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A rapid and inexpensive technique to separate the calcite and nacreous layers in Mytilus edulis shells
Marine Environmental Research 25 (1988) 125-129
A Rapid and Inexpensive Technique to Separate the Calcite and Nacreous Layers in Myti/us edulis Shells Bernard Patrick Bourgoin* McMaster University, Geology Department, Hamilton, Ontario, Canada L8S 4M1 (Received 30 April 1987; revised version received 30 August 1987; accepted 8 February 1988)
A BS TRA C T A method has been developedJor separating the calcite and aragonite shell layers ~f the blue mussel, Mytilus edulis L. Shells are h e a t e d to 400°C for 18 h. Upon cooling, the now-brittle and crumbly calcitic layer could easily be separated from the inner aragonite layer. The separation of these two layers occurs along the pallial myostracum. The importance of this technique in trace metal analyses of shells, as well as its application in palaeontological studies, is discussed.
INTRODUCTION The mussel, Mytilus edulis, is probably the most widely used bivalve for monitoring trace metal pollution. Most studies have analyzed the soft tissues, but trace metal shell analyses are increasingly popular (Bourgoin & Risk, 1987; Carell et al., 1987). Koide et al. (1982) have enumerated the advantages in using shells rather than soft tissues in trace metal analyses. The bivalve shell is a complex organic/inorganic system consisting of two valves covered by an outer organic sheet, the periostracum, and within this, the carbonate shell. In Mytilus edulis, the carbonate shell is composed of an outer calcite layer and an inner aragonite (nacreous) layer. Because all the constituents of the carbonate shell must ultimately have been assimilated by the organism (Wilbur & Saleuddin, 1983), trace metal levels in the shells can provide an index on their bioavailability. The periostracum should prevent adsorption of trace metals onto the * Present address: Trent Aquatic Research Centre, Trent University, Peterborough, Ontario. Canada K9J 7B8. 125
Marine Environ. Res. 0141-1136/88/$03"50 ~ 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain
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Bernard Patrick Bourgoin
carbonate shell. This proteinaceous layer is, however, rarely intact, leaving large portions of the calcite layer exposed to particulate and dissolved trace metals. The inner aragonite layer is therefore a better indicator of trace metal bioavailability. The object of this study was to develop a technique to separate the calcite and aragonite layers.
Fig. 1. Scanning electron micrograph of a fractured anteroposterior longitudinal section of the calcite and aragonite shell layers of Mytilus edulis L. The outer and inner shell surfaces are toward the top and bottom, respectively, of the micrographs. Scale bars are 1/~m for A, C, E, and 5/~m for B, D, F. [A3 extremely irregular aragonite prisms of the pallial myostracum. [B] myostracal band separating the outer (top) and inner (bottom) shell layers. I-C] polygonal calcite prisms of the outer shell layer. [D] sheet-like arrangement of the calcite prisms of the outer shell layer, [E] tabular aragonite crystals of the inner nacreous layer. IF] nacreous tablets arranged in steplike patterns characteristic of bivalve nacre.
Separating the calcite and nacreous layers in Mytilus edulis
127
M I C R O S T R U C T U R E OF SHELL LAYERS Taylor et al. (1969) defined seven main categories of shell structure in which the calcium carbonate crystals differ in their shape and orientation as seen through the electron microscope. These shell structures occur as distinct, monominerallic layers within the bivalve shell. The pallial myostracum consists of irregular aragonite prisms (Fig. 1A) and separates the calcitic and nacreous layers (Fig. 1B). This structure is relatively thin (4/~m) and is therefore considered as a band rather than a distinct shell layer. The outer layer of the shell is categorized as prismatic calcite and consists of columnar prisms (1-2/~m thick), polygonal in section and up to 50/~m long (Fig. 1C), arranged in sheet-like rows (Fig. 1D). The inner nacreous shell layer consists of tablet-like aragonite crystals 5/~m in length and 0"5 ~tm thick (Fig. 1E) deposited in regular layers parallel to the shell interior (Fig. 1F). Taylor & Layman (1972) noted that these shell microstructures conveyed different mechanical properties to the various shell layers. The nacreous material was observed to be more 'elastomeric' and could withstand cracking better than the prismatic calcite, ~¢hich was comparatively more brittle. Hence, large and rapid temperature fluctuations could perhaps cause the inner nacreous layer to separate from the brittle calcitic layer.
MATERIALS AND METHODS Frozen M y t i l u s edulis specimens ranging in length from 3 to 9 cm were thawed at room temperature. The soft parts were removed, the shells cleaned under a jet of tap water and dried at 60°C. Extraneous materials, such as barnacles and byssal threads, were scraped from the shells. Series of shells were then placed in a pre-heated muffle furnace at the following temperature settings and time intervals: 200 °, 300 °, 400 °, 500 ° and 600cC; 5 min, 10 min, 1 h, 6h, 12 h, 18 h and 24h. The samples were cooled at room temperature. after which the outer and inner shell layers were separated.
RESULTS A N D DISCUSSION The calcite and nacreous layers could consistently be separated, for all shell lengths, when heated to 400°C for 18 h. Most of the calcite layer would crack and readily fall off as the shell cooled, and the remaining calcite fragments could be gently scraped off the intact nacreous layer. Scanning electron microscope observations showed that parting of the shell layers occurred at the pallial myostracum band which remained associated with the calcitic
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layer. The calcite and aragonite layers were easily differentiated, the former being dull and brittle and the latter displaying a characteristic pearly lustre. In a study to separate annual shell layers in freshwater bivalves, Sterrett & Saville (1974) suggested heating the shells at 500°C for 10 min or 600°C for 5 min. At these settings, the calcite and nacreous layers in Mytilus edulis shells could only be separated in the smaller valves (between 3 and 4 cm). The nacreous layer crumbled easily and was difficult to manipulate when longer baking times were used. When shells were heated to lower temperatures, 200 ° and 300°C, the calcitic and nacreous layers would not readily separate and had to be pried apart with a scalpel. The nacreous component of the shell would usually fragment, especially in the smaller valves, because of the pressure required to separate the layers. The size of the microstructural units is the most significant factor in determining the mechanical properties of the shells (Taylor & Layman, 1972). Thus the prismatic structure consists of small crystals arranged into much larger sheet-like units (Fig. 1D), whereas in the nacreous structure the individual crystals are the largest units present (Fig. 1E). Cracks developing in the nacre would have their energy dissipated at the many crystal boundaries, whereas there will be a tendency for cracks to travel along the boundaries of the larger units in the prismatic structure. Although this technique has proven to be invaluable when establishing trends in trace metal contamination through shell analysis (Bourgoin, 1987), it can also be applied to other fields of study. Variations in aragonite:calcite ratios have always been an important parameter in paleontological studies. Until now, this particular aspect has been hampered by small data sets because the methods involved for determining this ratio, X-ray diffraction or thin sections, were expensive and time consuming. This method allows a large number of samples to be inexpensively and rapidly processed.
ACKNOWLEDGEMENTS Financial support from the Natural Sciences and Engineering Research Council of Canada is acknowledged. The author wishes to thank Dr D. M. Shaw for the use of his laboratory.
REFERENCES Bourgoin, B. P. (1987). Mytilus edulis shells as environmental recorders for lead contamination. PhD thesis, Geology Department, McMaster University, Hamilton, Ontario, 138 pp.
Separating the calcite and nacreous layers in Mytilus edulis
129
Bourgoin, B. P. & Risk, M. J. (1987). Historical changes in lead in the Eastern Canadian Arctic, determined from fossil and modern Mya truncata shells, Sci. Total Environ., 67, 287-91. Carell, B., Forberg, S., Grundelius, E., Henrikson, L., Johnels, A., Lindh, U., Mutvei, H., Olsson, M., Svardstrom, K. & Westermark, T. (1987). Can mussel shells reveal environmental history? Ambio, 16, 2 10. Koide, M., Lee, D. S. & Goldberg, E. D. (1982). Metal and transuranic records in mussel shells, byssal threads and tissues. Estuar. Coastal & Shelf Sci., 15, 679-95. Sterrett, S. S. & Saville, L. D. (1974). A technique to separate the annual layers of a naiad shell (Mollusca, Bivalvia, Unionacea) for analysis by neutron activation. Am. Malacol. Union Inc. Bull., 55 7. Taylor, J. D. & Layman, M. (1972). The mechanical properties of bivalve (Mollusca) shell structure. Palaeontology, 15, 73 87. Taylor, J. D., Kennedy, W. J. & Hall, A. (1969). Environmental and biological controls on bivalve shell mineralogy. Biol. Rev., 44, 499-530. Wilbur, K. M. & Saleuddin, A. S. M. (1983). Shell formation. In The MolluscaPhysiology. Vol. 4, ed. K. M. Wilbur, Academic Press, NY, 235-87.
A Rapid and Inexpensive Technique to Separate the Calcite and Nacreous Layers in Myti/us edulis Shells Bernard Patrick Bourgoin* McMaster University, Geology Department, Hamilton, Ontario, Canada L8S 4M1 (Received 30 April 1987; revised version received 30 August 1987; accepted 8 February 1988)
A BS TRA C T A method has been developedJor separating the calcite and aragonite shell layers ~f the blue mussel, Mytilus edulis L. Shells are h e a t e d to 400°C for 18 h. Upon cooling, the now-brittle and crumbly calcitic layer could easily be separated from the inner aragonite layer. The separation of these two layers occurs along the pallial myostracum. The importance of this technique in trace metal analyses of shells, as well as its application in palaeontological studies, is discussed.
INTRODUCTION The mussel, Mytilus edulis, is probably the most widely used bivalve for monitoring trace metal pollution. Most studies have analyzed the soft tissues, but trace metal shell analyses are increasingly popular (Bourgoin & Risk, 1987; Carell et al., 1987). Koide et al. (1982) have enumerated the advantages in using shells rather than soft tissues in trace metal analyses. The bivalve shell is a complex organic/inorganic system consisting of two valves covered by an outer organic sheet, the periostracum, and within this, the carbonate shell. In Mytilus edulis, the carbonate shell is composed of an outer calcite layer and an inner aragonite (nacreous) layer. Because all the constituents of the carbonate shell must ultimately have been assimilated by the organism (Wilbur & Saleuddin, 1983), trace metal levels in the shells can provide an index on their bioavailability. The periostracum should prevent adsorption of trace metals onto the * Present address: Trent Aquatic Research Centre, Trent University, Peterborough, Ontario. Canada K9J 7B8. 125
Marine Environ. Res. 0141-1136/88/$03"50 ~ 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain
126
Bernard Patrick Bourgoin
carbonate shell. This proteinaceous layer is, however, rarely intact, leaving large portions of the calcite layer exposed to particulate and dissolved trace metals. The inner aragonite layer is therefore a better indicator of trace metal bioavailability. The object of this study was to develop a technique to separate the calcite and aragonite layers.
Fig. 1. Scanning electron micrograph of a fractured anteroposterior longitudinal section of the calcite and aragonite shell layers of Mytilus edulis L. The outer and inner shell surfaces are toward the top and bottom, respectively, of the micrographs. Scale bars are 1/~m for A, C, E, and 5/~m for B, D, F. [A3 extremely irregular aragonite prisms of the pallial myostracum. [B] myostracal band separating the outer (top) and inner (bottom) shell layers. I-C] polygonal calcite prisms of the outer shell layer. [D] sheet-like arrangement of the calcite prisms of the outer shell layer, [E] tabular aragonite crystals of the inner nacreous layer. IF] nacreous tablets arranged in steplike patterns characteristic of bivalve nacre.
Separating the calcite and nacreous layers in Mytilus edulis
127
M I C R O S T R U C T U R E OF SHELL LAYERS Taylor et al. (1969) defined seven main categories of shell structure in which the calcium carbonate crystals differ in their shape and orientation as seen through the electron microscope. These shell structures occur as distinct, monominerallic layers within the bivalve shell. The pallial myostracum consists of irregular aragonite prisms (Fig. 1A) and separates the calcitic and nacreous layers (Fig. 1B). This structure is relatively thin (4/~m) and is therefore considered as a band rather than a distinct shell layer. The outer layer of the shell is categorized as prismatic calcite and consists of columnar prisms (1-2/~m thick), polygonal in section and up to 50/~m long (Fig. 1C), arranged in sheet-like rows (Fig. 1D). The inner nacreous shell layer consists of tablet-like aragonite crystals 5/~m in length and 0"5 ~tm thick (Fig. 1E) deposited in regular layers parallel to the shell interior (Fig. 1F). Taylor & Layman (1972) noted that these shell microstructures conveyed different mechanical properties to the various shell layers. The nacreous material was observed to be more 'elastomeric' and could withstand cracking better than the prismatic calcite, ~¢hich was comparatively more brittle. Hence, large and rapid temperature fluctuations could perhaps cause the inner nacreous layer to separate from the brittle calcitic layer.
MATERIALS AND METHODS Frozen M y t i l u s edulis specimens ranging in length from 3 to 9 cm were thawed at room temperature. The soft parts were removed, the shells cleaned under a jet of tap water and dried at 60°C. Extraneous materials, such as barnacles and byssal threads, were scraped from the shells. Series of shells were then placed in a pre-heated muffle furnace at the following temperature settings and time intervals: 200 °, 300 °, 400 °, 500 ° and 600cC; 5 min, 10 min, 1 h, 6h, 12 h, 18 h and 24h. The samples were cooled at room temperature. after which the outer and inner shell layers were separated.
RESULTS A N D DISCUSSION The calcite and nacreous layers could consistently be separated, for all shell lengths, when heated to 400°C for 18 h. Most of the calcite layer would crack and readily fall off as the shell cooled, and the remaining calcite fragments could be gently scraped off the intact nacreous layer. Scanning electron microscope observations showed that parting of the shell layers occurred at the pallial myostracum band which remained associated with the calcitic
128
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layer. The calcite and aragonite layers were easily differentiated, the former being dull and brittle and the latter displaying a characteristic pearly lustre. In a study to separate annual shell layers in freshwater bivalves, Sterrett & Saville (1974) suggested heating the shells at 500°C for 10 min or 600°C for 5 min. At these settings, the calcite and nacreous layers in Mytilus edulis shells could only be separated in the smaller valves (between 3 and 4 cm). The nacreous layer crumbled easily and was difficult to manipulate when longer baking times were used. When shells were heated to lower temperatures, 200 ° and 300°C, the calcitic and nacreous layers would not readily separate and had to be pried apart with a scalpel. The nacreous component of the shell would usually fragment, especially in the smaller valves, because of the pressure required to separate the layers. The size of the microstructural units is the most significant factor in determining the mechanical properties of the shells (Taylor & Layman, 1972). Thus the prismatic structure consists of small crystals arranged into much larger sheet-like units (Fig. 1D), whereas in the nacreous structure the individual crystals are the largest units present (Fig. 1E). Cracks developing in the nacre would have their energy dissipated at the many crystal boundaries, whereas there will be a tendency for cracks to travel along the boundaries of the larger units in the prismatic structure. Although this technique has proven to be invaluable when establishing trends in trace metal contamination through shell analysis (Bourgoin, 1987), it can also be applied to other fields of study. Variations in aragonite:calcite ratios have always been an important parameter in paleontological studies. Until now, this particular aspect has been hampered by small data sets because the methods involved for determining this ratio, X-ray diffraction or thin sections, were expensive and time consuming. This method allows a large number of samples to be inexpensively and rapidly processed.
ACKNOWLEDGEMENTS Financial support from the Natural Sciences and Engineering Research Council of Canada is acknowledged. The author wishes to thank Dr D. M. Shaw for the use of his laboratory.
REFERENCES Bourgoin, B. P. (1987). Mytilus edulis shells as environmental recorders for lead contamination. PhD thesis, Geology Department, McMaster University, Hamilton, Ontario, 138 pp.
Separating the calcite and nacreous layers in Mytilus edulis
129
Bourgoin, B. P. & Risk, M. J. (1987). Historical changes in lead in the Eastern Canadian Arctic, determined from fossil and modern Mya truncata shells, Sci. Total Environ., 67, 287-91. Carell, B., Forberg, S., Grundelius, E., Henrikson, L., Johnels, A., Lindh, U., Mutvei, H., Olsson, M., Svardstrom, K. & Westermark, T. (1987). Can mussel shells reveal environmental history? Ambio, 16, 2 10. Koide, M., Lee, D. S. & Goldberg, E. D. (1982). Metal and transuranic records in mussel shells, byssal threads and tissues. Estuar. Coastal & Shelf Sci., 15, 679-95. Sterrett, S. S. & Saville, L. D. (1974). A technique to separate the annual layers of a naiad shell (Mollusca, Bivalvia, Unionacea) for analysis by neutron activation. Am. Malacol. Union Inc. Bull., 55 7. Taylor, J. D. & Layman, M. (1972). The mechanical properties of bivalve (Mollusca) shell structure. Palaeontology, 15, 73 87. Taylor, J. D., Kennedy, W. J. & Hall, A. (1969). Environmental and biological controls on bivalve shell mineralogy. Biol. Rev., 44, 499-530. Wilbur, K. M. & Saleuddin, A. S. M. (1983). Shell formation. In The MolluscaPhysiology. Vol. 4, ed. K. M. Wilbur, Academic Press, NY, 235-87.