Dating the specimens

Amino acid racemization (AAR) analyses were undertaken on the intra-crystalline protein from four individual Bithynia tentaculata opercula from the Eemian type-site, Amersfoort (Cleveringa et al., 2000): Amersfoort-1, upper depth 27.71, lower depth 28.50 (NEaar 2982–3, 3972 and 4681) and compared with previously published data from a single horizon at Neumark-Nord 1 (15.5.87/2, Schluffmudde, 25 cm under Anmoor = surface of the lower shore area; NEaar 5698–5703 [Penkman, 2010]) and several horizons from Neumark-Nord 2 (Sier et al., 2011). All samples were prepared using procedures of isolating the intra-crystalline protein by bleaching (Penkman et al., 2008). Two subsamples were then taken from each shell; one fraction was directly demineralized and the free amino acids analyzed (referred to as the 'free' amino acids, FAA, F), and the second was treated to release the peptide-bound amino acids, thus yielding the 'total' amino acid concentration, referred to as the ‘total hydrolysable amino acid fraction (THAA, H*). Samples were analyzed in duplicate by RP-HPLC. During preparative hydrolysis, both asparagine and glutamine undergo rapid irreversible deamination to aspartic acid and glutamic acid, respectively (Hill, 1965). It is therefore not possible to distinguish between the acidic amino acids and their derivatives and they are reported together as Asx and Glx, respectively. The D/L values of aspartic acid/asparagine, glutamic acid/glutamine, alanine and valine (D/L Asx, Glx, Ala, Val) are then assessed to provide an overall estimate of intra-crystalline protein decomposition (Penkman et al., 2011).