Dear Editor, Letter to the Editor

Nitrogen stable isotope ratios (dN) have successfully been applied in the study of trophic linkages, as well as of human impacts in aquatic ecosystems. Anthropogenic wastewater input typically elevates dN values in dissolved inorganic nitrogen and this N enrichment subsequently propagates throughout the food chain. Bivalve mollusks are of interest for studies of this human influence since they are primary consumers and are known to trace environmental dN variability. The dN values of their soft tissues have, for example, been found to correlate with the fraction of residential development in watersheds around lakes and salt marshes. To determine the ’undisturbed’ dN values in an ecosystem, before anthropogenic nitrogen input, dN records need to be extended into the past. Bivalve shells can be useful for this, since they are often abundant in archaeological deposits as well as historic museum collections. A predictable relationship has been demonstrated between the dN values of shell organic matter and soft tissues and dN values of this organic matrix indeed trace anthropogenic influences. Various sample preparation techniques have been used to analyze dN values of skeletal organic matter, such as acidification or simple combustion of whole skeletal material (without prior acidification). These methods are also used in analysis of organic matter (e.g., soft tissues, particulate organic matter). Animal soft tissue samples contain varying amounts of CaCO3, which will introduce a bias in dC measurements. Therefore, samples are generally treated with an HCl solution before analysis. However, the acidification process in itself may influence dN values, although some authors found no effect of acidification on dN values. Nevertheless, authors typically avoid acidification of samples for dN analysis andwill run one set of non-acidified samples for dN and one for dC (e.g., Bouillon et al.). If there are no effects of CaCO3 on d N analysis, then avoiding acidificationwould be the method of choice for dN analysis of shell organic matter. Moreover, direct combustion of shell material is easier and less time-consuming than acidification. In museum collections bivalve shells are traditionally dry-stored, whereas soft tissues are preserved in 70% ethanol, sometimes after fixation with 10% formalin. However, often the whole animal is preserved in ethanol and shells are not stored separately. For the application of these preserved specimens in the investigation of past dN values it is essential to know if liquid preservation methods have an effect on the dN values of bivalve shells and if this effect is predictable. The effects of liquid preservation on the dN values of biological tissues have been examined in a variety of animals. Syväranta et al. [27] found that neither formalin nor ethanol had a significant effect on dN values of preserved zooplankton and macroinvertebrates. However, in fish muscle, enrichments of 0.5 to 1.4% have been found after fixation in formalin and subsequent preservation in ethanol (Table 1). Results on mollusks differ among studies, but generally preservation effects on tissue dN values are small in short-term studies. Sarakinos et al. found that ethanol preservation lowered dN values of the soft tissues of the freshwater bivalve Corbicula fluminea by 0.39% after 6 months. Similarly, in the freshwater mussel Amblema plicata, ethanol preservation for 1 year caused a change of 0.23% in tissue dN values (Table 1). In contrast, some other workers found higher dN values for liquid-preserved mollusk tissue samples in comparison to frozen or dried samples. Ethanol preservation for 12 weeks resulted in a non-significant enrichment in octopus and Littorinid tissues. In Mytilus galloprovincialis and Patella vulgata, tissue dN values increased up to 1.1% and 1.0%, respectively, after treatment with formalin for 2 days and ethanol for 6–24 months (Table 1). In summary, wet preserved specimens typically exhibit a small enrichment in N, but this effect is variable between studies (Table 1). We report herein the evaluation of the method of simple combustion without acidification by testing the influence of CaCO3 content on d N values of different mixtures of acetanilide [C6H5NH(COCH3)] and synthetic pure CaCO3. We also investigate the fractionation between tissue and shell organic matrix in the bivalve Mytilus edulis. Finally, we examine the effects of long-term (73 years) ethanol preservation on dN values of bivalve shell organic matrix. For the comparison of dN values of mantle tissue and shell organic matrix, three specimens of the blue mussel Mytilus edulis were collected in 2002 in Knokke, Belgium (salinity 30; see Gillikin et al. for site description). For the investigation of the long-term effect of ethanol preservation, six shells from the Royal Belgian Institute of Natural Sciences collected at Dudzele (Belgium, from a canal linked to the North Sea) on 27March 1936were selected. Three individuals were stored dry and three individuals were preserved in ethanol along with whole soft tissues. In addition, dry-stored shells from three individuals collected at a nearby site at Lissewege on 22 November 1938 were obtained from the same museum and one shell, collected on 3 June 1935 at Knokke, was obtained from the Dutch National Museum of Natural History, Naturalis. All shell samples were rinsed with deionized water (>18MV/cm) and left to dry. The periostracum was completely removed with a Dremel abrasive buff. Calcite samples were taken from the outside of the shell with a hand drill; the inner aragonite layer was avoided. Between 10 and 20mg of calcite powder was collected, covering an area of at least 1 year of the most recent growth. The mantles from the ethanol-preserved specimens were dissected, rinsed with Milli-Q grade water and dried overnight at 608C and pooled. An aliquot of the ethanol these specimens were preserved in Rapid Commun. Mass Spectrom. 2011, 25, 675–680 (wileyonlinelibrary.com) DOI: 10.1002/rcm.4905 Letter to the Editor