11:30 AM - 11:45 AM
[AOS20-04] Effects of pre-treatment on the isotopic composition of structural carbonate of teeth in ray-finned fishes
Keywords:Fish, Teeth, Isotope, Structural Carbonate
Although Actinopterygii is one of the most abundant taxa, its evolution and diversification remain largely unexplored. Fossil teeth are a powerful tool for understanding past Actinopterygii, and they are important samples abundant in marine sediments after the Cenozoic Era. In particular, stable carbon isotope ratios (δ13C・δ18O) of structural carbonate in teeth are expected to provide environmental, ecological, and physiological information. However, interpretations of the results of fossil sample analyses are difficult because the isotope analysis of modern samples has not been well studied, and the factors that cause variation in isotopic composition and the robustness as a proxy have not been adequately verified.
Compared to fossil tooth samples, modern tooth samples contain more organic matter. If organic matter decomposes in modern tooth samples stored at room temperature, not only will the sample deteriorate, but the CO2 gas and volatile organic matter generated may change the isotopic composition of the sample and contaminate the analyzer. Therefore, the consensus has been to remove organic matter by chemical treatment when conducting isotopic analysis of structural carbonates. However, no effective treatment for organic removal that preserves the isotopic composition of structural carbonate in dentin, which is the largest component of teeth, has been proposed. In this study, we developed a method to remove organic matter for isotopic analysis of structural carbonate in dentin.
In this study, half-smooth golden pufferfish(Lagocephalus spadiceus), which are widely distributed in the Indo-West Pacific and have large teeth, were used as a research target, and their teeth were collected as samples. To evaluate changes in the isotopic composition of structural carbonate due to pre-treatment, we measured changes in isotopic composition after various pre-treatments. In addition to physical cleaning, pretreatments involved chemical treatments with reagents like ethanol and acetone for fixation, hydrogen peroxide and sodium hydroxide solution for oxidation, and pancreatin and sodium hydroxide solution for hydrolysis of organic matter. Changes in carbonate amount(wt%) were also measured to determine the secondary formation of carbonate due to pre-treatment.
Although a large amount of organic matter remained in the samples that underwent physical cleaning(physically cleaned samples), the volatile organic matter did not affect the isotopic analysis. The samples that underwent physical cleaning did not experience any treatment that could cause the formation of secondary carbonate, and the amount of carbonate(wt%) detected in the analysis was close to the value reported in a previous study. Therefore, we concluded that CO2 gas emitted from the physically cleaned samples in the analysis was derived from structural carbonate and we analyzed the amount and isotopic composition of primary structural carbonate from the physically cleaned samples. Therefore, in this study, we used the amount(wt%) and isotopic composition of the structural carbonate in physically cleaned samples as references.
Treatment with neutral and alkaline aqueous solutions of pancreatin, composed of enzymes, apparently removed sufficient organic matter. The amount of carbonate(wt%) and δ13C did not change with treatment, indicating that the treatment with neutral/alkaline pancreatin solution is recommended to remove organic matter for the analysis of δ13C of structural carbonate in dentin.
On the other hand, no pre-treatment was identified that would sufficiently remove organic matter and retain the primary δ18O of structural carbonate. As the next best option, we recommend the treatment with the neutral aqueous pancreatin solution since the change in δ18O was relatively small. The change in δ18O(Δ18O) was relatively small in variance, and the degree of change was constant, suggesting the possibility of compensating for the altered δ18O by this treatment.
Compared to fossil tooth samples, modern tooth samples contain more organic matter. If organic matter decomposes in modern tooth samples stored at room temperature, not only will the sample deteriorate, but the CO2 gas and volatile organic matter generated may change the isotopic composition of the sample and contaminate the analyzer. Therefore, the consensus has been to remove organic matter by chemical treatment when conducting isotopic analysis of structural carbonates. However, no effective treatment for organic removal that preserves the isotopic composition of structural carbonate in dentin, which is the largest component of teeth, has been proposed. In this study, we developed a method to remove organic matter for isotopic analysis of structural carbonate in dentin.
In this study, half-smooth golden pufferfish(Lagocephalus spadiceus), which are widely distributed in the Indo-West Pacific and have large teeth, were used as a research target, and their teeth were collected as samples. To evaluate changes in the isotopic composition of structural carbonate due to pre-treatment, we measured changes in isotopic composition after various pre-treatments. In addition to physical cleaning, pretreatments involved chemical treatments with reagents like ethanol and acetone for fixation, hydrogen peroxide and sodium hydroxide solution for oxidation, and pancreatin and sodium hydroxide solution for hydrolysis of organic matter. Changes in carbonate amount(wt%) were also measured to determine the secondary formation of carbonate due to pre-treatment.
Although a large amount of organic matter remained in the samples that underwent physical cleaning(physically cleaned samples), the volatile organic matter did not affect the isotopic analysis. The samples that underwent physical cleaning did not experience any treatment that could cause the formation of secondary carbonate, and the amount of carbonate(wt%) detected in the analysis was close to the value reported in a previous study. Therefore, we concluded that CO2 gas emitted from the physically cleaned samples in the analysis was derived from structural carbonate and we analyzed the amount and isotopic composition of primary structural carbonate from the physically cleaned samples. Therefore, in this study, we used the amount(wt%) and isotopic composition of the structural carbonate in physically cleaned samples as references.
Treatment with neutral and alkaline aqueous solutions of pancreatin, composed of enzymes, apparently removed sufficient organic matter. The amount of carbonate(wt%) and δ13C did not change with treatment, indicating that the treatment with neutral/alkaline pancreatin solution is recommended to remove organic matter for the analysis of δ13C of structural carbonate in dentin.
On the other hand, no pre-treatment was identified that would sufficiently remove organic matter and retain the primary δ18O of structural carbonate. As the next best option, we recommend the treatment with the neutral aqueous pancreatin solution since the change in δ18O was relatively small. The change in δ18O(Δ18O) was relatively small in variance, and the degree of change was constant, suggesting the possibility of compensating for the altered δ18O by this treatment.