Pre-treatment methods for stable oxygen and carbon isotope analyses of structural carbonates of bones in marine teleost fishes

Rationale: Although the proportion of structural carbonates in vertebrate bones is low, the values of isotopes, namely stable oxygen (delta O-18) and carbon (delta C-13), in structural carbonates provide environmental and physiological information, which can be beneficial for estimating the palaeontological and ecological parameters of vertebrates. However, a few studies have analysed the isotopes of structural carbonates in modern teleost fishes, and a well-developed protocol for sample preparation is lacking.Methods: We examined different pre-treatment methods of preparing bone samples of three marine teleost fishes (Japanese flounder, Pacific bluefin tuna and yellowtail) and investigated the effects of the cleaning methods on the stable isotope values of structural carbonates among vertebrae in the same individual. Isotope values were analysed using isotope ratio mass spectrometry.Results: Physical cleaning was the most promising pre-treatment method and resulted in delta O-18 values that were comparable to those of otoliths. Chemical treatments with NaOH and H2O2 changed the percentage of structural carbonates in the bone and affected delta O-18 and delta C-13 values. High-temperature treatments, such as boiling and roasting, altered delta O-18 values due to the exchange of oxygen with environmental water or vapour.Conclusions: Our results suggest that chemical cleaning methods used to prepare bone phosphate or collagen samples for isotope analyses are not suitable for structural carbonates. Physical cleaning is the appropriate pre-treatment method for analysing the isotopes of structural carbonates. Also, we emphasise that standardising the vertebral number is necessary to make delta C-13 values comparable between specimens in the same species.