DOI: 10.5061/dryad.cvdncjtcp
title: Data from: Bivalve shells reflect 15N enrichment in a fertilizer-dominated estuary

description: Agricultural nitrogen (N) contributes a dominant percentage to global N
 pollution in the coastal zone. Emerging research on N isotopes in bivalve
 shells has shown value for reconstructing historical increases in
 estuarine wastewater inputs. However, applications for fertilizer N are
 understudied. Here, we integrate the study of organic N isotopes, in
 concert with δ18O and δ13C, in estuarine bivalve shells to investigate
 spatial and long-term change in nitrogen inputs and sources. Modern,
 museum collection, and subfossil specimens of the genera Mytilus and
 Ostrea were profiled in a California estuary with an intensely
 agricultural watershed. Spatial patterns in bivalve isotopic composition
 reflected gradients in watershed nutrient inputs and productivity
 parameters. Furthermore, comparison of modern and historical periods
 revealed changes in nutrient source or processing over the last 1000
 years. The N isotope values from shells offer perspective on agricultural
 pollution in estuaries.
description: Elkhorn Slough (36° 48' 25.18"N, 121° 47'
 23.24"W) is a shallow estuary (2.5 m deep) that extends inland 10 km
 from Monterey Bay in central California, USA. Elkhorn Slough drains a
 watershed of 585 km<sup>2</sup>, and while a portion has been
 protected, 24% of the watershed is cultivated, generally with crops that
 have moderate to high fertilizer demands and/or are multi-cropped. Outside
 of tidally restricted areas or during infrequent winter storms, the
 salinity and temperature of the Elkhorn Slough’s estuarine waters are
 similar to those found in Monterey Bay (S=30 PSU;
 T=14<sup>o</sup>C). It serves as a model system for studying
 agricultural N inputs. Water sampling has frequently documented high
 nitrate levels exceeding 1000  μM, an order of magnitude greater than many
 other well-studied eutrophic estuaries. Furthermore, historical changes in
 the hydrology of the estuary, including creation of an artificial oceanic
 inlet at the harbor in 1946, may have also altered marine nutrient
 exchange. In October of 2021, live
 <em>Mytilus</em> specimens were collected at 11 sites
 throughout the estuary, while recently deceased <em>Ostrea
 lurida</em> shells were collected from one location near the mouth
 of the estuary. <em>Mytilus</em> specimens were not identified
 to the species level because <em>Mytilus</em>
 <em>galloprovincialis</em> and <em>Mytilus</em>
 <em>trossulus </em>species hybridize in coastal
 California<em>.</em> Despite the scarcity of
 <em>Ostrea,</em> they were sampled due to extensive prior
 research on δ<sup>15</sup>N<sub>shell</sub> values
 in oysters. Five specimens were collected at each site, based on prior
 research suggesting this as an optimal sample size for
 reproducibility. Modern bivalve isotopes were related
 to water chemistry measured in grab samples collected monthly from
 November 2020 to October 2021 by the National Estuarine Research Reserve.
 Several parameters were measured in situ, including temperature, dissolved
 oxygen, and salinity, using EXO2 multiparameter sondes (YSI, Yellow
 Springs, OH, USA). Grab samples were stored on ice, filtered within 48
 hours, and analyzed for chlorophyll <em>a </em>using
 fluorometry and dissolved inorganic nitrogen using a Lachat QC8000 Flow
 Injection Analyzer at Moss Landing Marine Laboratories, following standard
 methods. Historical specimens of <em>Mytilus
 </em>and <em>Ostrea </em>species (collected between 1933
 and 1999) were obtained from the California Academy of Sciences museum.
 Fragments of <em>Mytilus </em>shells (dating from
 approximately 964 to 1760) were extracted from three archived sediment
 cores collected in 2010, which were previously measured for sedimentary
 isotopes on the top 50 cm. Chronological control for subfossil specimens
 was established using AMS radiocarbon<sup> </sup>dating of
 organic material (seeds and peat), generating a composite chronology from
 seven cores collected throughout the estuary to mitigate complications
 from the marine reservoir effect. The chronology was developed using a
 Bayesian age model called “<em>Plum” </em>that combines
 lead-210, radiocesium, and radiocarbon dating. We
 selected moderately sized <em>Mytilus</em> specimens (~40mm)
 to control for age impacts on increasing
 δ<sup>15</sup>N<sub>shell</sub> values. We sampled
 a narrow portion of recent calcite growth (average 4.2mm) at the ventral
 margin of the valve from the outer shell layer (OSL) of each specimen.
 Based on estimated growth rates, this sample size corresponds to
 approximately one year of growth. <em>Ostrea</em> shells were
 sampled from the inner shell surface. For both species, sampling was
 conducted on calcite to control for fractionation between mineral forms.
 Nitrogen content of the historical specimens was compared to modern
 samples to investigate degradation of organic matter over time.
 Shells were sampled using a dental drill with a 1 mm tungsten
 carbide conical bit at a low speed of 1000 rotations
 min<sup>-1</sup> to minimize isotopic fractionation. Subfossil
 <em>Mytilus</em> material preserved in sediment cores was
 sampled using the same approach as modern samples, grinding 4-5 mm
 portions along the margin where preserved, or the full sample for
 fragments less than 5 mm long. Samples (300-400 mg)
 were analyzed for δ<sup>18</sup>O<sub>shell</sub>
 and inorganic δ<sup>13</sup>C<sub>shell</sub>
 using an Elementar Isotope Ratio Mass Spectrometer (IRMS) (Elementar
 Americas Isoprime100) coupled with a Multiflow carbonate prep system.
 Carbonates were digested with 103% orthophosphoric acid at 70°C for four
 hours. The same samples (20-50 mg) were analyzed for
 δ<sup>15</sup>N<sub>shell</sub> on the Elementar
 IRMS coupled with a Pyrocube elemental analyzer equipped with a carbon
 trap (NaOH) between the reduction and absorption tubes to remove excessive
 CO<sub>2</sub> and reduce interference of the carbonate matrix
 with organic isotope analysis. Standard materials represented 20% of the
 samples (run precision ± 0.19 ‰ for
 δ<sup>13</sup>C<sub>shell</sub>, ± 0.41 ‰ for
 δ<sup>18</sup>O<sub>shell</sub>, ± 0.71 ‰ for
 δ<sup>15</sup>N<sub>shell</sub>). For inorganic
 samples, internal standards were calibrated against the international
 standards NBS-18 and NBS-19, achieving a precision of ± 0.09‰ (C) and ±
 0.16‰ (O) based on long-term replication. For organic samples, calibration
 was performed with IAEA N1, IAEA N3, and USGS 26 standards, with precision
 of ± 0.32 ‰. Internal standard “Elk” (1.05% N) closely matches the N
 content of the <em>Mytilus</em> and
 <em>Ostrea</em> samples.<strong> </strong>Every
 tenth sample was duplicated. We also measured
 δ<sup>15</sup>N<sub> tissue</sub> (freeze-dried
 and ground soft<em> </em>tissues of
 <em>Mytilus</em>),
 δ<sup>15</sup>N<sub>SPM</sub> (suspended
 particulate matter), and
 δ<sup>15</sup>N<sub>NO3</sub> (water column
 nitrate) values at several select sites to verify the
 δ<sup>15</sup>N<sub>shell</sub> patterns (Fig.
 S8). Isotopic data are expressed in parts per thousand (‰) using the delta
 notation (δ<sup>15</sup>N, δ<sup>18</sup>O, and
 δ<sup>13</sup>C), where δ =
 [(<em>R<sub>sample</sub>−R<sub>standard</sub>)</em>/<em>R<sub>standard</sub></em>] × 1000. We used a Partial Least Squares (PLS) model in R version 4.3.1 to relate modern δ<sup>15</sup>N<sub>shell</sub> values with spatial patterns of modern water chemistry (‘plsr’ package). This modeling approach was chosen for its ability to handle multicollinearity, which is typically present in water chemistry data. The isotopic signatures were compared to one year of water chemistry data, represented as median values, which are temporally comparable to amount of shell material sampled. Predictors of δ<sup>15</sup>N<sub>shell</sub> values included δ<sup>18</sup>O<sub>shell</sub>, δ<sup>13</sup>C<sub>shell</sub>, NO<sub>3</sub>, NH<sub>4</sub><sup>+</sup>, temperature, salinity, dissolved oxygen, turbidity, and distance to the oceanic inlet. Statistical analysis of change over time was performed by grouping data into bins according to time: (1) modern samples collected in 2021; (2) museum specimens from 1933 − 1999 C.E.; and (3) core specimens from circa 964 to 1760 C.E. The time bins were compared using Analysis of Variance (ANOVA) because the data are normally distributed, as confirmed by the Shapiro–Wilk Test and histograms.
description: # Data from: Bivalve shells reflect 15N enrichment in a
 fertilizer-dominated estuary
 [https://doi.org/10.5061/dryad.cvdncjtcp](https://doi.org/10.5061/dryad.cvdncjtcp) ## Description of the data and file structure This dataset includes nitrogen, carbon, and oxygen stable isotopes measured on modern and historical bivalve shells from Elkhorn Slough estuary, CA. Datasets include the age-depth relationship of sediment cores used to date historical shells, the isotopic data from modern shells and historical shells as well as shell parameters, and also includes the code to generate figures and statistical analysis (ANOVA and PLS) of the change of isotopes over time and relationship to water column chemistry. ### Files and variables #### File: Water\_Column\_Isotopes.csv **Description:** : Isotopes of the water column nutrients and suspended particulate material collected across a tide cycle from low to high tide (hourly for 6 hours) on 16 August 2022 and 20 February 2023 at four sites throughout Elkhorn Slough, Missing values were below the 4uM detection limit (labeled as “bd”), and “NA” are null indicating that these samples were not collected. ##### Variables * Sample_ID: the code given to each sample labeled as 1 to 6 across the tidal cycle * Site: the site name * Date: sample collection date * Time: sample collection time); * NO3_um: NO_um (the nitrate concentration in micromolar in the water column * d15NSPM: the organic nitrogen isotopic signature of suspended particulate material * d13CSPM: the organic carbon isotopic signature of suspended particulate material * d15NNO3: the nitrogen isotopic signature of water column nitrate * d18ONO3: the oxygen isotopic signature of water column nitrate #### File: Modern\_Mussels.csv **Description:**  Data from the modern mussels including all five replicates at each site in Elkhorn Slough, dataset includes isotopic signatures of the shell and tissue for select speciments, and other size parameters of the shells. "NA” means null values were not measured for the specimen. ##### Variables * Site: site name * Code: First letter of the site name * Sample: sample number 1 to 5 of the five replicate individuals at each site * d15N: the nitrogen isotopic signature of the shell * d13C: the carbon isotopic signature of the shell * d18O: the oxygen isotopic signature of the shell * d15N_tissue: the nitrogen isotopic signature of the tissue * Size_mm: length of the shell umbo to margin in mm); * Bands_count: number of growth bands on the shell * Growth_rate: size of the shell divided by annual growth bands * Sampled_mm: the length of shell ground for the isotope sample * Sampled_months: the amount of time that the sampled area corresponds to #### File: Historical\_Isotope\_Timeseries.csv **Description:** All bivalve specimens sampled for isotopes including subfossil specimens from sediment cores, historical specimens from museums, and modern samples for comparison, “NA” are null indicating that these specimens were not sampled.** ** ##### Variables * Code: Descriptor of the sample including the core identifier, museum identifier, or site location of the modern samples in Elkhorn Slough * Type: Origin of the sample whether from a sediment core, natural history museum, or modern collection * Sample_ID: lab identifier for the replicate samples * Depth: Depth in the sediment that a sample was collected in a core) * Species: Variety of bivalve either “Mussel” or “Oyster” * Year: Date of the historical samples generated from the Age-Depth Model for the core sample or the date of collection for the museum samples * d18O: the oxygen isotopic signature of the shell * d13C: the carbon isotopic signature of the shell) * d15N: the nitrogen isotopic signature of the shell * N_Perc: the percent of nitrogen in the shell #### File: Mean\_Modern\_Isotopes.csv **Description:**  : Isotope data from the shells of modern bivalve samples shown as averages of five replicates at each site, Mean values in this data frame are used as the import of the PLS model relating N isotopes to water chemistry, Water quality data collected was monthly from November 2020 to October 2021 and is shown as the annual median. ##### Variables * d15N: the nitrogen isotopic signature of the shell, mean of 5 replicate individuals * d13C: the carbon isotopic signature, mean of 5 replicate individuals * Distance: Distance of the site to the marine inlet in km * d18O: the oxygen isotopic signature, mean of 5 replicate individuals * Temp: Water temperature in Celsius, annual median * Chla: Chlorophyll-a in ug/L in the water column, annual median * DO: dissolved oxygen in mg per L in the water column, annual median * Salinity: Salinity in ppt in the water column, annual median * Turb: Turbidity in NTU in the water column, annual median * NH4: Ammonium concentration in mg per L in the water column, annual median * NO3: Nitrate concentration in mg per L in the water column, annual median * d15N_std: standard deviation of the nitrogen isotopes of 5 replicate individuals * d13C_std: standard deviation of the carbon isotopes of 5 replicate individuals * d18O_std: standard deviation of the oxygen isotopes of 5 replicate individuals * Type: Variety of organism whether “M” for mussel or “O” for oyster * Site_code: First letter of the site name #### File: Composite\_Chronology.csv **Description:** Output of the Bayesian Age-Depth model that combines 7 cores collected throughout Elkhorn Slough, the cores span a depth of 0 to 510 cm and radiocarbon, radiocesium, and lead dating techniques are combined in the model ##### Variables * Depth_cm: Depth of sediment in the core units= cm * Year_min: the minimum possible year corresponding to that depth lower confidence interval * Year_max: the maximum possible year corresponding to that depth upper confidence interval * Year_median: the median of possible years * Year_mean: the mean of possible years, used for dating the shell material ## Code/software “Code_Stats and Figures.R”; Software: Run using coding language R version 4.3.1. Use: R file containing the code for generating the graphs in Figures 2 and 3 in the manuscript, and supplementary Figures S3, S5, S10, and S12. Statistical analysis of variance over time as shown in Figure 3 and the PLS model relating N isotopes to water chemistry and shown in Figure 2. Input data: Original data generated in the study: “Modern Mussels.csv”; combined with publicly available water quality data input in “Mean Modern Isotopes.csv”. Packages: “pls” (Liland K, Mevik B, Wehrens R, 2023); “tidyverse” (Wickham H, Averick M, Bryan J, Chang W, McGowan LD, François R, Grolemund G, Hayes A, Henry L, Hester J, Kuhn M, Pedersen TL, Miller E, Bache SM, Müller K, Ooms J, Robinson D, Seidel DP, Spinu V, Takahashi K, Vaughan D, Wilke C, Woo K, Yutani H, 2019); “egg” (Auguie B, 2019); ggplot2” (H. Wickham, 2016); “ggrepel” (Slowikowski K, 2024); “dplyr” (Kucheryavskiy S, 2020). ## Access information Other publicly accessible locations of the data: * Data is described in the publication titled “Bivalve shells reflect 15N enrichment in a fertilizer-dominated estuary”. Data was derived from the following sources: * Additional publicly available datasets used in the paper and R code including Water quality and Nutrients monitoring data can be accessed from the National Estuarine Research Reserve Systemwide Monitoring Program (NOAA, 2021), at [http://www.nerrsdata.org](http://www.nerrsdata.org).