does not enable an unequivocal determination.
4. Conclusions stretching vibrations of NH and CH bonds of the purinic ring of the urate group. The strong absorption bands at 1624 and 1550 cm1 can be assigned to the
In the present study we have shown that the granules isolated from the barnacle Tetraclita squamosa are rich
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in metals which are mainly bound to phosphates. It is difficult to discern which form of phosphate predominates, however IR spectroscopy analysis showed that a certain amount is in the form of pyrophosphate. Carbonates, oxalates and urates are also present in smaller quantities. Sulfur has been also found occasionally in considerable amounts, mainly bound to carbon. Different trace metals are present in the granules with calcium, iron, potassium and zinc being the most abundant. These features are common to the barnacles Elminius modestus and Semibalanus balanoides [11]. In these species metal-rich granules are mainly composed by phosphates (with pyrophosphate being the major species), along with significant amounts of sulfur in organic form. Such a relationship is not surprising. Barnacles are known to be strong accumulators of metals over time and one of the dominant detoxification processes involves the incorporation of accumulated trace metals into phosphate deposits. The role of pyrophosphate and other forms of phosphates as candidates for metal detoxification has been already discussed [8,30,31]. Since phosphates (and in particular pyrophosphate) form insoluble salts with most metal ions, they act as ideal sequestering agents for potentially toxic trace metals. In this work, we have also shown that variations may occur between granule and body metal accumulation. The granules appeared to be enriched with particular trace metals relative to body concentrations and this is in accordance with the theory that granules act as detoxification sites for toxic metals [7]. Such variations make the choice between studying the fate of metals at body levels or into inorganic granules a delicate issue for any reliable biomonitoring programme. A detailed knowledge of granule metal incorporation by barnacles is essential to understand the process by which these living organisms accumulate trace metals and is the basis of a reliable biomonitoring programme.
Acknowledgements We thank La Regione Autonoma della Sardegna (Italy) for a Ph.D. studentship to Ombretta Masala. We are also grateful to Graham Blackmore for collecting the barnacles.
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