Michael Raessler, Jan Rothe and Ines Hilke Determination of Al, Cd, Cr, Cu, Fe, Mn, Ni and Zn in woodlice and their skins – Is moulting a means of decontamination? Michael Raessler, Jan Rothe and Ines Hilke Max-Planck-Institut fuer Biogeochemie, Hans-Knoell-Strasse 10, D-07745 Jena, Germany

Why analyzing woodlice and their skins? They are: good bioindicators; widespread throughout rural and urban areas in Central Europe; relatively large, conspicous and easily collected; show a high tolerance for heavy metals; accumulate heavy metals efficiently in their hepatopancreas; can be reared under standardized conditions;

Sample preparation Woodlice were cultivated under standardized conditions in a climatic chamber at T= 22°C and a humidity of > 90% Animals were fed with carrots and lettuce from the same supplier; no additional water was provided The density of the population was regularly checked to avoid trophic enrichment of minerals by cannibalism After 1 year, 15 animals (30 – 40 mg dry weight) per species of the same age and size were picked out manually Animals were killed by cooling to – 18°C Animals were freeze-dried, pooled and ground

Skin of Animals Separately and manually collected after moulting every 2 – 3 weeks Pooled samples from 6 months of skin collection were dried and ground Subsamples of 50 mg (10 – 12 skins of adult woodlice) were used for digestion and analysis

Sample Digestion Microwave-assisted high pressure digestion (Multiwave, Anton Paar, Graz, Austria) Sample weight: 100 mg (woodlice and reference material ) 50 mg (skin samples) 3 mL nitric acid (HNO3), 65%, suprapur (Merck, Damrstadt, Germany) A blank was run with each series to check for possible contamination of reagents and vessels Digested samples were transferred to 50 mL glass vessels for analysis by ICP-OES

Reference Materials Dorm-2, Dogfish muscle (Squalus acanthias) SRM 1577b, BOVINE LIVER were selected to respond the following requests: To match the animal matrix as closely as possible To contain the elements under consideration in a concentration range which was both expected in the animals and also accessible to ICP-OES after digestion and dilution To contain certain elements in both standards for double control (Cu, Fe, Mn, Zn)

Analysis by ICP-OES (1) Plasma Gas 15 L/min Auxiliary Gas 0,5 L/min RF power 1300 watts Plasma Gas 15 L/min Auxiliary Gas 0,5 L/min Nebulizer Gas 0,8 L/min Pump Rate 1,3 L/min Nebulizer Cross-flow Spray Chamber Scott-type, Ryton Plasma View Axial Viewing Distance 15 mm Optima 3300 DV, Perkin Elmer, Norwalk, CT, USA

Analysis by ICP-OES (2) Element WL [nm] LOD [µg/L] LOQa [µg/kg] Al 396,152 0,8 400 Cd 226,502 0,4 200 Cr 267,716 Cu 224,700 1,0 500 Fe 259,940 0,3 150 Mn 259,372 0,1 50 Ni 231,604 Zn 213,857 0,5 250

Results of Dorm-2 Element Certified Concentration [mg/kg] Tolerance [mg/kg] Analysis [mg/kg] SD [mg/kg] Al 10,9 1,7 12,1 0,9 Cr 34,7 5,5 29,6 1 Cu 2,34 0,16 2,32 0,23 Fe 142 10 135,9 5 Mn 3,66 0,34 4,01 0,18 Ni 19,4 3,1 19,5 0,3 Zn 25,6 2,3 26,6 0,4

Results of Bovine Liver Element Certified Concentration [mg/kg] Tolerance [mg/kg] Analysis [mg/kg] SD [mg/kg] Cd 0,50 0,03 0,51 0,02 Cu 160 8 163 1 Fe 184 15 187 3 Mn 10,5 1,7 10,7 0,2 Zn 127 16 2

Analyses of P. scaber and their Skins Element Animal [mg/kg] SD [mg/kg] Skins [mg/kg] Al 229 0,5 631 6,4 Cd 2,58 0,04 < 0,2 Cr 2,02 0,08 3,04 0,2 Cu 171 2,8 8,4 0,1 Fe 249 1,4 502 2,1 Mn 77,6 0,4 142 0,8 Ni 4,02 9,9 Zn 170 28,2 1,1

Analyses of P. dilatatus and their Skins Element Animal [mg/kg] SD [mg/kg] Skins [mg/kg] Al 621 2,1 922 6,3 Cd 0,99 0,06 < 0,2 Cr 4,63 0,1 13,8 0,6 Cu 116 2,8 9,1 0,8 Fe 642 7,1 969 7,0 Mn 77 1,4 125 1,3 Ni 4,16 28,3 1,5 Zn 97,6 0,5 32,3

Comparison of P. scaber and P. dilatatus

Conclusions Enrichment in skins for Al, Cr, Fe, Mn and Ni No enrichment in skins observed for Cd, Cu and Zn Moulting is a possible means of decontamination in both P. scaber and P. dilatatatus for Al, Cr, Fe, Mn and Ni, but not for Cd, Cu and Zn

Acknowledgement Many thanks to Silja Ursel and Kristin Lober, FSU Jena And to all