Methodological comparison for the isolation of shell-bound organic matter for carbon, nitrogen and sulfur stable isotope analysis

Shell-bound organic matter (SBOM) is present in the shells of biomineralizing organisms and can act as an isotopic proxy for nutrition. Stable isotope analysis of SBOM generally requires its isolation from the mineral component of the shell, and this study shows that various shell removal techniques (cation exchange resin, ethylenediaminetetraacetic acid (EDTA), hydrochloric acid (HCl), and acetic acid) can influence the carbon (δ¹³C), nitrogen (δ¹⁵N) and sulfur (δ³⁴S) stable isotope values of both total SBOM and intra-crystalline SBOM to varying extents. In addition, isotopic and compositional differences are reported here between the different SBOM pools in the shell: total SBOM and intra-crystalline SBOM. Total SBOM isolated from Mytilus edulis, Ruditapes decussatus and Cerastoderma edule show minor differences in δ¹⁵N values between methods, but all treated samples have slightly higher δ¹⁵N values when compared to untreated shell powder. Methodological differences for δ¹⁵N values of intra-crystalline SBOM are also limited to ~1‰, with the exception of cation exchange resin (max. −4‰ compared to mean values). Use of the cation exchange technique is also discouraged for obtaining δ¹³C and δ³⁴S values for total and intra-crystalline SBOM, due to large deviations from mean values (to a maximum of −2‰ and −10‰, respectively). The other tested methods produce data with a 2‰-range for δ¹³C values for total SBOM, although for intra-crystalline SBOM δ13C values the use of acetic acid produced negative outliers. For sulfur stable isotope analysis extraction by EDTA is recommended, as acidification methods produce 1–2‰ lower δ³⁴S values for total SBOM, and using HCl can result in extremely negative intra-crystalline SBOM δ³⁴S values.