DOI: 10.5061/dryad.ngf1vhj4s
title: Data from: Paralytic shellfish toxin concentrations measured in alaskan arctic clams using ELISA and HPLC methods

description: Clams are efficient vectors of potent algal neurotoxins, a suite of
 saxitoxin (STX) congeners collectively called paralytic shellfish toxins
 (PSTs), to higher trophic levels. The Alaskan Arctic is a region facing an
 expanding threat from PSTs due to ocean warming, yet little is known about
 PSTs in clams from this region. Quantifying total toxicity in bivalves
 requires analytical techniques, such as high-performance liquid
 chromatography (HPLC). Enzyme-linked immunosorbent assays (ELISAs) are an
 efficient but only semi-quantitative method for measuring clam toxicity.
 PSTs (STX eq.) were measured in split clam samples (n = 16) from the
 Alaskan Arctic using ELISA and HPLC methods to develop a preliminary
 linear model for conservatively estimating total toxicity in clams from
 ELISA toxin values (R2adj = 0.95, p &lt; 0.001). Profiles of PST
 congeners and total toxicity using HPLC were also assessed in additional
 clams (n = 36 additional, n = 52 total). Clams contained mostly potent PST
 congeners, and over half of the clams had PST concentrations above the
 seafood regulatory limit. These data will help assess the exposure risks
 of PSTs in Arctic marine food webs, as harmful algal bloom activity is
 predicted to increase as the Arctic continues to warm.
description: <em>Sample Collection</em> Clams
 were collected during research cruises of opportunity (<em>n
 =</em> 5) during 2019, 2020, and 2022 along routinely sampled survey
 transects (Figure 2A). Van Veen Grabs were deployed at various stations
 along survey transects to collect clams from the sea floor. Grab contents
 were sieved with seawater to isolate clams, which were then sorted to the
 genus or species level, collected in sterile whirl-paks or zip-lock
 plastic bags, and stored at −20 °C until toxin analysis.
 Clams were analyzed for PSTs using ELISA and HPLC methods based
 on sample availability at research cruise stations throughout the Alaskan
 Arctic (Figure 2A). Initially, one clam at each station was analyzed for
 PSTs (STX eq.) using an Abraxis<sup>®</sup> ELISA kit. (Gold
 Standard Diagnostics, Horsham, PA, USA) Based on ELISA results, stations
 throughout the Alaskan Arctic with clams containing low (BDL) to high
 toxins (≥80 µg STX eq. 100 g<sup>−1</sup>) were selected, and
 an additional clam sample (<em>n =</em> 1) was submitted for
 HPLC analysis. Additionally, one or more clams were pooled from either the
 same station or among similar stations along the same transect,
 homogenized using metal scissors and a spatula, and then the homogenate
 (<em>n =</em> 1) was split equally to perform ELISA (target
 sample mass; 1.0 g, minimum; 0.4 g) and HPLC (target mass; 1.0 g, minimum;
 0.5 g) analyses (“split” sample, Figure 1A). These “split” samples were
 limited (<em>n</em> = 16) due to sample availability and
 achieving soft tissue mass required to perform both ELISA and HPLC
 analyses. A total of 52 samples were submitted for toxin analysis
 (<em>n =</em> 36, HPLC-only; <em>n =</em> 16,
 split samples for ELISA and HPLC analyses).
 <em>Paralytic Shellfish Toxin
 Measurements</em> <em>Abraxis
 ELISA</em> Saxitoxin (STX eq.) was measured in
 clam samples using an Abraxis<sup>®</sup> ELISA kit (product
 no: 52255B) following Lefebvre et al. [22,23]. Solvent (50% methanol/50%
 water) was added to homogenized samples using a 4:1 ratio (solvent
 (mL)/sample mass (g)), which were then homogenized for 1 min at 2100 rpm
 using a hand-held probe (GLH 850, 10 mm; Omni-International, Kennesaw, GA,
 USA). Samples were then centrifuged at 3063 × <em>g</em> for
 20 min at 4 °C (Jouan CR3i centrifuge, Thermo Electron Corp., Waltham, MA,
 USA), extract transferred to 4 mL amber vials and stored at 4 °C prior to
 toxin analysis. Aliquots (200 µL) of extracts were filtered through a spin
 filter (Millipore Sigma Ultra-Free Centrifugal filters, 0.22 μm; Merck
 KGaA, Darmstadt, Germany) and further diluted to 1:50 prior to measuring
 STX concentrations (STX eq.) using the Abraxis<sup>â</sup>
 ELISA kit. All manufacturer instructions were followed to obtain STX eq.
 concentrations (µg STX eq. 100 g<sup>−1</sup>) from extracts.
 The cross-reactivity of the ELISA antibodies with PST congeners according
 to the Abraxis user manual are listed in Table 2. ELISA values that were
 below detectable limits (BDL) (<em>n</em> = 2 clams) were
 included in linear model development by assigning half the lowest
 quantifiable limit (LQL) (LQL = 0.70 µg STX eq. 100
 g<sup>−1</sup>, BDL= 0.35 µg STX eq. 100
 g<sup>−1</sup>) [44,45]. <em>HPLC
 Methods</em> HPLC analysis of samples for
 paralytic shellfish toxins followed the post-column oxidation (PCOX) AOAC
 Official Method 2011.02 [46]. Extractions involved using equal volume of
 extraction acid to sample weight (1:1 ratio). Briefly, extraction acid
 (0.1N HCl) was added to homogenized sample in a 50 mL Falcon centrifuge
 tube (Falcon – BD, Franklin Lakes, NJ) and placed in boiling water for 5
 min. Afterwards, the cooled sample was spun down via centrifugation, and
 proteins were removed by adding 30% trichloroacetic acid (TCA) followed by
 pH adjustment with NaOH. After filtration of the processed sample, it was
 ready for analysis. The HPLC instrumentation was a
 Waters Acquity Arc system equipped with a refrigerated autosampler (4 °C)
 and a Waters 2475 FLR fluorescence detector (Waters, Milford, MA, USA).
 All extracts were injected at 10 µL and a flow rate of 0.8 mL/min.
 Attached to the HPLC was a Pickering Laboratories ONYX PCX post-column
 oxidation instrument (Pickering Laboratories Inc., Mountain View, CA,
 USA). Flow rates for both oxidant and acid were 0.4 mL/min, and column
 temperature was 30 °C. Standards used were Certified Reference Material
 (CRM) obtained from the National Research Council Canada (NRCC, Ottawa,
 ON, Canada). Toxin analogs analyzed were the following: GTX1, GTX4, GTX2,
 GTX3, dcGTX2, dcGTX3, GTX5, NEO, dcSTX, STX, C1, and C2. The saxitoxin
 standard used was saxitoxin dihydrochloride, and all results are expressed
 as STX·diHCl equivalents. Detection limit was established as 2 µg
 STX·diHCl 100 g<sup>−1</sup>. Quality control of instrument
 and method performance was evaluated in several ways. Upon instrument
 startup, upper-level and lower-level calibration mix standards were run to
 ensure instrument response was sufficient. Then, after analysis of 4–6
 samples, a mid-level standard mix was run to check that instrument
 performance was being maintained and not degraded. Post analysis, again,
 included analysis of upper-level and lower-level quality control standard
 mixes. Also, once a zero toxin sample was obtained, it was spiked with a
 standard mix and carried out through extraction and analysis to ensure no
 matrix effects were occurring by comparing those results with previous
 quality control standard mixes. Chromatograms for standard mixes and
 detectable congeners in a clam sample can be found in the Supplementary
 Material. Toxicity equivalency factors (TEFs) used in the equation below
 are listed in Table 2. Calculations were carried out
 using the following equation to yield µg STX·diHCl eq. 100
 g<sup>−1</sup>: μg STX∙diHCl eq= μM toxin
 ×TEF ×(372.2 g)/(1000 mL)×Fvol/(Ext.vol)×((Wt+Vol)/Wt)×100
 where: µM = concentration of toxin in
 extract; Fvol = final volume of deproteinated extract
 (560 µL); Ext. vol = volume of crude extract used (500
 µL); Wt = weight of sample used (g);
 Vol = volume of acid extractant used (mL); TEF
 = toxicity equivalence factor (Table 2). The µg
 STX·diHCl eq. 100 g<sup>−1 </sup>concentrations from HPLC
 analysis are referred to as µg STX eq. 100 g<sup>−1
 </sup>throughout the manuscript (including figures and tables) for
 ease in comparison of total toxicity concentrations across studies (e.g.,
 EFSA [4]). <em>Statistical
 Analysis</em> All statistical analyses were
 conducted using the software programs R version 4.4.2 [47] and R Studio
 version 2024.09.01+394 [48]. All maps were generated using the software
 QGIS version 3.34 [49]. All samples (<em>n</em> = 52) analyzed
 for PSTs via HPLC were included in total toxicity analysis; however, one
 clam had zero measured toxins and was excluded from PST congener profile
 analyses (<em>n =</em> 51). An alpha of 0.05 was used as a
 significance threshold for all relevant analyses.
 <em>Correction Model—ELISA and HPLC PST
 Measurements</em> An initial linear regression
 was performed using split clam samples (<em>n</em> = 16) to
 predict total PST concentrations (µg STX eq. 100
 g<sup>−1</sup>, HPLC PST values) from ELISA STX eq. values.
 One clam was BDL for STX eq. using ELISA, but had a paired HPLC value of
 over 80 µg STX eq. 100 g<sup>−1</sup>. This clam was a clear
 outlier and was removed from linear regression analysis, resulting in a
 total of <em>n</em> = 15 clams for model development (Table
 1). Linear models were compared with square root transformed and
 non-transformed data using AIC criteria to determine best model fit. The
 selected model was verified for underlying assumptions by plotting model
 residuals with fitted values, covariates, and assessing the distribution
 of residuals. <em>Paralytic Shellfish Toxin
 Profiles and Total Toxicity Analyses</em>
 Paralytic shellfish toxin profiles for clams were constructed by
 using relative abundances (µM, molarity) of each congener measured during
 HPLC analysis. Profiles were assessed for similarities among regions using
 distance-based multi-variate analysis. Specifically, toxin profiles of
 clams (<em>n =</em> 51) were compared among different regions
 (Bering Strait [<em>n =</em> 16], Chukchi Sea [<em>n
 =</em> 22], and Beaufort Sea [<em>n =</em> 13]) using
 non-metric multidimensional scaling (NMDS), analysis of similarities
 (ANOSIM), and similarity percentage (SIMPER) analyses from the R package
 <em>vegan</em> . A Bray–Curtis transformation was applied to
 congener proportions in clams to calculate a distance matrix for NMDS,
 ANOSIM, and SIMPER analysis, which allowed for the assessment of which
 proportional differences of specific congeners contributed to overall
 dissimilarities in PST profiles among regions.
 Differences in total toxicity (µg STX eq. 100
 g<sup>−1</sup>, HPLC PST values) in clams collected from
 different regions were tested by comparing linear models with and without
 (i.e., null model) region as a main effect and selecting the model that
 had the lowest AICc value and highest AIC weight. Models were generated
 using the R package <em>lme4</em> ]. A square root
 transformation was applied to toxicity data to achieve normality
 (Shapiro–Wilks test, <em>p =</em> 0.08) for linear model
 analysis. The full and selected models were verified as described above
 for linear regression analysis. If the main effect (region) was selected,
 a Type II ANOVA (F-tests for linear models) with subsequent Tukey HSD
 tests compared estimated marginal mean (EMM) toxicity concentrations using
 the R package <em>emmeans</em>. Estimated marginal means were
 compared because they are dependent on model results (compared to ordinary
 means based on empirical data) and, therefore, take into account
 unbalanced sample sizes across regions .
description: # Data from: Paralytic shellfish toxin concentrations measured in alaskan
 arctic clams using ELISA and HPLC methods
 [https://doi.org/10.5061/dryad.ngf1vhj4s](https://doi.org/10.5061/dryad.ngf1vhj4s) ## Description of the data and file structure ### Files and variables There are two csv files and one table that includes all the raw data and metadata used for the manuscript #### File: Toxins\_SampleMetadata.csv **Description:** This csv file contains the metadata for the clams analyzed for PSTs in this study ##### Variables * ID: individual clam ID label * Species: species of clam analyzed * Cruise: research cruise of opportunity from which each clam was collected * Station: The station name from where each clam was collected during their respective research cruise of opportunity * Latitude: approximate latitude coordinates from which clams were collected * Longitude: approximate longitude coordinates from which clams were collected * Region:region in the Alaskan Arctic clams were collected from * Date_collected:month/day/year clam was collected #### File: Toxins\_HPLCdata.csv **Description:** This spreadsheet includes the ID of all n = 52 clams that were analyzed for paralytic shellfish toxin (PST) congeners using high-performance liquid chromatography (HPLC) methods and the resulting PST data. NOTE: The µg STX·diHCl eq 100g-1 concentrations from HPLC analysis are referred to as µg STX eq. 100 g-1 throughout the manuscript (including figures and tables) for ease in comparison of total toxicity concentrations across studies. ##### Variables * ID: individual clam ID label * Toxin:indicates which PST congener was measured * Peak_area: the area under the curve for the targeted toxin during HPLC analysis * Tox_conc_uM: resulting toxin concentration of the targeted congener in micromoles [uM] * Weight_g: weight of clam sample analyzed for PSTs using HPLC methods * VolExt_ml: volume of acid extractant used in milliliters [mL] * STX_uM: toxin concentration of the targeted toxin converted to micromoles of saxitoxin (STX) equivalent units * TEF: toxicity equivalency factors used for each PST congener that converts all congeners to STX equivalent units (see equation for µg STX·diHCl eq 100g-1 in Methods) * STXeq_ugper100g: the resulting targeted toxins' concentration in migrograms of STX equivalent units per 100 grams of tissue #### **File: Toxins\_LMdata.docx** **Description**: This file contains the contents of "Table 1" from the manuscript. Specifically, it contains all data to reconstruct the linear model that can estimate total PST toxicity (i.e., HPLC concentrations) from ELISA PST concentrations (STX Eq.) in clams (n = 15).  **Variables** *  Species: species of clam  * Region: region in the Alaskan Arctic where clams were collected from  * Date: date clams were collected * ELISA (ug STX eq./100g): PST concentration (STX eq.) from clam using ELISA methods * HPLC (ug STX eq./100g): PST concentration (STX eq.) from clam using HPLC methods  * ELISA% of HPLC: the percentage of PSTs in clams using ELISA: HPLC methods  * ELISA +/- to HPLC: Whether ELISA methods underestimated ("-") or overestimated ("+") PSTs in clam samples.  ## Code/software N/A ## Access information Other publicly accessible locations of the data: * N/A Data was derived from the following sources: * N/A