1 INTRODUCTION

Cold seeps are seafloor manifestations of methane-rich fluids which are mainly composed of methane and hydrogen sulfide and are globally distributed on the seafloor of continental margins, transporting these fluids from sediments into the ocean and even the atmosphere (Campbell, 2006; Boetius and Wenzhöfer, 2013; Suess, 2014; Ceramicola et al., 2018). Now, cold seeps, one of the important sinks for carbon and sulfur, are recognized as a significant part of the global carbon and sulfur cycles (Peckmann and Thiel, 2004; Yoshinaga et al., 2015; Zhang et al., 2017; Wang et al., 2018; Bowles et al., 2019; Sun et al., 2019; Dong et al., 2020). In cold seeps, seepage of the methane-rich fluids results in the formation of the authigenic carbonates and chemoautotrophic biocoenoses which mainly depend on a specific mechanism of anaerobic oxidation of methane (AOM) (Boetius et al., 2000). Meanwhile, these authigenic carbonates and chemoautotrophic biocoenoses also become faithful recorders of the methane-rich fluids seepage (Callender and Powell, 1999; Feng et al., 2016). Peckmann et al. (2009) first stated that the cement content in authigenic carbonates could be used as an approximation of seepage intensity since more intense seepage appears to favor cement precipitation. After that, a series of indicators, including the Mg/Ca and Sr/Ca ratios and Mo content of submarine sediment (Chen et al., 2016; Wang et al., 2018), S isotopes of sulfur compound (Zhang et al., 2018), and biomarker contents in authigenic carbonates (Guan et al., 2016), have been proposed to trace the seepage history and even the seepage intensity of the methane-rich fluids in cold seeps. These pioneer studies have provided important efforts to assess the seepage history of cold seeps.

As the important parts of these chemoautotrophic biocoenoses, bivalve species, e.g., Gigantidas platifrons, Akebiconcha marissinica, and Archivesica marissinica, are common in cold seeps. The growth processes of these bivalves are influenced by the methane-rich fluids and the surrounding seawater, which might be geochemically and isotopically recorded by bivalve shells (e.g., Lietard and Pierre, 2008, 2009; Yang et al., 2014; Feng et al., 2018a; Riekenberg et al., 2018). Previous studies have shown that some specific elements in bivalve shells in marine and freshwaters, e.g., Mg, Ba, S, and Sr, are greatly influenced by variations in temperature and salinity of waters, making they were widely used in reflecting the palaeoenvironment (Putten et al., 2000; Gillikin et al., 2006; Schöne, 2008; Poulain et al., 2015). However, rare studies were involved in the bivalves in cold seeps. Hence, a series of geochemical analyses, including LA-ICP-MS elemental and mapping, in-situ XRD, and SEM analyses, were conducted on a bivalve shell fossil of the G. platifrons, collected in the ROV2 site of Haima cold seep, South China Sea (SCS) (Fig. 1). We examine the Mg/Ca, Ba/Ca, and S/Ca ratios of the profile, aiming to assess the physical environment variation and provide insight into the relationship between the S/Ca ratios and growth rate and/or seepage intensity variation of the cold seep activity history.

2 MATERIAL AND METHOD 2.1 Description and preparation of the shell fossils

The active Haima cold seeps with an area of 350 km², first discovered in 2015, are located at approximately 1 370–1 390-m water depth on the lower continental slope of the northwestern SCS. The Haima cold seeps host abundant chemosynthetic communities, methane-derived authigenic carbonates, and massive gas hydrates (Liang et al., 2017). The bivalve shell fossils were collected from the Haima cold seep of the southern Qiongdongnan Basin, SCS, at a water depth of 1 381 m by the Remote Operated Vehicle (ROV) "Haima" during the Haiyang-6 cruise conducted by Guangzhou Marine Geological Survey in 2016. These bivalve shell fossils are cemented by the authigenic carbonates (Fig. 2a). The ¹⁴C ages of these shell fossils were 5.1–6.1 ka. BP, reported by Liang et al. (2017). These authigenic carbonates, mainly white to grey in color, are composed of aragonite, foraminifer fossils, and microcrystalline carbonate matrix with minor Fe-sulfides (Fig. 2a–d).

The shell piece, with a length of 1 cm and a width of 2 mm, was cut by a handheld cutting machine (Fig. 2b). Firstly, the sample was soaked in the sodium hydroxide solution with a mass fraction of 20% for 1 h in a water bath of 65 ℃ to remove the organic materials (Zhao et al., 2015). Secondly, it was both cleaned with deionized water and then dried for 24 h. Thirdly, this shell piece was mounted into an epoxy resin block. Finally, this cross-section was polished to obtain a flat surface. But before the analysis, the surface of the sample was etched by using the acetic acid with a mass fraction of 10%.

2.2 Scanning electron microscope (SEM) imaging for the bivalve shell fossil

The SEM imaging for this sample was carried out on the TESCAN MIRA 3 SEM instrument at the Guangzhou TuoYan Analytical Technology Co., Ltd., Guangzhou, China. This instrument equips a high-intensity Schottky field emission gun with electron energy of 200 eV–30 keV, beam current of 2 pA–100 nA, and resolution ratio of 1.2 nm@30 keV. Before the analyses, the samples were first cleaned in an ultrasonic cleaner which is filled with deionized water. Then, it was plated with gold to enhance its electrical conductivity. The location of these SEM images was marked in Fig. 3a.

2.3 In-situ X-ray diffraction (XRD) analyses for the bivalve shell fossil

The XRD analyses for the shell section were carried out on a Rigaku Rapid IIR micro-diffractometer at the Key Laboratory of Metallogenic Prediction of Nonferrous Metals and Geological Environment Monitoring, Ministry of Education, Central South University, Changsha. The X-ray was emitted by a copper target. The measuring process was set as the following parameters, i.e., a voltage of 40 kV and electricity of 250 mA. In addition, the measuring duration was 20 min for each analysis. The raw data was conducted by Jade 6.5.

2.4 In-situ LA-ICP-MS trace element analysis and mapping of the bivalve shell

Trace element analysis and map scanning of bivalve shells were conducted by LA-ICP-MS at the Guangzhou TuoYan Analytical Technology Co., Ltd., Guangzhou, China. Laser sampling was performed using an NWR 193 laser ablation system. An iCAP RQ ICP-MS instrument was used to acquire ion-signal intensities. The trace element composition analysis was conducted with helium as a carrier gas. Argon was used as the make-up gas and mixed with the carrier gas via a Y-connector before entering the ICP. The spot size, frequency, and energy of the laser were set to 50 µm, 5 J/cm², and 6 Hz, respectively. In this study, analyses were carried out in the middle of the calcite layer of the bivalve shell fossil with a spot spacing of 100 μm along with the growth profile. Glass standards reference NIST 610 and NIST 612 and carbonate standard MACS-1 were used as external standards for trace element calibration. Each analysis incorporated a background acquisition of approximately 30 s followed by 40 s of data acquisition from the sample. Raw data were calibrated against multiple silicate reference materials without using an internal standard (MRM-NoIS) by using an Excel-based software ICPMSDataCal (Liu et al., 2008). Analytical accuracy was recommended values within 10% for trace elements and better than 5% for major elements by using the MRM-NoIS calibration strategy, which was described in detail by Chen et al. (2011). The LA-ICP-MS trace element mapping was carried out, using the laser size, frequency, energy, and scanning speed of 30 µm, 3.5 J/cm², 6 Hz, and 150 µm/s, respectively. A detailed measuring procedure was described by Paton et al. (2011).

3 RESULT 3.1 SEM imaging and XRD analyses

Based on the observation, the shell can be subdivided into two major parts: unaltered zones and altered zones. The former mainly contains two microstructures, i.e., prismatic structure and stratified structure (Fig. 3b–e). The prismatic structure is mainly distributed in clusters with a series of uneven prismatic materials (Fig. 3b–e). The stratified structure material, the major part of the nacreous layer, is composed of the laminated lamella, with each lamella of 1–2 μm (Fig. 3d–e). In addition, the altered zones are mainly characterized by amorphous features (Fig. 3f–g), indicating that these zones might be influenced by the authigenic carbonates and/or the methane-derived fluids.

XRD analyses on the prismatic layer of the bivalve shell were carried out in this study. As shown in Fig. 4, the legible spectral peaks of the fibrous prismatic layers indicate a high degree of crystallinity. In addition, the prismatic layers of the bivalve shell show similar diffraction peaks of each lattice plane to the standard aragonite, i.e., (111), (021), (102), (130), (220), (221), and (041). The result illustrates that the mineralogical composition of the prismatic layer of the bivalve shell is composed of aragonite.

3.2 LA-ICP-MS mapping and Mg/Ca, Ba/Ca, and S/Ca ratios

LA-ICP-MS mapping results for the shell profile were represented in Fig. 5. Generally, the prismatic layer has lower Ba/Ca, S/Ca, and Mg/Ca ratios than the nacreous layer (Fig. 5), which is confirmed by the detailed studies from Takesue et al. (2008). However, some altered zones of this profile, marked in Fig. 5, contain abnormal high Ba/Ca, Mg/Ca, and S/Ca ratios which are ten times more than these unaltered zones.

Eighty-three analyses were carried out on the prismatic layer along with the growth profile (Fig. 5). The results of Mg/Ca, Ba/Ca, and S/Ca ratios were represented in Table 1. Mg/Ca, Ba/Ca, and S/Ca ratios range 0.56–0.97 mmol/mol (mean value of 0.84 mmol/mol), 0.01–0.11 mmol/mol (mean value of 0.04 mmol/mol) and 0.04–23.2 mmol/mol (mean value of 4.11 mmol/mol), respectively. However, in order to avoid the influences from the altered zones, a series of data (spots Nos. 1–39) were selected, with Mg/Ca, Ba/Ca and S/Ca ratios of 0.72–0.97 mmol/mol (mean=0.87 mmol/mol), 0.01–0.06 mmol/mol (mean= 0.04 mmol/mol) and 0.04–4.16 mmol/mol (mean=1.32), respectively. Generally, the selected Mg/Ca, Ba/Ca, and S/Ca ratios show wave-type variability (Fig. 6). However, due to the relatively low Mg/Ca and Ba/Ca ratios, Mg/Ca and Ba/Ca ratios did not show obvious variations along the section, implying that Mg/Ca and Ba/Ca ratios remain constant (Fig. 6). In addition, Mg/Ca ratios generally correspond to the S/Ca ratios, whereas the Ba/Ca did show a positive relationship with the Mg/Ca and S/Ca ratios (Fig. 6).

4 DISCUSSION 4.1 Mg/Ca constraints on the temperature of the cold-seep province

Bivalves can incorporate some specific elements into their shells during the growth process which are sensitive to the physical and biological properties of the surrounding seawater (Poulain et al., 2015). These elements will be enriched and/or depleted in response to the physical and biological variations of the surrounding seawater and organisms, therefore, they can be used as efficient tracers to reflect these variations (Schöne et al., 2002, 2010, 2011; Schöne, 2008; Gillikin and Dehairs, 2013). Previous studies proposed that the Mg/Ca ratios of carbonate minerals in solids and liquids show a close relationship with the temperatures (Mucci, 1987). In addition, it is well accepted that Mg/Ca ratios in seawater remain invariable if the seawater salinity is over 10% (Dodd and Crisp, 1982). Therefore, the Mg/Ca ratios of shells mainly reflect the temperature of the surrounding seawater (e.g., Mucci, 1987; Putten et al., 2000). In this study, Haima bivalve shell fossils show low Mg/Ca ratios of the 0.56–0.97 mmol/mol (mean= 0.84 mmol/mol), remarkably lower than the Mg/Ca ratios of Mytilus edulis reported by Putten et al. (2000), Wanamaker et al. (2008), and Freitas et al. (2008). In addition, the low Mg/Ca variations along with the section further imply that the surrounding seawater temperatures keep constant. Based on this positive correlation between the Mg/Ca ratios and temperature, a series of Mg/Ca-temperature calibration curves have been successfully developed to estimate the water temperature (e.g., Putten et al., 2000; Freitas et al., 2012; Mouchi et al., 2013; Tynan et al., 2017). The temperatures for these selected data, calculated by the model from Mouchi et al. (2013), range from 3.8 to 4.1 ℃ (mean=4.0 ℃), consistent with the measured data of 3.9 ℃ which was conducted by a conductivity-temperature-depth system (CTD) during the same cruise. In addition, the LA-ICP-MS mapping results also show that the Mg/Ca ratios of the section show a rising trend in these altered zones, but the detailed Mg/Ca ratios vary in a limited range which has a minor impact on these calculated temperatures (Fig. 5). Therefore, these calculated temperatures show minor variations, indicating that the surrounding seawater temperatures likely remain constant, which is consistent with the low temperatures of the deep-sea environment.

4.2 Ba/Ca constraints on the salinity of the cold-seep province

A previous study proposed that the Ba concentration profile of aragonite biominerals was regarded as a forceful tool for productivity which is mainly governed by the Ba concentration of ambient water (e.g., Elliot et al., 2009). A further study reported that the shell Ba/Ca ratios were controlled by Ba/Ca ratios of the surrounding waters (Poulain et al., 2015). This property makes the shell Ba/Ca ratio an efficient tool to track the Ba/Ca ratio of waters and then rebuild the salinity of waters (Gillikin et al., 2006, 2008). However, Rosenheim et al. (2005) hypothesized that Ba incorporation into shell may be restricted by ambient water with high Ba concentration, implying that Ba/Ca be not a reliable paleoenvironmental proxy where the Ba concentration is high. Other factors might also influence the Ba/Ca ratios of shells, e.g., type of bivalve species, shell mineral compositions, and growth rate (Rosenberg et al., 2001; Gillikin et al., 2008; Izumida et al., 2011; Hatch et al., 2013; Marali et al., 2017). However, Ba/Ca in carbonate skeletons, as a palaeoenvironment proxy, plays a significant role in the reconstruction of the paleosalinity (Gillikin et al., 2006; Sarimin and Mohamed, 2014; Poulain et al., 2015; de Nooijer et al., 2017). In this study, the section of bivalve shell is featured by relatively low Ba/Ca ratios of 0.01–0.11 mmol/mol (mean=0.04 mmol/mol) which is consistent with the unaltered zones of 0.01–0.06 mmol/mol (mean=0.04 mmol/mol). In addition, the LA-ICP-MS mapping also reveals that this section hosts invariable Ba/Ca ratios (Fig. 5). Consequently, these minor variations of Ba/Ca ratios indicate that the surrounding seawater salinity remains constant during the bivalve growth.

4.3 S/Ca: a potential tracer for recording the growth rate?

Previous studies indicated that sulfur (S) might be served as organic S and inorganic S in bivalve species (Dauphin et al., 2005; Yoshimura et al., 2013, 2014; Tamenori and Yoshimura, 2018). The former exists in the organic tissues as amino acids and sulfated polysaccharides (Dauphin et al., 2005; Tamenori and Yoshimura, 2018), whereas the latter is incorporated in the shell CaCO₃ as inorganic sulfate (Yoshimura et al., 2013, 2014). In addition, Yoshimura et al. (2014) further illustrated that inorganic sulfate is the primary species in coral calcite skeletons, with a minor distribution of organic sulfurs. Some researchers argued that those higher shell organic contents might be associated with high sulfur concentrations in biogenic mollusk carbonates (Rosenberg et al., 2001; Dauphin et al., 2005; Lazareth et al., 2007). One conceivable reason for his debate is the difference in the nature of the bivalve species (Tamenori and Yoshimura, 2018). Previous studies have proposed that S in bivalve shell cross-sections is correlated with the annual lines, which could be used as a tracer to record the growth process of shells (Dauphin and Cuif, 1999; Cuif et al., 2008; Takesue et al., 2008). In general, element substitution in CaCO₃ is governed by equilibrium partitioning or kinetically controlled inorganic partitioning, therefore, the S content tends to increase with the CaCO₃ precipitation rate (Busenberg and Plummer, 1985). Commonly, shallow sea bivalves will have a higher growth rate in summer than in other seasons since the high temperature will promote CaCO₃ precipitation. Inversely, the higher growth rate of these bivalves will result in the elevation of the CaCO₃ precipitation and the assimilation of some specific elements, e.g., Mg and S (Lorens and Bender, 1980; Poulain et al., 2015). As aforementioned, growth rate have a powerful impact on the Mg/Ca ratios of the bivalve shells, Mg in shell carbonate is highly regulated by the organism during shell precipitation, which means that a high growth rate likely promotes the compatibility for Mg in the shells (Lorens and Bender, 1980). In addition, Shirai et al. (2008) reported a positive relationship between Mg and S contents in shells of the Bathymodiolus platifrons. In addition, detailed fields with Mg and S contents did not conform to the locations of growth rims (Shirai et al., 2008), indicating that locations of growth rims, representing a low growth rate, are negatively related to the high Mg and S contents. Therefore, the growth rate might affect the variations of not only the Mg/Ca ratios but also the S/Ca ratios. In order to avoid the negative influence on the S/Ca ratios by the authigenic carbonate rocks and/or methane-derived fluids. A series of data for the unaltered zones of the shell section was intercepted for further discussion. As shown in Fig. 6, the selected S/Ca ratios have regular variations which generally correspond to the variations of the Mg/Ca ratios, confirming that the S/ Ca ratios of bivalve shells can also reflect the growth rate of the G. platifrons. Although the sample was firstly conducted by removal procedures of organic materials in this study, it is not certain whether the organic S was removed thoroughly. Therefore, the shell aragonite mainly contains inorganic S in the shell of the G. platifrons, whereas the residual organic S might also be a potential S source for the data in this study. However, the influences from the residual organic sulfurs might be negligible for the following reasons: 1) the sample was firstly conducted by removal procedures of organic materials in this study; and 2) the shell mainly contain inorganic sulfurs with a few organic sulfurs as proposed by Tamenori et al. (2014).

To our knowledge, G. platifrons, living in the cold-seep province, are parts of these chemosynthesis-based species and they get energy from the co-existing chemoautotrophic bacteria which may use a variety of vent or seep gases e.g., H₂S and CH₄ (Petersen and Dubilier, 2009; Ponnudurai et al., 2017; Wang et al., 2020; Ip et al., 2021). In addition, the extraordinary gene makes that these bivalves use energy, provided by these chemoautotrophic bacteria, rather than phytoplankton (Ip et al., 2021). Nix et al. (1995) found that the G. platifrons would have a faster growth rate in the methane-rich site. This perspective was confirmed by the study that the number of chemoautotrophic bacteria in G. platifrons is significantly influenced by the concentrations of methane (Riou et al., 2008). In this way, concentrations of H₂S and CH₄ might have a significant impact on the growth condition for bivalves in the cold-seep province, rather than physical conditions of the surrounding seawater, e.g., temperature and salinity, since temperature and salinity in deep-sea will remain constant within the transitory life-circle of these species which was supported by the low Mg/Ca and Ba/Ca ratios in this study. In addition, it was proposed that the S in carbonate associated sulfate (CAS) and shell organic matter (SOM) were likely sourced from the seawater and methane fluids modified by the chemosynthetic bacteria, respectively (Newton et al., 2018), which confirms that the ocean- and methane-derived S are both involved in the growth process of these bivalves in the cold-seep province. Since the SOM and CAS share a symplastic growth relationship, the SOM growth will promote the CAS growth, which will affect the S compatibility with the CAS. However, we do not know if any other factors also affect the growth rate, e.g., oxygen fugacity, pressure, current, and so on, since rare studies were carried out on these bivalve shells in the cold-seep province, constraining the further understanding of the intrinsic links between growth rate and element contents. In conclusion, the current data highlight that the S/Ca of the shell can reflect the growth rate of the G. platifrons, however, more works need to be done to confirm the direct correlation between the S/Ca ratios and seepage intensity of cold-seep fluids.

5 CONCLUSION

We investigated Mg/Ca, Ba/Ca, and S/Ca profiles of a bivalve shell fossil by using an LA-ICP-MS technique and evaluated them as environmental and growth proxies for bivalve shells in Haima cold seep of Qiongdongnan Basin, SCS. This study strengthens the idea that Mg/Ca and Ba/Ca ratios in reflecting the environmental temperature and salinity of the surrounding water not only in the deep sea but also in the shallow sea. In addition, the invariable Mg/Ca and Ba/Ca ratios indicate that the surrounding seawater temperatures and salinity remain constant. The S/Ca ratios of the bivalve shell can be an efficient tracer to reflect the growth rate, however, further researches need to be done to evaluate if the S/Ca ratios could be the potential tracer to record the seepage intensity of cold-seep fluids.

6 DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

7 ACKNOWLEDGMENT

We appreciate the constructive suggestions and comments from the editor Dr. Yang CHEN and two anonymous reviewers.