Artículos

 

A technique for the separation of the most recently deposited nacreous layer in Mytilus californianus shells for trace metal analysis

 

Técnica para la separación de la capa de nácar recientemente depositada en la concha de Mytilus californianus para el análisis de metales traza

 

L.E. Rivero and M.L. Lares*

 

Departamento de Ecología Centro de Investigación Científica y de Educación Superior de Ensenada Ensenada, CP 22800, BC, México. * E-mail: llares@cicese.mx

 

Recibido en febrero de 2003;
aceptado en febrero de 2004.

 

Abstract

A new method to separate a section of the most recently deposited nacreous layer from the rest of the shell of Mytilus californianus was developed. Techniques described for other mussel species proved unsuitable for this species. In this new method, the shells are soaked in 30% hydrogen peroxide for 10-15 min at 80-85°C to eliminate the periostracum, a potential source of trace metal contamination. Next, the shells are heated in a muffle furnace at 350°C for 1 h to separate the nacreous layer from the calcite layer. Finally, the nacreous band is easily separated from the rest of the shell by gently tapping it with a stainless steel needle. This is a simple and fast method that is proposed for trace metal monitoring purposes usingM. californianus shells. To illustrate the application of the method, results from an experiment performed in our laboratory, in which mussels were exposed to two different Cd concentrations for 60 days and their shells processed by this technique, are included. The most recently deposited nacreous layer showed significant increases in Cd concentrations, which were proportional to the exposed ambient concentrations.

Key words: Mytilus californianus, shell, nacreous layer, cadmium.

 

Resumen

Se desarrolló un nuevo método para separar una sección de la capa de nácar recientemente depositada del resto de la concha de Mytilus californianus. Los métodos descritos para otras especies de mejillones demostraron no ser apropiados para las conchas de esta especie. En este nuevo método, las conchas son sumergidas en peróxido de hidrógeno al 30% por 10-15 min a 80-85°C para eliminar el periostraco, una fuente potencial de contaminación por metales traza. Enseguida, las conchas son calentadas en una mufla a 350°C por 1 h para separar la capa de nácar de la calcítica. Finalmente, la banda de nácar recientemente depositada es fácilmente separada del resto de la concha, al golpearla suavemente con una aguja de acero inoxidable. Éste es un método simple y rápido propuesto para el monitoreo ambiental de metales traza utilizando las conchas deM. californianus. Como un ejemplo de la aplicación de este método, se presentan los resultados de un experimento realizado en laboratorio, en el que los mejillones fueron expuestos a dos diferentes concentraciones de Cd por 60 días y sus conchas fueron procesadas por medio de esta técnica. La capa de nácar más recientemente depositada mostró incrementos significativos en sus concentraciones de Cd, los cuales fueron proporcionales al aumento en las concentraciones ambientales de exposición.

Palabras clave: Mytilus californianus, concha, capa de nácar, cadmio.

 

Introduction

Mytilus californianus is a characteristic organism of high-wave energy areas on the Pacific coast, where it is abundant, while other mussel species are absent; hence, this species could represent a good alternative for trace metal monitoring in these environments. Usually, mussel soft tissues are analyzed for their metal content, but in the case of M. californianus they show high variability, even in time spans of days (Lares and Orians, 1997), and their usefulness as a medium-term indicator has been questioned. For this reason, their shells could be a better alternative for medium-term (months) studies, since they are expected to retain metals for longer periods of time once the metals are incorporated into the calcium carbonate matrix.

Mytilus californianus has a shell composed of four principal layers (fig. 1a): the periostracum (entirely organic), nacreous layer (aragonite), and outer and inner prismatic layers (calcite) (Dodd, 1964). In order to provide an index of bio-availability, it is essential that the shell structure chosen for the analysis has not been exposed to either particulate or dissolved metals in the water column. Several authors (Sturesson, 1978; Bourgoin, 1990; Puente et al., 1996) have pointed out the advantage of analyzing metals associated with the nacreous layer, instead of the periostracum or calcite layer. This layer is located in the internal part of the shell and is isolated from the external medium (seawater); therefore, metals incorporated into the nacreous layer will represent only those accumulated by the organism metabolism (Bourgoin, 1990). On the other hand, the periostracum has the disadvantage of having direct contact with seawater, and could leave the outer calcite layer exposed if it is eroded.

The separation of the internal shell layer of M. californianus for its use in trace metal analyses has not been previously reported, although Sturesson (1976) and Bourgoin (1988) developed a similar approach using shells of M. edulis. However, it is known that M. edulis and M. californianus shells present several differences; while M. edulis has a carbonate shell composed of an outer prismatic (calcite) layer and an inner nacreous (aragonite) layer, the M. californianus carbonate shell has an extra inner prismatic layer (Dodd, 1964). Therefore, the aim of the present study was to develop a suitable clean technique for the separation of a nacreous layer section that represents the most recent growth of M. californianus shells.

 

Materials and methods

Mussels of 50-80 mm in length collected from Amphitrite Point, Canada, and from Ensenada, Mexico, were used in this study. Their shells were separated from the soft tissue with a stainless steel scalpel, cleaned from all extraneous material with a plastic brush under a flow of deionized distilled water (DDW) and, finally, air-dried for 2 days. To remove the perios-tracum, experiments were run using 30% H2O2 (ACS reagent, Sigma-Aldrich) at different temperatures (18, 60, 65, 70, 75, 80, 85, 90, 95 and 100°C) and time intervals (5, 10 and 15 min, 12 and 24 h). Once the periostracum was removed, a final rinse with 30% H2O2 (puriss. pa, Fluka) and DDW was performed. Once the valves were clean, they were heated in a muffle furnace to separate the nacreous layer from the calcitic layer. To find the optimum conditions for this treatment, several temperatures (300, 350, 400 and 500°C) and different time intervals (0.5, 1.0, 1.5, 2.0, 2.5 and 18.0 h) were tested on a minimum of five valves. Finally, the most recently deposited nacreous band was separated by gently tapping with a stainless steel needle. This band is located in the internal face of the valve, in the direction of the growth of the organism and adjacent to the internal part of the calcite layer (5 and 2 of fig. 1a, b, respectively).

In order to analyze Cd for the experiment presented as an example, the material of 10 valves was pooled and digested with 0.5 mL of 6M HCl and 1 mL of 7M HNO₃ (both Trace-Metal) at 75°C, evaporated until nearly dry and redissolved in 1 mL of 1M HNO₃ (TraceMetal). Cadmium was determined by graphite furnace atomic absorption spectrophotometry (GFAAS; Varian AA300/400) with Zeeman background correction. The standard additions technique was used to reduce matrix effects.

 

Results and discussion

Bourgoin (1988) developed a technique that uses only heat to separate the calcite from the nacreous layer. However, with this method the periostracum ashes remain next to the shells after they have been processed. Since the periostracum can accumulate several metals (e.g., Cd and Pb) to levels higher than the nacreous layer (Sturesson, 1976, 1978), these ashes could represent a potential contamination source if they ever come in contact with the nacreous layer. Hence, one of the improvements of the present method is the removal of the protein matrix that comprises the periostracum before the physical separation of the nacreous layer is carried out. According to our experiments, the best alternative to remove the perios-tracum from the shell, without affecting the calcium carbonate matrix (Babukutty and Chacko, 1992), was soaking the valves in 30% H₂O₂ at 80-85°C during 10-15 min. The rapidness with which the periostracum is separated has the additional advantage of allowing the processing of a large number of shells in a short period of time. At lower temperatures the periostracum was not removed (60-70°C), or was only partially eliminated (70-80°C). Increasing the H₂O₂ digestion time (12 and 24 h) at room temperature (18°C) only partially removed the perios-tracum, whereas at temperatures higher than 85°C the recently deposited nacreous layer band was partially lost.

The section of the nacreous layer isolated in this work (fig. 1) was different from that proposed by Bourgoin (1988) for M. edulis. This author obtained the sample by scraping the entire internal surface of the nacreous layer at a maximum depth of 0.1 mm. In the present work, it was observed that most of the valves of M. californianus exhibited a certain degree of acidification (chalky appearance) as well as internal repairs (purple coloration), in addition to the extra inner prismatic layer that M. californianus presents (fig. 1a); therefore, the scraping of the entire nacreous layer surface was not suitable for the analysis of shells from this species. Acidification and repairs were not observed in the zone of interest where the highest incorporation of CaCO3 to the aragonite layer occurs (Crenshaw, 1980). A consequence of acidification is the dissolution of the recently deposited CaCO3 (Wilbur and Saleuddin, 1983) and, hence, of the metals associated with this material as well. Presence of repairs may indicate that the seawater could have been in contact with the internal face of the shell, and the trace elements deposited in the nacreous layer may then represent a passive incorporation of metals into this layer, in addition to that resulting from the metabolism of the organism. For this reason, it was decided to separate only a band instead of scraping the entire internal surface of the nacreous layer, as reported by Bourgoin (1988).

The procedure for the optimum separation of the most recently deposited section of the nacreous layer involves heating the shells in a muffle furnace at 350°C for 1 h. With this treatment the valves preserved their integrity (which facilitated the handling) and the nacreous section of interest remained essentially intact, which allowed its easy separation from the rest of the shell by gently tapping it with a stainless steel needle. The use of higher temperatures and longer times (400°C for 18 h, as suggested by Bourgoin, 1988) made them fragile and, consequently, their handling was difficult. When they were heated at 300°C for 1 h, or at 400°C for 30 min, the targeted zone did not separate from the rest of the shell. Although the material of interest was also separated at 400°C for 1 h, a better shell consistency was obtained at 350°C for 1h.

The isolated section of one valve supplied an average of 4.2 mg of material. However, depending on the detection limits of the available analytical instrument, one or several valves can be pooled for the analysis (Rivero, 1999). The maximum length in the growth direction of the separated nacreous band was 2-3 mm (fig. 1b). Measurements performed in our laboratory indicate that the nacreous layer of shells 50-80 mm in length grows 90% lengthwise with respect to the total length of the shell. Hence, the 2-3 mm separated section represents a total growth of 2.2 to 3.3 mm in length. The period of time that the isolated section represents, in terms of mussel metal accumulation, will depend on the average growth rate of the organisms in the selected site which, in turn, will depend on the environmental conditions (Behrens-Yamada and Dunham, 1989), and the initial lengths of the organisms (Coe and Fox, 1942). Based on literature values (Coe and Fox, 1942; Behrens-Yamada and Dunham, 1989), we estimate that for the organisms analyzed in the present study, each isolated section probably represents 1 to 3 months in growth.

The method developed in this study to separate a section of the most recently deposited nacreous layer from the shell of M. californianus is fast and simple. This technique was applied to shells from organisms used in an experiment performed in our laboratory, where M. californianus were exposed for 60 days to three different dissolved Cd concentrations: 0.05 (control), 1 and 20 µg L^-1. Analysis of the most recently deposited nacreous layers showed significant increases in their Cd concentrations (0.078 ± 0.016 and 0.162 ± 0.018 µg g^-1, respectively) relative to the control (0.033 ± 0.006 µg g^-1) (fig. 2). The proposed method represents a valuable tool for processing large number of valves and, more important, it can be applied even in shells that are not in good external and internal conditions due to the high-energy environment where M. californianus species is normally found.

 

References

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