Nitrogen Isotope Sclerochronology—Insights Into Coastal Environmental Conditions and Pinna nobilis Ecology

Introduction

Pinna nobilis is a large bivalve endemic to the Mediterranean Sea. In early autumn of 2016, P. nobilis mass mortality event was detected in the Spanish part of the Mediterranean Sea (Vázquez-Luis et al., 2017), which soon spread to other parts of this enclosed sea (Cabanellas-Reboredo et al., 2019), including the Adriatic Sea (Čižmek et al., 2020; Šarić et al., 2020). By the end of 2020, all P. nobilis populations in the Croatian part of the Adriatic Sea have been affected by the mass mortality and very few individuals survived (Šarić, personal observation). At the first occurrence of a mass mortality event in Spain, the parasite Haplosporidium pinnae was considered. as the likely cause (Darriba, 2017; Catanese et al., 2018). However, further research has indicated that other pathogens, such as bacteria from the genus Mycobacterium and Vibrio, were also involved (Carella et al., 2019; Šarić et al., 2020; Scarpa et al., 2020; Lattos et al., 2021). Moreover, according to these reports, the mass mortality events were presumably caused by polymicrobial disease associated with various abiotic factors, and the pathogenesis has not yet been fully elucidated.

Conservation efforts require comprehensive knowledge on the biology and ecology of species as well as environmental variations, including those related to nitrogen in coastal marine habitats. Pinna nobilis was listed as an endangered and protected species under the European Council Directive 92/43/EEC (EEC, 1992), is recorded in the ANNEX II of the Barcelona Convention, and is under local law protection in all European Union Mediterranean countries. Concerns for the status of P. nobilis were raised even before 2016 and the start of mass mortality event, because it declined due to exposure to a number of cumulative stressors including habitat degradation, food web alteration and contaminant burden (Basso et al., 2015). Due to the devastating effects of mass mortality event on the population of P. nobilis, IUCN changed the status of this species to “critically endangered” (Kersting et al., 2019).

Over the past decade, methods for the analysis of nitrogen stable isotopes in carbonate-bound organic matter (δ¹⁵N_CBOM) using direct combustion of carbonates have been developed and optimized enabling the investigation of δ¹⁵N_CBOM in bivalve shells from freshwater, estuarine, and marine habitats (e.g., Versteegh et al., 2011; Darrow et al., 2017; Gillikin et al., 2017; Graniero et al., 2021; Kukolich and Dettman, 2021; Schöne and Huang, 2021). Nitrogen isotopes measured in carbonate-bound organic matter are a function of δ¹⁵N values of dissolved nitrogen in the ambient water as well as the physiology and diet of the bivalve (e.g., Gillikin et al., 2017; Whitney et al., 2019; Das et al., 2021; Graniero et al., 2021). Bivalves are considered as low-level consumers, and as such, they can provide information on the nitrogen isotope baseline of the environment (e.g., Jennings and Warr, 2003; Thibault et al., 2020). In many previous studies, δ¹⁵N_CBOM was measured in marine bivalve species including Mercenaria sp. (O’Donnell et al., 2003), Mercenaria mercenaria (Carmichael et al., 2008; Oczkowski et al., 2016), Ruditapes philippinarum (Watanabe et al., 2009), Mytilus edulis (Versteegh et al., 2011), Crassostrea virginica (Kovacs et al., 2010; Oczkowski et al., 2016; Black et al., 2017; Darrow et al., 2017), Pecten maximus (Gillikin et al., 2017), Rangia cuneata (Graniero et al., 2021), Spisula solidissima (Das et al., 2021), and Arctica islandica (Whitney et al., 2019; Schöne and Huang, 2021). An increase in δ¹⁵N_CBOM has been related to nutrient enrichment reflecting anthropogenic perturbation (e.g., Carmichael et al., 2008; Kovacs et al., 2010; Oczkowski et al., 2016). As yet, only a limited number of bivalves were sampled at high-resolution to generate δ¹⁵N_CBOM time-series, e.g., Pecten maximus (Gillikin et al., 2017), Spisula solidissima (Das et al., 2021), and Arctica islandica (Schöne and Huang, 2021). Due to its size (>1 m, Zavodnik et al., 1991) and very fast shell growth rate (Richardson et al., 2004), Pinna nobilis presents an interesting taxon for high-resolution δ¹⁵N_CBOM research. High-resolution δ¹⁵N_CBOM can provide an important insight into temporal variation in bivalve diet, physiology (including ontogenetic changes), as well as background nitrogen isotopic signatures (Gillikin et al., 2017; Das et al., 2021; Schöne and Huang, 2021).

Previous studies on the chemical properties of P. nobilis shells include research on oxygen (δ¹⁸O_shell) and carbon (δ¹³C_shell) stable isotopes (Richardson et al., 1999, 2004; Kennedy et al., 2001a; Freitas et al., 2005; García-March et al., 2011), as well as element ratios including Mg/Ca and Sr/Ca (Richardson et al., 2004; Freitas et al., 2005). García-March et al. (2011) suggested that P. nobilis can be used to reconstruct environmental, ecological and climate changes in the Mediterranean Sea. Following previous analyses of δ¹⁸O and δ¹³C in P. nobilis shells, the present study focuses on δ¹⁵N_CBOM values of this species. The main objectives were to test if P. nobilis shells (i) can be used as an indicator of the nitrogen isotope baseline of the system, and (ii) provides high-resolution data on environmental δ¹⁵N variability. Due to the multiple mass mortality events spreading throughout the Mediterranean, including the Adriatic Sea, we also tested if (iii) the chemical properties of P. nobilis and shell deposition process changed in response to the diseases.

Materials and Methods

A preliminary study was conducted in 2018 to test the feasibility to obtain δ¹⁵N_CBOM data from shells of several bivalve species from the Adriatic Sea using the standard combustion technique on an elemental analyzer (e.g., Versteegh et al., 2011).

Sample Collection

For the purpose of this study, P. nobilis shells were opportunistically collected by skin diving from four shallow coastal localities in the eastern Adriatic (Figure 1), as a part of a project on mortality monitoring. More information on sample collection and epidemiological status of live-collected specimens is available in Šarić et al. (2020). Three specimens from Lim Bay (October 2019; 45°07′55′′N, 13°43′55′′E), Kaštela Bay (January 2020, 43°33′01′′N, 16°21′36′′E), and Mali Ston Bay (November 2019, 42°52′01′′N, 17°41′51′′E) were collected alive, while in Pag Bay (44°26′35′′N, 15°03′01′′E), shells of three recently dead specimens were collected in September 2020. In addition, one dead empty shell collected from Kaštela Bay in January 2020 was also analyzed. Lim Bay and Mali Ston Bay are important bivalve aquaculture areas. Lim Bay is located in the North of the eastern Adriatic and Mali Ston Bay in the South. Kaštela Bay is semi-enclosed bay that is under strong anthropogenic influence including agriculture as well as municipal and industrial effluents (Anđelić et al., 2020). Of the four localities, Pag Bay is the most pristine and least polluted area (Kušpilić, personal communication).

FIGURE 1

Figure 1. Map of the Adriatic Sea showing sample localities (red dots).

Sample Preparation and Stable Isotope Analysis

In the laboratory, tissue and epibionts were physically removed and shells carefully cleaned and air-dried, and shell heights were measured. Due to the P. nobilis mass mortality event and opportunistic sampling strategy it was not possible to target specific shell sizes. The height of the studied P. nobilis shells ranged from 15 cm (Pag Bay, Pag3) to 58 cm (Mali Ston Bay, Sto1) (Table 1). The average shell height was 50.7 ± 4.0, 30.3 ± 14.6, 39.8 ± 7.9, and 56.7 ± 1.5 cm for Lim Bay, Pag Bay, Kaštela Bay, and Mali Ston Bay, respectively. Muscle tissue samples from Lim Bay and Kaštela Bay were frozen for later isotope analysis.

TABLE 1

Table 1. Overview of Pinna nobilis specimens and number of samples per shell used in this study.

Shell powder (∼20–30 mg) was collected by milling sample swaths by hand using a DREMEL Fortiflex drill equipped with a 300 μm tungsten carbide drill bit. For the analysis of spatial δ¹⁵N_CBOM variations, three samples were collected from each of these shells by milling three shallow lines parallel to the growth axis from the internal shell surface (Figure 2A and Table 1). Sample tracks measured 130 ± 37 mm in length. These sample swaths started, on average, 40 ± 11 mm from the shell edge. For the analysis of temporal variability, high-resolution δ¹⁵N_CBOM data were collected for the last deposited shell material from two shells collected at three localities including Lim Bay, Kaštela Bay, and Mali Ston Bay. Milling swaths were arranged perpendicular to the major growth axis on the external shell surface (Figure 2B). For shell Kas1 from Kaštela Bay, a total of 40 samples were collected, while between 14 and 17 samples were obtained from each of the five other shells. Shell powder (∼80 μg) was also used for δ¹⁸O_shell and δ¹³C_shell analysis. An overview of shells and samples is given in Table 1. Gillikin et al. (2017) previously established that vigorous chemical cleaning of bivalve shell carbonate is not required for CBOM analysis in bivalves due to the dense structure of the shell.

FIGURE 2

Figure 2. Illustration of milling lines on (A) inner shell surface and (B) external shell surface of Pinna nobilis Kas1 collected at Kaštela Bay. Scale bar 1 cm.

Soft tissues were dried for ∼24 h at 55°C, then ground and homogenized using a mortar and pestle. ∼1 mg powdered samples were packed into tin capsules. 20–30 mg of shell carbonate were packed into tin capsules (signal intensities on the mass spectrometer were similar between soft tissues and shell). Samples were analyzed on a Thermo Delta V Advantage isotope ratio mass spectrometer (IRMS) in continuous flow mode connected to a Costech Elemental Analyzer (EA) via ConFlo IV at Union College (Schenectady, NY) following methods in previous studies (Versteegh et al., 2011; Graniero et al., 2016, 2021; Black et al., 2017; Darrow et al., 2017; Gillikin et al., 2017; Whitney et al., 2019). An in-house acetanilide (δ¹⁵N = –0.96‰), ammonium sulfate (IAEA-N-2) (δ¹⁵N = + 20.3‰), and caffeine (IAEA-600) (δ¹⁵N = + 1.0‰) reference standards were used for regression-based corrections, and to assign the data to the appropriate isotopic scale. Percent N was calculated using additional acetanilide standards of varying mass. Corrections were done using a regression-based method. The reproducibility for δ¹⁵N (AIR) was ± 0.04‰, based on 12 acetanilide standards over three analytical sessions. Stable carbon isotope analysis used similar techniques, see Graniero et al. (2021) for complete details.

Oxygen and carbon isotopes of carbonate powders (∼80 μg) were analyzed on a Gas Bench II coupled to the aforementioned mass-spectrometer. Powders were reacted with > 100% phosphoric acid in helium-flushed Exetainer vials at 55°C for at least 3 h. Carbonate isotope results are reported in δ notation (‰) relative to the VPDB carbonate standard. NBS-18 and IAEA 603 were used for regression-based corrections; LSVEC was used as a check standard. The following values (VPDB) were used for δ¹³C and δ¹⁸O, respectively: IAEA 603: + 2.46‰, −2.37‰, NBS-18: −5.014‰, −23.2‰, and LSVEC: −46.6‰, −26.7‰. The reproducibility (1σ) for δ¹³C_shell and δ¹⁸O_shell was ± 0.03 and ± 0.04‰, respectively, based on eight IAEA 603 standards over two analytical sessions.

Environmental Data and Statistical Analysis

In this study we used results based on Regional Ocean Modeling System (ROMS), a 3D numerical ocean model for the Adriatic Sea with a spatial resolution of 2 km and 20 vertical levels in terrain following coordinates (Janeković et al., 2014). The model was previously validated in studies using available observations including CTD profiles and satellite SST data (e.g., Janeković et al., 2010, 2014; Vilibić et al., 2016) and was used to estimate daily bottom salinity and temperature data—corresponding to the first vertical level in the model. These data were used to calculate predicted δ¹⁸O_shell values and temporally align the measured δ¹⁸O_shell data of Kaštela Bay. An earlier study by García-March et al. (2011) on Pinna nobilis used Friedman and O’Neil (1977) paleothermometry equation for calcite and water and observed ∼1.4‰ offset relative to measured seawater temperature. To enable aligning of data obtained from shell with expected δ¹⁸O_calcite, in this study we used the paleothermometry equation by Killingley and Newman (1982) (Eq. 1).

$$T=22.14-4.37\left({\mathrm{\delta }}^{18}{O}_{calcite}-{\mathrm{\delta }}^{18}{O}_{water}\right)+0.07{({\mathrm{\delta }}^{18}{O}_{calcite}-{\mathrm{\delta }}^{18}{O}_{water})}^2(1)$$

δ¹⁸O_water values were estimated from the modeled salinity values using the equation of Purroy et al. (2018) for the coastal areas in the eastern Adriatic Sea:

$${\mathrm{\delta }}^{18}{\text{O}}_{water}=0.23\times salinity-7.54(2)$$

including a VPDB-SMOW scale correction for δ¹⁸O_water of -0.27‰ (Gonfiantini et al., 1995). Calculated δ¹⁸O values were converted from SMOW to VPDB scale using the equation from Coplen et al. (1983) to temporally align δ¹⁸O_shell values with predicted shell oxygen isotope values obtained from the ocean model:

$${\mathrm{\delta }}^{18}{\text{O}}_{PDB}=0.97002{\mathrm{\delta }}^{18}{\text{O}}_{V-SMOW}-29.98(3)$$

Differences in δ¹⁵N_CBOM between localities were tested using one-way ANOVA. A Shapiro-Wilk test was used to test for normality, and the Brown-Forsythe test for equal variances. The Holm-Sidak method was applied for pairwise multiple comparison. Kruskal-Wallis test was applied for the analysis of percent nitrogen. Spearman correlation coefficient was applied for testing correlation between different variables.

Results

A preliminary study of seven bivalve species from the Adriatic Sea, showed pronounced interspecies variations in %Ns_hell (Table 2). The lowest values (<0.03%) were noted for Venus verrucosa, and Glycymeris pilosa specimens. Shells of the commercially important European flat oyster (Ostrea edulis) and Mediterranean mussel (Mytilus galloprovincialis), had among highest values, 0.22 and 0.16%, respectively. However, the highest value (0.47%) was obtained for a Pinna nobilis shell, clearly indicating potential of this species for the analysis of δ¹⁵N_CBOM data.

TABLE 2

Table 2. Results of preliminary study of δ¹⁵N_CBOM on bivalve species from the Adriatic Sea.

Spatial Variations in δ¹⁵N_CBOM Data

Values of δ¹⁵N_CBOM in samples milled from the internal shell surface of Pinna nobilis shells had pronounced spatial variations (Figure 3 and Supplementary Material 1). Lowest values were obtained for shells from Pag Bay, with a range of 3.06–.47‰ (average ± 1σ = 3.72 ± 0.41‰). Samples from Mali Ston Bay had intermediate values ranging from 5.43 to 5.96‰ (5.72 ± 0.22‰), while values between 6 and 8‰ were characteristic for P. nobilis from Lim Bay and Kaštela Bay. Observed differences were statistically significant between localities (one-way Anova F = 27.36, p < 0.001). According to pairwise comparison tests between locations, differences were not statistically significant, except for Lim Bay and Kaštela Bay. Percent N ranged from 0.2 to 0.7%. Percent nitrogen was higher in shells from Lim Bay (0.43 ± 0.16%), while Pag Bay shells had intermediate amounts (0.35 ± 0.06%), and Mali Ston Bay (0.30 ± 0.03%) and Kaštela Bay (0.28 ± 0.05%) had lower amounts. Observed differences were statistically significant between localities [Kruskal-Wallis test, H (chi²): 13.3, p < 0.005]. According to pairwise comparison tests, samples from Lim Bay had significantly higher percent nitrogen than samples from Mali Ston Bay and Kaštela Bay. Statistically significant difference was also noted between Kaštela Bay and Pag Bay. The localities with highest δ¹⁵N_CBOM had both the highest (Lim Bay) and lowest percent N (Kaštela Bay).

FIGURE 3

Figure 3. δ¹⁵N_CBOM values of the inner shell surface of Pinna nobilis. (A) Data of the three milling lines per specimen, (B) differences between locality averages. Mean values ± 1 standard deviation (often smaller than the symbol).

High-Resolution δ¹⁵N_CBOM Data

Samples from the external shell surface of Pinna nobilis show spatial and temporal variations in δ¹⁵N_CBOM values. The largest number of samples came from the shell Kas1 (Kaštela Bay) and corresponded to the last 6 cm of shell growth (Figure 4). Earlier deposited shell material had δ¹⁵N_CBOM values between approx. 4.5 and 5.5‰ and showed cyclic variations, while an increase in δ¹⁵N_CBOM values (up to approx. 10‰) was identified closer to the shell margin. This increase in δ¹⁵N_CBOM was accompanied by a pronounced δ¹³C_shell decrease. The δ¹⁸O_shell provided an insight into the timing and rate of shell growth. Accordingly, the sampled shell portion was formed between spring 2018 and summer 2019 (Figure 5). As this shell was collected alive in January 2020 and the last sample was milled 1.6 mm from the shell margin, these results indicate that the shell Kas1 slowed down its growth or even ceased to grow for several months prior to collection. The increase in δ¹⁵N_CBOM values occurred in spring and summer of 2019. Temporal resolution of δ¹⁵N_CBOM data was ∼10 days, with seasonal variations ranging between 1 and 3 weeks.

FIGURE 4

Figure 4. High resolution δ¹⁵N_CBOM, δ¹⁸O_shell, and δ¹³C_shell data of Pinna nobilis specimen Kas1 collected alive at Kaštela Bay in January 2020.

FIGURE 5

Figure 5. Predicted shell oxygen isotope values (gray line) obtained from the temperature (ROMS model) and δ¹⁸O_water estimates based on salinity (ocean model) using the paleothermometry equation by Killingley and Newman (1982). Gray circles represent temporally aligned δ¹⁸O_shell values of Pinna nobilis specimen Kas1 collected alive at Kaštela Bay in January 2020; green diamonds represent corresponding δ¹⁵N_CBOM values. Scale on y1-axes is reversed.

The δ¹⁸O_shell values from shell Kas1 ranged between −0.21 and 3.10‰, while those for δ¹³C_shell values varied between −1.48 and 0.55‰ (N = 46). There was a statistically significant positive correlation between δ¹⁸O_shell and δ¹³C_shell (Spearman r = 0.74, p < 0.001). Shell δ¹⁵N values were negatively correlated to both δ¹⁸O_shell (r = −0.344, p = 0.03) and δ¹³C_shell (r = −0.438, p = 0.005). However, this relationship was largely driven by the last eight samples. Without these data points, the only statistically significant correlation exists between δ¹⁸O_shell and δ¹⁵N_CBOM values (r = 0.667, p < 0.001).

We also completed a high-resolution analysis of five additional P. nobilis shells, two live-collected specimens from Lim Bay (Lim1 and Lim2) and Ston Bay (Sto1 and Sto3), and one dead, empty shell from Kaštela Bay (Kas4). In these shells, 15 mm (Lim2) to 23 mm (Sto3) transects were sampled, with the last samples coming from approx. 10 mm before the shell margin. In these specimens, sampling closer to the shell margin was not possible because of contamination: foreign particles, mostly sediment, were trapped in the shell.

δ¹⁵N_CBOM values ranged from 3.43 to 7.84‰ (Figure 6). Lowest values were characteristic for shells from Mali Ston Bay, with an average value of 4.30 ± 0.42‰ for shell Sto1 4.09 ± 0.52‰ and for shell Sto3. Shells from Lim Bay had highest mean values, i.e., 7.00 ± 0.57‰ (Lim1) and 6.24 ± 0.36‰ (Lim2). These spatial variations are consistent with δ¹⁵N_CBOM values of the inner shell surface. Shell Kas4 from Kaštela Bay showed intermediate average values for samples obtained from the outer shell (5.46 ± 0.16‰), and the least variable range of 5.22–5.74‰. In the case of this shell, average δ¹⁵N_CBOM values obtained from samples milled from the outer shell were lower than those obtained from the inner shell (7.84 ± 0.10‰).

FIGURE 6

Figure 6. High-resolution δ¹⁵N_CBOM values of Pinna nobilis shells collected at Lim Bay (Lim1 and Lim2), Kaštela Bay (Kas4), and Mali Ston Bay (Sto1 and Sto3).

As noted above, muscle tissues were only available for shells from Lim Bay and Kaštela Bay (Figure 7). The δ¹⁵N_tissue data of these two localities varied from each other, with higher values for Lim Bay (range: 7.22–7.28‰) and lower values for Kaštela Bay (5.74–6.19‰). In the present study, δ¹³C_tissue data had a very narrow range at both localities (Lim Bay: −19.88 to −19.70‰; Kaštela Bay: −20.06 to −19.57‰).

FIGURE 7

Figure 7. Relationship between δ¹³C and δ¹⁵N in (A) muscle tissue and (B) shell of Pinna nobilis.

Discussion

Shell δ¹⁵N Values

Results of this study clearly indicate that Pinna nobilis shells contain a sufficient amount of N-bearing organic matter to enable reliable analyses of δ¹⁵N_CBOM values using direct combustion. For the analysis of site-specific/temporal δ¹⁵N_CBOM variations of P. nobilis, carbonate material can be milled from the inner or outer shell surface (Figures 3, 6). Pronounced site-specific δ¹⁵N_CBOM variations were observed in specimens collected in the eastern Adriatic Sea. δ¹⁵N_CBOM values from the inner shell surface of specimens from Lim Bay and Kaštela Bay were almost two times higher than those from P. nobilis from the Pag Bay (Figure 3). Similar site-specific δ¹⁵N_CBOM differences existed in samples from the external shell surface of the specimens collected from three localities.

Kaštela is a semi enclosed coastal bay that was identified as one of the most polluted coastal areas in the eastern Adriatic in the late 20th century (Barić et al., 1992). The situation has improved since 1990 onward when most chemical industries located in the area were closed and again after mid-2000 when a modern wastewater treatment plant was completed (Kušilić et al., 2009). However, due to large urban population in the area, including cities of Split, Kaštela and Trogir, Kaštela bay is still one of the areas with the highest anthropogenic impacts in the eastern Adriatic. Due to the number of freshwater springs, Lim Bay, an important bivalve aquaculture site, is characterized with high nutrient input (Bosak et al., 2009). Other than impacts associated with aquaculture activities, anthropogenic influences in this area are limited. A similar situation exists in Mali Ston Bay, as it is also relatively sparsely populated area. The Mali Ston Bay may be classified as a moderate natural eutrophicated system based on the phytoplankton community structure (Viličić et al., 1998). The least data are available for Pag Bay. The Island of Pag, including the Pag Bay area, is characterized by bare rocks, and scarce terrestrial vegetation. There is no industrial activity in the area, and agriculture is very limited. Previous studies (e.g., Carmichael et al., 2008; Kovacs et al., 2010; Gillikin et al., 2017) showed that δ¹⁵N_CBOM data relate to nitrogen in the environment and can be used for reconstructing environmental biochemical conditions. Our results are consistent with this, as samples from the more pristine Pag Bay had the lowest δ¹⁵N_CBOM values.

According to Gillikin et al. (2017) bivalve shells can provide high-resolution δ¹⁵N_CBOM data because they are capable of recording the δ¹⁵N signal of metabolically-acquired N with suitably short time-averaging. The target species of the present study, P. nobilis is the largest marine bivalve in the Mediterranean Sea (Vicente, 1990; Zavodnik et al., 1991) and is characterized by rapid shell growth during early ontogeny. It attains a shell height of 20–30 cm during first 2 years of life (Richardson et al., 2004; Kožul et al., 2012; García-March et al., 2020). In this study, average temporal resolution of δ¹⁵N_CBOM from the external shell samples was 10 days, and this was obtained by milling ∼20–30 mg of carbonate material. Resolution was estimated from temporal alignment of measured δ¹⁸O_shell values with predicted δ¹⁸O_shell values obtained from the ocean model. The sample mass of milled carbonate in this study was rather large, as we targeted an easy to analyze mass with a robust signal to noise ratio. The sample mass could be smaller (e.g. Gillikin et al., 2017; Graniero et al., 2021) in future studies, which would greatly increase temporal resolution; down to a few days in early ontogeny and during seasons of faster shell growth. Such extremely highly resolved δ¹⁵N_CBOM has great potential for reconstructing environmental changes in coastal marine ecosystems. Gillikin et al. (2017) reported a resolution of δ¹⁵N_CBOM down to 2 days in Pecten maximus shells, and P. nobilis has more than three times the N content and faster growth rates, so daily resolved δ¹⁵N_CBOM should be attainable using this technique.

In this study, we conducted high-resolution analysis of δ¹⁵N_CBOM on specimens ranging in size from 42 to 58 cm. Due to the mass mortality event it was not possible to collect more shells and select animals of similar size and age limiting comparison and temporal alignment of geochemical data. As Pinna nobilis does not show periodic growth marks on its shell margin, adductor muscle scars have been used to estimate its age (Richardson et al., 1999). However, later studies pointed out bias and limitation of this method including the difficulty in determining first and second scar and obscuring of scars by nacre in older shells (Richardson et al., 2004; Garcia-March and Márquez-Aliaga, 2007). Furthermore, shells from Lim Bay and Mali Ston Bay did not have distinct muscle scars on the inner shell surface (Peharda, personal observation). Hence, estimation of their age was beyond the scope of the present study. However, it is interesting to note that largest shells came from the Mali Ston Bay, and they appear to have a larger number of muscle scars than those sampled from Kaštela Bay (see Supplementary Material 3). Furthermore, shell Lim1 from Lim Bay had a similar size (47 cm) as shell Kas1 from Kaštela Bay (49 cm), but based on muscle scars visible on the internal shell surface, this shell was older and slower growing thereby likely leading to more time averaging in data from stable nitrogen isotope analysis this shells. In addition, shell layering near the shell margin was the most fragile and friable part of the shell, which resulted in trapping of particles from the environment preventing proper sampling for isotope analysis.

Taking into account the limitations described above, high-resolution δ¹⁵N_CBOM data were presented according to sample distance from the shell margin (Figure 6). These data revealed strong temporal variations in specimens from same site (Lim2: Δ = 1.24‰; Lim 1: Δ = 1.76‰). In a recent study, Das et al. (2021) also observed inter- and intra- specimen variations in S. solidisima shells. Gillikin et al. (2017) analyzed Pecten maximus and noted differences of up to 2.5‰ between shells growing at the same site and time. Despite variations between specimens, data presented here and in previous studies seem to reflect that the nitrogen isotope baseline changes seasonally. This change is most likely caused by seasonal variation in nutrients and food sources available to bivalves, including P. nobilis, in shallow coastal marine habitats. According to Gillikin et al. (2017) and Das et al. (2021), bivalve shells have a high potential to reveal short-term nitrogen cycle dynamics and this is confirmed in our study.

Precise temporal alignment was only performed for one specimen (Kas1) collected from Kaštela Bay. In this specimen, it was possible to collect carbonate samples from very close to the shell margin, i.e., the last formed shell portion prior to collection. Based on modeled temperature and salinity it was possible to calculate expected δ¹⁸O_shell values. In January 2020, when sampling was conducted, Pinna nobilis population in Kaštela Bay has been impacted by a mass mortality event, and strong infiltrative inflammation that included Haplosporidium pinnae and Mycrobacterium was observed in Kas1 specimen (Šarić et al., 2020). The δ¹⁸O_shell data indicated that shell material was deposited until late summer (see Figure 5). According to results of previous studies (Kennedy et al., 2001a; Richardson et al., 2004; Freitas et al., 2005; García-March et al., 2020), P. nobilis deposits shell material in late summer and autumn period, what was not the case with our shell Kas1. As this specimen was collected in January, it is possible that it was experiencing starvation and/or infection for several months.

An increase in δ¹⁵N_CBOM by up to approx. 10‰ most likely occurred in early summer and was coupled with a decrease in δ¹³C_shell likely indicating a stressed animal and an increase in the amount of metabolic-sourced carbon in the shell (see Lorrain et al., 2004; McConnaughey and Gillikin, 2008). Changes in δ¹⁵N have been linked to variations in diet and physiological state of the animals, and according to a recent experimental study by Prado et al. (2021), on feeding of P. nobilis. The increase in δ¹⁵N can be a consequence of starvation. As the nitrogen balance of bivalves drops below nitrogen demands, they start to break down their own tissues, thereby preferentially losing ¹⁴N and retaining ¹⁵N (see Fry, 2006 for examples). Both δ¹³C_shell and ¹⁵N_CBOM suggest the animal was sick. This is further supported by the lack of shell growth in the second half of 2019 (Figure 5).

δ¹⁵N in Pinna nobilis Tissue

Previous studies conducted on other bivalve species found that bulk δ¹⁵N_CBOM correlates with δ¹⁵N of soft tissues (e.g., O’Donnell et al., 2003; Carmichael et al., 2008; Kovacs et al., 2010; Gillikin et al., 2017; Graniero et al., 2021). In the present study, only a limited number of samples was available for tissue analysis, and due to mass mortality event and conservation status of the species, it was not possible to collect additional samples. Hence, a direct comparison of shell and tissue δ¹⁵N was not possible, and the tissue data need to be interpreted with care. Yet, the tissue δ¹⁵N values were much higher than such determined in the few previous studies on P. nobilis from relatively undisturbed settings suggesting increased anthropogenic and/or natural inputs at the study localities.

To the best of our knowledge, Kennedy et al. (2001b) provided the first stable isotope data of Pinna nobilis muscle tissue. Respective specimens came from the southeastern Spanish Mediterranean coast. These authors reported a mean value of −18.3‰ for δ¹³C_tissue and 3.3‰ for δ¹⁵N_tissue. In a study by Cabanellas-Reboredo et al. (2009), average stable isotope values for P. nobilis muscle tissue samples were −19.30‰ for δ¹³C_tissue and 3.51‰ for δ¹⁵N_tissue. According to data presented in their study, δ¹⁵N_tissue values higher than 5‰ were found only at one sampling site. Similar results were found in a third study conducted in Spain by Alomar et al. (2015). According to their results, P. nobilis tissue samples from non-Marine Protected Areas were characterized by higher δ¹⁵N_tissue and δ¹³C_tissue, than those from Marine Protected Areas. Deudero et al. (2017) analyzed muscle tissue of monthly collected P. nobilis and found a 0.93‰ annual valuation in δ¹⁵N_tissue, with minima recorded in March (on average, 2.54‰) and maxima in May (3.47‰). All the above studies were conducted prior to mass mortality events and their values are lower than those obtained for δ¹⁵N_tissue in the present study (5.74–7.28‰). Possible explanations for observed differences are variations in anthropogenic perturbations that suggest eutrophication and/or physiological response of animal to the mass mortality event. From the conservation perspective, it would be informative if future studies are conducted on the disease spread involving live-collected bivalves and analysis of stable isotopes in different P. nobilis tissues.

Pinnindae and Shell Chemistry

According to Huber (2010), the cosmopolitan family Pinnidae comprises more than 50 species, inhabiting tropical and temperate seas. Pinnids likewise inhabit shallow coastal regions (e.g., P. nobilis, Zavodnik et al., 1991) and deep waters (e.g., Pinna cellophana, Matsukuma and Okutani, 1986). Despite their characteristic shape and size, new species are still being discovered, e.g., in 2016, Araya and Osorio (2016) described Pinna rapanui from Easter Island, South Pacific Ocean.

Results of this study as well as earlier works conducted on the shell chemistry of P. nobilis (e.g., Richardson et al., 1999, 2004; García-March et al., 2011; Gilbert et al., 2017), clearly indicate the great potential of P. nobilis as well as related taxa for sclerochronological research. Over the past decade, research on shell chemistry has been conducted on several species of Pinnidae including Atrina fragilis (Valentine et al., 2011), Atrina rigida (Gilbert et al., 2017), and Pinna carnea (Gilbert et al., 2017). Respective studies have been conducted on modern and fossil specimens presenting possibilities to reconstruct past environmental conditions. High-resolution analysis of δ¹⁵N_CBOM in different pinnids from coastal environments would provide powerful insights into anthropogenic caused perturbations of the environment, especially in areas with limited instrumental data.

Conclusion and Recommendations

Results of this study clearly indicate the potential of Pinna nobilis to provide reliable high-resolution δ¹⁵N_CBOM time-series. Such analyses can be conducted on museum stored specimens for which the collection date is known, thereby extending valuable δ¹⁵N reconstructions further into the past. Furthermore, P. nobilis shells collected in the mid twentieth century at different locations within the Adriatic Sea, as well as probably in other parts of the Mediterranean, are available from private collections. Although for many of such specimens exact date of collection is not known, data on approximate year of collection is often available. Challenges of high-resolution δ¹⁵N_CBOM analysis of P. nobilis shells include the crowding of growth patterns in shell material deposited during late ontogeny that can result in large time averaging, trapping of different particles from the environment (organic and inorganic) within the shell, and difficulties associated with milling samples from the margin where shell is very thin and fragile. Spatial δ¹⁵N_CBOM variations can be a valuable indicator of the nitrogen isotope baseline. However, due to the current P. nobilis mass mortality event in the Mediterranean, further research on this species is very restricted and attempts should be made to identify other bivalve taxa that are relatively fast growing and whose shells contain sufficient amounts of N-bearing organic matter to enable high-resolution δ¹⁵N_CBOM analyses. Results of our preliminary study indicate that two commercially important and relatively fast growing bivalves, the European flat oyster (Ostrea edulis) and Mediterranean mussel (Mytilus galloprovincialis), have high shell nitrogen content, making them interesting taxa for high resolution δ¹⁵N_CBOM analysis in the Mediterranean. At a global level, other species of the Pinnidae likely serve as valuable δ¹⁵N_CBOM archives as well.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

Author Contributions

MP: obtained the funding, designed the experiment, analyzed the data, and wrote the manuscript. DG: designed the experiment, conducted laboratory analysis of stable isotopes in soft tissues and shell carbonate material, analyzed the data, and wrote the manuscript. BS: designed the experiment, analyzed the data, and wrote the manuscript. AV: conducted laboratory analysis of stable isotopes in soft tissues and shell carbonate material, helped with writing, and revision of the manuscript. HU: milling of carbonate material, help with data analysis, and writing and revision of the manuscript. KM: milling of carbonate material, help with writing and revision of the manuscript. TŠ and IŽ: field work, help with writing and revision of the manuscript. IJ: 3D numerical ocean model data, help with writing and revision of the manuscript. All authors gave final approval of the version to be published.

Funding

Research has been funded by the Croatian Science Foundation under the project BivACME (IP-2019-04-8542). The U.S. National Science Foundation funded Union College’s isotope ratio mass spectrometer and peripherals (NSF-MRI #1229258).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

We are grateful to Ivica Matijaca for his help with collection of samples and Madelyn Miller for assistance in the isotope lab (Union College). Sampling was performed under permission from the Croatian Ministry of Environmental Protection and Energy (CLASS UP/I-612-07/18-48/145; N°517-05-1-1-18-3 and CLASS UP/I-612-07/19-48/205; N° 517-05-1-1-19-3).

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars.2021.816879/full#supplementary-material

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