High-resolution nitrogen stable isotope sclerochronology of bivalve shell carbonate-bound organics

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Abstract

Nitrogen stable isotope ratios (δ15N) of organic material have successfully been used to track food-web dynamics, nitrogen baselines, pollution, and nitrogen cycling. Extending the δ15N record back in time has not been straightforward due to a lack of suitable substrates in which δ15N records are faithfully preserved, thus sparking interest in utilizing skeletal carbonate-bound organic matter (CBOM) in mollusks, corals, and foraminifera. Here we test if calcite Pecten maximus shells from the Bay of Brest and the French continental shelf can be used as an archive of δ15N values over a large environmental gradient and at a high temporal resolution (approximately weekly). Bulk CBOM δ15N values from the growing tip of shells collected over a large nitrogen isotope gradient were strongly correlated with adductor muscle tissue δ15N values (R2 = 0.99, n = 6, p < 0.0001). We were able to achieve weekly resolution (on average) over the growing season from sclerochronological profiles of three shells, which showed large seasonal variations up to 3.4‰. However, there were also large inter-specimen differences (up to 2.5‰) between shells growing at the same time and location. Generally, high-resolution shell δ15N values follow soft-tissue δ15N values, but soft-tissues integrate more time, hence soft-tissue data are more time-averaged and smoothed. Museum-archived shells from the 1950s, 1965, and 1970s do not show a large difference in δ15N values through time despite expected increasing N loading to the Bay over this time, which could be due to anthropogenic N sources with contrasting values. Compiling shell CBOM δ15N data from several studies suggests that the offset between soft-tissue and shell δ15N values (Δtissue-shell) differs between calcite and aragonite shells. We hypothesize that this difference is caused by differences in amino acids used in constructing the different minerals, which should be specific to the CaCO3 polymorph being constructed. Future work should use compound specific isotope analyses (CSIA) to test this hypothesis, and to determine whether certain amino acids could specifically track N sources or possibly identify amino acids that are more resistant to diagenesis in fossil shells. In conclusion, bivalve shell CBOM δ15N values can be used in a similar manner to soft-tissue δ15N values, and can track various biogeochemical events at a very high-resolution.

Introduction

Nitrogen stable isotope signatures (δ15N) of organic matter are a powerful tool for studying food webs and tracking nitrogen dynamics in terrestrial and aquatic systems (Fry, 1988, Cabana and Rasmussen, 1996, Cole et al., 2011). Nitrogen in consumers is usually enriched in 15N relative to their diet, typically by +2‰ to +4‰, or on average 3.4‰, which allows estimations of trophic positions of consumers relative to the base of the food web (DeNiro and Epstein, 1981, Minagawa and Wada, 1984, Vander Zanden and Rasmussen, 2001, Post, 2002, Caut et al., 2009). It can however be difficult to estimate the isotopic composition of N sources at the base of the food web (isotopic baseline) since ecosystem biogeochemistry is dynamic and multiple nutrient sources available to phytoplankton can have distinct stable isotope signatures that vary temporally and spatially (McMahon et al., 2013). Bivalve soft-tissues have been proposed as good proxies of this isotopic baseline as they are sessile and integrate this variability (Jennings and Warr, 2003, Vokhshoori and McCarthy, 2014). Natural variations in δ15N values of particulate N caused, for example, by upwelling (e.g., Mollier-Vogel et al., 2012) could thus be preserved in tissues, potentially serving as an upwelling/El Niño Southern Oscillation proxy in certain locations. In addition, δ15N values of organics can be used as a wastewater pollution indicator (e.g., Costanzo et al., 2005). Processed wastewaters are 15N enriched, with δ15N values of particulate N typically around +15‰ (Heaton, 1986), but much higher values have been recorded (e.g., Schlacher et al., 2007, Marwick et al., 2014; see Bouillon et al., 2012, for a review).

Extending organic δ15N records back through time to develop isotopic baselines, or gather data on past pollution events, is challenging. Sediment δ15N records can be used to track relative changes, but sediment trap and surface sedimentary δ15N values suggest alteration during early burial, cautioning against the use of sediment cores for reconstructing isotope baselines (reviewed in Robinson et al., 2012). Although there are excellent archives of preserved organic material in museums (e.g., animal soft-tissues), the effect of long-term preservation in formalin and/or ethanol are not well characterized. Delong and Thorp (2009) recorded a −0.2‰ shift in freshwater mussel tissue δ15N values after 12 months in ethanol. Whereas, Carabel et al. (2009) reported a positive shift in δ15N values of bivalve tissues of about +1‰ after storage in ethanol for two years. While these are not large effects considering the strong 15N enrichment associated with anthropogenic N loading, it is not clear if longer time periods would result in larger isotopic shifts. For example, Versteegh et al. (2011) found more than a 5‰ difference in shell organic δ15N values between shells stored dry and shells stored in ethanol for 73 years. Clearly, dry stored specimens would be the safer option for extending δ15N data back in time. Typically, shells are stored dry in museum collections making them ideal archives for reconstructing past δ15N values. Moreover, pristine unaltered fossils may also provide insights into the nitrogen cycle into the geological past (e.g., O'Donnell et al., 2003, O’Donnell et al., 2007).

The study of nitrogen isotopes in carbonate bound organics has recently received renewed attention. Studies investigating N in carbonate bound organics in foraminifera (Ren et al., 2012a, Ren et al., 2012b), corals (Marion et al., 2005, Williams and Grottoli, 2010, Yamazaki et al., 2011a, Yamazaki et al., 2011b, Yamazaki et al., 2013, Wang et al., 2015), fish otoliths (Vandermyde and Whitledge, 2008, Rowell et al., 2010, Grønkjær et al., 2013), and bivalves (O’Donnell et al., 2007, Carmichael et al., 2008, Watanabe et al., 2009, Kovacs et al., 2010, Versteegh et al., 2011, Dreier et al., 2012, Graniero et al., 2016) have been increasing. Continuous long-term (>150 years) N isotope records have been developed from coral skeletons (Erler et al., 2016), and foraminiferal N isotope records have been extended back 30 Ka (Ren et al., 2009), both illustrating the power of this technique. Bivalves have a global distribution in many environments, generally withstand pollution, are common, and are not mobile over large distances allowing for spatial reconstructions. In addition, the dense ‘closed’ shells of bivalves (Marin et al., 2007) exclude foreign organic material and make them relatively diagenetically resistant (c.f., Engel et al., 1994). Nevertheless, both corals and mollusk shells have been shown to maintain their carbonate bound organics for hundreds to thousands of years (Engel et al., 1994, Ingalls et al., 2003). Therefore, similar to corals and foraminifera, bivalve shell carbonate is a suitable structure to preserve high-resolution carbonate bound N.

As previously noted, bivalves are a good substrate to target for N isotope studies because they are low-level consumers and therefore record baseline δ15N values (Jennings and Warr, 2003). Finally, bivalves can be long-lived and have high growth rates allowing both high-resolution environmental reconstruction (down to daily) and provide records extending back in time (e.g., Arctica islandica shell chronologies have been extended back more than 1300 years; Butler et al., 2013).

Although bivalve shells have higher %N than corals and foraminifera, they typically have only a few percent organic matter bound within the carbonate (typically 1–5% organic matter; Marin and Luquet, 2004). This presents analytical challenges when determining such small amounts of nitrogen in a large carbonate matrix, especially in a sclerochronological context. To circumvent this problem, several studies have removed the carbonate via acidification (e.g., Carmichael et al., 2008, Watanabe et al., 2009, Kovacs et al., 2010). However, several studies have shown that acidifying organic matter with high CaCO3 content alters the δ15N value (Jacob et al., 2005, Ng et al., 2007, Mateo et al., 2008, Serrano et al., 2008), likely because acidification removes all or part of the acid soluble N, leading to analysis of an unknown part of the bulk organic N. This has prompted other groups to directly combust the bulk shell to release the entire organic N pool, with good results (e.g., Vandermyde and Whitledge, 2008, Rowell et al., 2010, Versteegh et al., 2011, Graniero et al., 2016).

The aims of this study are to (1) illustrate that low %N samples can be accurately measured on a standard elemental analyzer - isotope ratio mass spectrometer (EA-IRMS) configuration, (2) determine if bivalve shells can be used as an archive of δ15N values, providing the same or parallel information as soft-tissues, and (3) determine if shells can provide an ultra-high-resolution δ15N record (i.e., at a daily or weekly resolution).

Section snippets

Shell collection

This study presents data from three sets of Pecten maximus shell samples collected from the French coast: shells collected along a depth gradient in 2008, shells from the Bay of Brest collected in 2000, and three archived shells collected from the Bay over the past century. Shells were collected along a transect from the Bay of Brest, France (40 m depth, 4° 40′ W, 48° 18′ N), to the edge of the continental shelf (220 m depth, 8° 15′ W, 48° 12′ N) in 2008 (Fig. 1; shell collection details and other data

Depth-transect shells

Carbonate bound organics in shells collected along the depth transect exhibited a wide range of δ15N values, 1.0‰ to 10.3‰, with a general trend of lower δ15N values with increasing depth (Fig. 5). The range of values in each shell (intra-shell variability) was highest in the shells from the deepest sites (both = 4.2‰) and lower in shells from shallower sites (1.3‰ to 2.1‰) (Fig. 5). Shell δ15N values were similar to soft-tissue (adductor muscle) δ15N values, with the data from the shell edge

Discussion

Our data illustrate that shells can be used as archives for nitrogen isotope studies (Fig. 7). The strong correlation between shell and soft-tissue δ15N values show that shells can be utilized in a similar manner as soft-tissues, which have been extensively utilized to trace pollution, food webs, metabolism, and developing baseline isoscapes (see Fry, 1988, Fry, 1999, Lorrain et al., 2002, Paulet et al., 2006, Carmichael et al., 2012, Nerot et al., 2012, Vokhshoori and McCarthy, 2014).

Conclusions

In conclusion, we show that simple combustion of shell material in an EA permits very low %N samples to be easily analyzed with minimal processing, allowing increased temporal resolution up to approximately one week. Our high-resolution results suggest that metabolism and/or energy allocation can affect shell δ15N values (as it does soft-tissue δ15N values), but that seasonal and spatial variation shows good correlation with soft-tissue δ15N values. These carbonate bound organic δ15N values

Acknowledgements

We thank Jean-Marie Munaron for milling shells for this study, David Dettman for useful discussions on combusting shells for δ15N analysis, Ruth Carmichael for discussions on CBOM δ15N analysis methods, and shells collectors (YM Paulet, J. Grall, and M. Glemarec). We also thank Alan Wanamaker, Anouk Verheyden, three anonymous reviewers, and Ethan Grossman for constructive comments on earlier versions of this manuscript, and Roger Hoerl who helped with statistical tests. This study was funded by

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