Mussel tissue The batch of mussels (Mytilus edulis) was collected in the Oosterschelde estuary (The Netherlands) in October 1996 (total of 1000 kg). The material was obtained from a commercial supplier. After transport to the TNO institute, the outer surface of the mussels was cleaned by brushing with a nylon brush; then the mussels were placed in circular tanks (2.5 m diameter) supplied with fresh aerated clear coastal seawater for a period of 24 h. During this period, the mussels were allowed to defecate and depurate from possible sedimentary matter in the gut. After depuration, the mussels were packed in polythene bags in portions of about 10 kg wet-weight, immediately frozen at -20 °C and stored at a temperature to -20 °C until further processing. All further sample handling was carried out in a clean bench to minimise contamination. Titanium knives were used for cutting the tissue from the shells. Glassware was cleaned with 2 M HCl and demineralised water subsequently. All staff wore dust-free garments and polythene gloves. An amount of the frozen material that could be handled within a working day was allowed to thaw by natural action. Neither heating (nor microwave oven) was applied. The mussel shells were cut open and placed on a slightly tilted glass plate for about 20 minutes in order to drain the adhered seawater from the tissue (mantle water). The tissues were taken out from the shells and collected in a 2 litre glass beaker. The content of the beaker was transferred to a numbered polythene bag. The polythene bags were sealed and frozen at -20 °C until transportation. About 348 kg wet tissue weight were shipped to the IRMM for further treatment and bottling. There freeze-drying was carried out using an Epsilon freeze-drier with a shelf surface of 5.4 m². The freeze-drying programme consisted of a series of sequential steps: 45 h at -20 °C, 30 h at -10 °C, 25 h at 0 °C and 45 h secondary drying at +23 °C. After completion of the freeze-drying process the samples were collected in polyethylene containers and stored. The freeze-dried mussel tissue was ball-milled in a modified and upside Pulverisette planetary mill containing four 1.25 L Teflon bowls. Each bowl contained 26 Teflon balls of 20 mm diameter and 11 balls of 30 mm diameter with 120 g dry mussel tissue on top. The freeze-dried and ground powder was sieved through a 125 µm sieve and homogenised for 2 h in a Turbula mixer. Finally, the mussel tissue powder was bottled in well cleaned 30 ml brown glass vials capped with Teflon protected rubber stoppers with aluminium caps (10 g of dry mussel powder). Before closing, the vials were put into the freeze-drying chamber for a vacuum treatment; they were then filled with argon and stoppered using an hydraulic device. The water content was determined at 1.71 ± 0.30 % (mass fraction, Karl Fischer, n=20). The particle size analysis showed a top particle size of 305 µm, but with 99% of the material < 200 µm. The mean moisture content (as calculated from 12 sets of data) found in this certification campaign was (2.5 ± 1.5)%
keywords: biological material , animal tissue , foodstuff , fish/shellfish , trace elements , rare earth elements (REEs)
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