Age Determination of Modern Opercula of Turbo sp.

Age determination of Turbo sp. using the growth rings of calcified opercula is difficult because they lack clear periodic cycles in their rings that can be related to seawater temperature. We measured Mg/Ca and Sr/Ca ratios in calcified opercula of Turbo sp. but could not recognize any clear seasonal cycles (Figure S4) such as coral skeletal Sr/Ca (Hirabayashi et al. Reference Hirabayashi, Yokoyama, Suzuki, Kawakubo, Miyairi, Okai and Nojima2013; Zeng et al. Reference Zeng, Yokoyama, Hirabayashi, Miyairi, Suzuki, Aze and Kawakubo2022). Hence, the age assumptions of KTN-1 and K-HATE-1 were based on relationships between shell height and age.

We used modern and fossil calcified opercula, rather than shells, for age determinations. This approach requires that we first calculate their shell height/length based on the maximum calcified opercula lengths. Linear regressions comparing shell height/length, and maximum length of calcified opercula have been reported by Kinoshita (Reference Kinoshita2007) for Turbo marmoratus and Uno (Reference Uno1962) and Fujii (Reference Fujii1998) for Turbo sazae (Figures S2 and S3). Kinoshita (Reference Kinoshita2007) reported a linear relationship between shell height and maximum length of calcified opercula in Turbo marmoratus from the Amami Islands and Ryukyu Islands. Likewise, Uno (Reference Uno1962) and Fujii (Reference Fujii1998) reported a linear relationship for Turbo sazae. For modern samples, we used pairs of shells and their opercula to confirm that these samples fit the linear regression lines of Kinoshita (Reference Kinoshita2007), Uno (Reference Uno1962), and Fujii (Reference Fujii1998) (Figures S2a and S3a). Subsequently, we estimated calcified opercula lengths for each subsampling band used for radiocarbon dating based on imagery. Using the estimated opercula lengths in the subsampled bands, we calculated the shell height/length of these bands at their time of formation based on the linear regression lines of Kinoshita (Reference Kinoshita2007), Uno (Reference Uno1962), and Fujii (Reference Fujii1998).

We then estimated the ages at which the subsampling bands were formed using the relationships between shell height/length and age of Turbo marmoratus collected from Tokunoshima Island, the Amami Islands, reported by Igari et al. (Reference Igari, Matsumoto and Kitaue2001), and for Turbo sazae in Chiba reported by Midorikawa (Reference Midorikawa1986). The results are shown as “expected year” in Table 1. We could not estimate the ages at which the maximum length of a calcified operculum was smaller than 30 mm because of the lack of data in Kinoshita (Reference Kinoshita2007). Both KTN-1 and K-HATE-1 are estimated to have lived for at least three years based on the height and length of their shells. In the following section, we use the expected year of modern samples to compare their ¹⁴C variability with seawater radiocarbon data.

Table 1 Measured radiocarbon data of calcified opercula from living Turbo sp.

Radiocarbon Dating of Opercula of Turbo sp.

The ages of fossil calcified opercula of Turbo sp. were 871 ± 93 cal year BP for K-ARA-1, 647 ± 79 cal year BP for K-ARA-3 and, 642 ± 80 cal year BP for K-NISH-I. The ¹⁴C concentration (Δ¹⁴C as defined by Stuiver and Polach Reference Stuiver and Polach1977) of modern calcified opercula of Turbo sazae KTN-1 ranges from 14.36‰ to 32.03‰, averaging 21.73‰. The averaged ¹⁴C concentration of modern calcified opercula of Turbo marmoratus, K-HATE-1, was 32.26‰, ranging from 24.34‰ to 41.25‰.

The ¹⁴C concentration (Δ as defined by Stuiver and Polloch Reference Stuiver and Polach1977) of seawater samples collected at a depth of 10 m in 2019 at Kume Island was 22.03‰. Due to a lack of seawater radiocarbon datasets around the Ryukyu Islands since 2008, we assumed the radiocarbon variability from 2009 to 2019 in the Ryukyu Islands using coral skeletal radiocarbon data reported from Ishigaki Island (Hirabayashi et al. Reference Hirabayashi, Yokoyama, Suzuki, Miyairi and Aze2017; Yokoyama et al. Reference Yokoyama, Tims, Froehlich, Hirabayashi, Aze, Fifield, Koll, Miyairi, Pavetich and Kuwae2022) and seawater data measured at Kume Island in September 2019 (Table 2) and compared these values with the ¹⁴C concentrations recorded in Turbo sp. in this study.

Table 2 Measured radiocarbon data of seawater collected at Kume Island in 2019.

As mentioned above, we estimated the radiocarbon ages of each subsample based on the size of modern Turbo sp. opercula, using expected ages to calculate Δ and compare with seawater ¹⁴C datasets for the Ryukyu Islands (Tables 1 and 2; Figure 2). ¹⁴C variability in Turbo sp. was found to closely match with seawater ¹⁴C, suggesting that ¹⁴C in the calcified opercula of Turbo sp. reflect the ¹⁴C content in ambient seawater; this suggests that our age assumptions for KTN-1 and K-HATE-1 are reasonable. Both Turbo sazae and Turbo marmoratus ingest marine algae, whose dissolved inorganic carbon reflects ¹⁴C variability in ambient seawater (Satoh et al. Reference Satoh, Fukuda, Miyairi, Yokoyama and Nagata2019). Ota et al. (Reference Ota, Yokoyama, Miyairi, Hayakawa, Satoh, Fukuda and Tanaka2021) reconstructed ¹⁴C variability in Otsuchi Bay based on ¹⁴C data recorded in abalone shells, which also ingest marine algae; as such, their shells also reflect ¹⁴C variability in ambient seawater. ¹⁴C data in KTN-1 were always ≤10‰ higher than those of K-HATE-1, suggesting that differences in the depth and location of the organism’s habitat between Chiba and the Ryukyu Islands have some influence.

Figure 2 Radiocarbon profile of living Turbo sazae (KTN-1, blue) collected from Katsuura, living Turbo marmarotus (K-HATE-1, red), and seawater at Kume Island (black). The radiocarbon content of Kuroshio seawater was estimated based on coral skeletal radiocarbon data from Ishigaki (Hirabayashi et al. Reference Hirabayashi, Yokoyama, Suzuki, Miyairi and Aze2017; Yokoyama et al. Reference Yokoyama, Tims, Froehlich, Hirabayashi, Aze, Fifield, Koll, Miyairi, Pavetich and Kuwae2022) and seawater in Kume Island measured in this study.

Uranium Distribution

Averaged uranium concentrations of modern samples range from 0.003 to 0.115 ppm (Table 3). Our results show that uranium in the opercula of modern Turbo sp. were unevenly distributed and that the concentrations recorded are 1000 times less than that measured in coral skeletons (Figure 3), while radiocarbon in opercula samples reflect the radiocarbon values of the ambient seawater as well as corals. The U/Ca ratios of both modern and fossil Turbo sp. samples were lower than those of corals and seawater (1.3×10⁻⁶) reported by Edwards et al. (Reference Edwards, Gallup and Cheng2003). U/Ca in modern Turbo sazae samples ranges from 10⁻¹⁰ to 10⁻⁶, while that of Turbo marmoratus ranges from 10⁻⁸ to 10⁻⁷. U/Ca ratios in fossil Turbo sazae and Turbo marmoratus samples were 10⁻⁷ to 10⁻⁶. These results suggest that either biological control or the vital effect of marine gastropods influences uranium incorporation from ambient seawater more strongly than in corals. Based on differences in their U/Ca ratio, the vital effect in Turbo marmoratus was possibly stronger than that in Turbo sazae, although the number of measurements from which this suggestion is drawn are limited, and more investigation is needed to fully clarify the mechanism responsible for this observation.

Table 3 Measured radiocarbon, U, Mg, Sr, and Ca contents of calcified opercula from Turbo sp.

* This sample was cut K-ARA-1 to measure uranium destribution in the cross section of the Turbo sp.

Figure 3 Uranium distributions in calcified opercula of (a) modern Turbo sazae samples KTN-1 and KTN-3, (b) fossil Turbo sp. samples K-ARA-1 and K-ARA-3, (c) modern Turbo sazae sample KTN-5 (cross section), (d) modern Turbo marmoratus sample K-HATE-1, (e) fossil Turbo marmoratus sample K-NISHI, and (f) fossil Turbo sp. sample K-ARA-1 (cross section). Measurement lines are shown in Figure S1.

In all samples, uranium is most concentrated in the central area, where the age of the organism is likely less than 1 year based on the radiocarbon results discussed above. Uranium was not concentrated into the opercula (more than one year old) of either Turbo sazae or Turbo marmoratus. The highest uranium values, approximately 2.2 ppm, were recorded in the central regions of the Turbo sazae samples, KTN-1 and KTN-5, which are almost the same as the uranium content of corals. Modern Turbo marmaoratus also contained higher uranium concentrations in the central area than other areas of the opercula, although the highest uranium values in Turbo marmoaratus were one order of magnitude lower than those in Turbo sazae (Table 3).

During the larval stage of Turbo sp., the organism adopts a lecithotrophic life mode with a short non-feeding pelagic stage. Shell formation in such non-feeding veligers, including Turbo sazae, is thought to be completed in the veliger stage (Onitsuka et al. Reference Onitsuka, Kimura, Ono, Takami and Nojiri2014). Their non-feeding larval stage is usually finished within a few days, after which they settle as juveniles to start feeding on microalgae such as diatoms in the case of Turbo marmorotus; and on coralline algae in the case of Turbo sazae. Large juveniles and adults feed on the red algae Gracilaria, Hypnea, and Echeuma and brown algae, such as Sargassaum. Uranium concentrations in seawater in Ibaraki and Mie were reported as 3.1 ± 0.3 and 2.9 ± 0.3 ng/ml, respectively (Matsuba et al. Reference Matsuba, Ishii, Nakahara, Nakamura, Watabe and Hirano2000). Miyake et al. (Reference Miyake, Sugimura and Mayeda1970) measured the uranium contents of the open ocean water and in marine algae, showing that concentrations in marine algae were approximately 500–600 ng/g and that the uranium activity ratio in marine algae reflected that of ambient seawater. Ishii et al. (Reference Ishii, Nakahara, Matsuba and Ishikawa1991) reported uranium distributions in marine organisms, finding higher uranium concentration in soft tissues relative to shells in the case of Tridacna squamosa. The highest uranium concentrations of 370 ng/g were identified in the soft tissues of the liver in Tridacna squamosa. We therefore propose that the high uranium concentrations recorded in the central opercular regions in modern samples are related to their non-feeding pelagic stage, as opposed to post-settlement. Uranium concentration has not yet been reported in the yolk of the organism, but it is possible that these concentrations would also be high. Additional investigations will be needed to evaluate this.

The average values of uranium concentrations in all fossil samples were higher than in modern samples. However, the maximum uranium concentration values in fossil samples were lower than modern samples with the exception of Turbo marmoratus (Table 3). The highest uranium values in fossil Turbo sazae samples were located approximately 5–10 mm from the center, which differs from modern samples (Figure 3). Likewise, the highest uranium concentrations in fossil Turbo marmoratus were also located 5–10 mm from the center (Figure 3e). These results suggest that uranium uptake occurred especially in the area of ∼5 mm outside from the center, with subsequent uranium loss from the central opercular area after the organism’s death and deposition.

Alying et al. (Reference Ayling, Eggins, McCulloch, Chappell, Grün and Mortimer2017) measured uranium concentrations in modern and fossil Tridacna gigas samples from MIS 5e and MIS 11 using LA-ICP-MS, showing that uranium uptake occurred mostly in the outer zones of the fossils. Additionally, Alying et al. (Reference Ayling, Eggins, McCulloch, Chappell, Grün and Mortimer2017) developed simple uranium uptake and loss models for T. gigas, suggesting that uranium adsorption occurred within the first 200,000 years after death and that loss occurred after 50,000 years. In case of two Turbo sazae samples, K-ARA-1 and K-ARA-3, the areas with the highest uranium values differ although their age difference is only 230 years. In the case of K-ARA-3, areas with high uranium concentrations were 1.5 mm and 6 mm from the center; for K-ARA-1, this concentrated region was 2–6 mm from the center. These differences might reflect uranium uptake and loss processes in the opercula of Turbo sazae during the aforementioned 230-year period. Older fossil Turbo sp. samples are required to fully investigate uranium input and output processes over time.

Considering other elements, such as Sr, Mg, and Ca measured simultaneously with U by LA-ICP-MS (Figure S4), we found no correlation between U and Mg/Ca either in the modern or fossil samples (Figure 4a). Unlike U, concentrations of Sr and Mg in fossil and modern samples did not significantly change. Because calcic opercula of Turbo sp. consist of aragonite, Mg/Ca ratio would be increased if their opercula underwent diagenesis. Distribution profiles of U/Ca ratios in the fossil calcified opercula was different from that observed for Mg/Ca ratio (Figure S4), suggests that heterogenous uranium distribution in fossil samples did not result from diagenesis. We also observed crystals of modern and fossil samples using scanning electron microscopy−energy dispersive X-ray spectroscopy (SEM-EDS) (Figure S5, Figure S6, Figure S7). We did not identify any recrystallization at the surface of fossil Turbo marmoratous. Alying et al. (Reference Ayling, Eggins, McCulloch, Chappell, Grün and Mortimer2017) suggested that uranium uptake may be partly controlled by diagenetic changes. We note however, that since the uranium distributions in K-NISHI, KTN-1, and KTN-3 are similar, areas in which uranium was easily adsorbed were not primarily controlled by diagenesis.

Figure 4 Relationship between uranium concentrations and (a) Mg/Ca and (b) Sr/Ca ratios in modern and fossil Turbo sp. measured using laser-ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS).

Positive correlations were found between U and Sr/Ca in both modern samples and fossil samples with the exception of modern Turbo marmoratus (Figure 4b). Elliot et al. (Reference Elliot, Welsh, Chilcott, McCulloch, Chappell and Ayling2009) suggested that Sr/Ca ratio in T. gigas exhibited no correlations with temperature, growth rate, and metabolic rate. On the other hand, studies using other species, such as S. giganteus and M. mercenaria, have suggested that Sr incorporation into the shell was influenced by growth rates (Takesue and van Geen Reference Takesue and van Geen2004; Gillikin et al. Reference Gillikin, Lorrain, Navez, Taylor, André, Keppens, Baeyens and Dehairs2005). In case of Turbo sp., Sr/Ca in the calcified opercula did not have any correlations with temperature and growth rates judging from the similarly heterogeneous distribution of U and Sr.

We estimated the age difference reflected by the heterogenous distribution of uranium in fossil Turbo sp. samples. The U/Th ages were determined using following equation (Edwards et al. Reference Edwards, Chen and Wasserburg1987):

(1) $${\left[ {{{{}_\;^{230}Th\;} \over {{}_\;^{238}U}}} \right]_{act}} - 1 = \; - {e^{ - {\lambda _{230}}T}} + {{\delta {}_\;^{234}{U_m}} \over {1000}}\left( {{{{\lambda _{230}}} \over {{\lambda _{230}} - {\lambda _{234}}}}} \right)\left( {1 - {e^{ - \left( {{\lambda _{230}} - {\lambda _{234}}} \right)T}}} \right)$$

(2) $$\delta^{234}U = \left(\left[\frac{^{234} U}{^{238} U}\right]_{act} - 1\right) \times 1000 \; (\unicode{x2030})$$

(3) $$\delta {}_\;^{234}{U_m} = \delta {}_\;^{234}{U_i}\;{e^{ - {\lambda _{234}}T}}\;$$

where T is sample age, [²³⁰Th/²³⁸U]_act and [²³⁴U/²³⁸U]_act are the activity ratios for each isotope, and λ₂₃₀ and λ₂₃₄ are the decay constants for ²³⁰Th and ²³⁴U, respectively. We used decay constants of λ₂₃₀ = 9.1705 ×10⁻⁶ year⁻¹ and 2.82206 ×10⁻⁶ year⁻¹ as reported by Cheng et al. (Reference Cheng, Edwards, Shen, Polyak, Asmerom, Woodhead, Hellstrom, Wang, Kong, Spötl, Wang and Alexander2013). δ²³⁴U_m is the uranium isotope ratios corresponding to the time at which U/Th was measured and δ²³⁴U_i is the initial value. We assumed a constant ²³⁰Th concentration in the calcified opercula of Turbo sp. and that ²³⁴U/²³⁸U in the calcified opercula of Turbo sp. was as same as that of seawater. Therefore, we used a δ²³⁴U_initial of 146.8 following Andersen et al. (Reference Andersen, Stirling, Zimmermann and Halliday2010) and calculated δ²³⁴U_m using Eq. (3) for each fossil sample. We calculated ²³⁰Th concentrations using Eq. (1) with the minimum ²³⁸U concentrations in each fossil sample. We used calibrated ¹⁴C ages using Marine20 shown in Table 3 as the value of T in Eq. (1). After evaluating the ²³⁰Th concentration, we calculated U/Th ages using the maximum ²³⁸U values in each sample and the estimated ²³⁰Th. The age differences were found to be 800 years, 602 years, and 605 years in KTN-1, KTN-3, and K-NISHI, respectively (Table S1). Although some assumption were made in determining the distribution of ²³⁰Th and δ²³⁴U in the calcified opercula of Turbo sp., our estimations suggest that uranium uptake and loss processes should be considered when using fossil marine mollusks for U/Th dating. Contrary to Arslanov et al. (Reference Arslanov, Tertychny, Kuznetsov, Chernov, Lokshin, Gerasimova, Maksimov and Dodonov2002) and Cheong et al. (Reference Cheong, Choi, Khim, Sohn and Kwon2006), who suggested that U/Th dating may be performed using Holocene samples, our results warn against using Holocene Turbo sp. samples from the Japanese archipelago for U/Th dating without careful scrutiny.