DOI: 10.5061/dryad.12jm63z48
title: Bulk Carbon and Amino Acid nitrogen isotope data from Baltic cod (Gadus morhua) and European flounder (Platichthys flesus) muscle tissue samples from the western and central Baltic Sea

description: Eutrophication, increased temperatures and stratification can lead to
 massive, filamentous, N2-fixing cyanobacterial (FNC) blooms in coastal
 ecosystems with largely unresolved consequences for the mass and energy
 supply in pelagic and benthic food webs. Mesozooplankton adapt to not
 top-down controlled FNC blooms by switching diets from phytoplankton to
 microzooplankton, resulting in a directly quantifiable increase in its
 trophic position (TP) from 2.0 (herbivore) to as high as 3.0 (carnivore).
 If this process in mesozooplankton, we call trophic lengthening, was
 transferred up to higher trophic levels of a food web, a large loss of
 energy could result in massive declines of fish biomass. We used
 compound-specific nitrogen stable isotope data of amino acids (CSIA) to
 estimate and compare the nitrogen (N) sources and TPs of cod and flounder
 (mesopredators) from areas with influence of FNC blooms (central Baltic
 Sea) and without it (western Baltic Sea). We tested if FNC-caused trophic
 lengthening in mesozooplankton is carried over to fish. The TP of cod from
 the western Baltic, feeding mainly on decapods, was equal to the global
 mean value (4.1, secondary carnivore). Only cod from the central Baltic,
 mainly feeding on zooplanktivorous pelagics, had a higher TP (4.8,
 near-tertiary carnivore), indicating a strong carry-over effect of
 FNC-caused trophic lengthening from mesozooplankton. In contrast, the TP
 of molluscivorous flounder (3.2 ± 0.2 in both areas), associated with the
 benthic food web, was unaffected by trophic lengthening. This suggests
 that FNC blooms cause a large loss of energy in zooplanktivorous but not
 in molluscivorous mesopredators. If FNC blooms continue to detour energy
 at the base of the pelagic food web, the TP of cod will not return to
 global mean values and the fish stock not recover. Monitoring the TP of
 key species can identify fundamental changes in ecosystems and provide
 useful information for resource management.
description: The fishes were collected during four cruises to
 the western Baltic Sea (ICES subdivision (SD) 22) and central Baltic Sea
 (SD 24-25) in January/February 2019 and 2020 and caught by bottom trawl, with a Bacoma
 cod end (2019) and a TV30 #520 trawl gear (2020) following the standards
 of the Baltic International Trawl Survey (ICES, 2017).
 On board, the fish were killed,
 identified to species level, counted, and measured. The otoliths were
 extracted and a sample of white
 muscle tissue was taken from behind the third dorsal fin
 of 30 cod individuals and from the tail
 muscle of 21 flounder individuals. All samples were frozen
 immediately at −20°C for later stable isotope
 analyses. The frozen cod
 und flounder muscle tissue samples were cleaned mechanically and with
 distilled water to remove surface contaminants, freeze dried (Christ Alpha
 1-4), and then ground and homogenized for further processing. For CSIA,
 ~10 mg of each dried sample was transferred into a heat-resistant
 borosilicate vial, mixed with 5 ml of 6M HCl solution and 1 ml of internal
 standard (trans-4 (amino methyl)-cyclohexane carboxylic acid), and
 hydrolyzed for 24 h at 110°C. The samples were then filtered through
 cellulose-acetate filters, dried under a nitrogen flow at 50°C, and then
 derivatized to TFA-isopropyl amino esters (Silfer et al., 1991; Hofmann et al., 2003),
 which included an additional purification step using a
 chloroform-phosphate buffer solution (Veuger et al., 2005). The derivatized samples were dissolved
 in 500 µl of methylene chloride and stored in GC-vials at −20°C until
 analyzed as described below. The measurements
 of the so-called bulk δ<sup>13</sup>C were carried out
 according to Loick et al.
 (2007), using Elemental
 Analyzer Isotope Ratio Mass Spectrometry (EA-IRMS) analyses of the fish
 muscle tissue samples (Thermo Finnigan Delta Plus + Thermo Flash EA 1112).
 For this purpose, the dried and powdered fish muscle tissue samples were
 weighed (~0.5 mg per sample), packed in tin boats and measured by EA-IRMS.
 Calibration for the total carbon determination was done daily with an
 acetanilide standard (Merck). All isotope abundances are expressed in δ
 notation as follows: δ X (‰) =
 [(R<sub>sample</sub>/R<sub>standard</sub>) - 1)] *
 103, where X is <sup>13</sup>C, and R is the
 <sup>13</sup>C:<sup>12</sup>C ratio. The internal
 laboratory reference gas for the C-analyses was ultrapure
 CO<sub>2</sub>, which was calibrated against the materials
 from the International Atomic Energy Agency (IAEA): NBS 22 (mineral oil
 δ<sup>13</sup>C = −29.74 ‰) and USGS 24 (graphite
 δ<sup>13</sup>C = −15.99 ‰). In addition, peptones (Merck)
 were analyzed as in-house standards after every sixth fish sample run. The
 analytical error for stable isotope ratios indicated by the peptone
 standards was less than ± 0.2 ‰ for carbon
 isotopes. The
 TFA-isopropyl-derivatized samples were analyzed for their content of
 δ<sup>15</sup>N in 13 AAs; an external standard (16 AA) was
 also included. The 13 AAs were: alanine (Ala), glycine (Gly), threonine
 (Thr), serine (Ser), valine (Val), leucine (Leu), isoleucine (Ile),
 proline (Pro), aspartic acid (Asp), glutamic acid (Glu), phenylalanine
 (Phe), tyrosine (Tyr), and lysine (Lys). The concentrations of cysteine
 (Cys), arginine (Arg), and methionine (Met) in the samples were below the
 qualitative detection limit of the measurement device and could therefore
 not be determined in the fish muscle tissue samples. During the initial
 hydrolysis step, glutamine and asparagine were converted into glutamic
 acid and aspartic acid, respectively, such that they were considered together as Glu + Gln
 (referred to herein as Glu) and Asp + Asn (Asp) (Brault et al.,
 2019). Amino-acid-specific δ<sup>15</sup>N values were measured using an isotope ratio mass spectrometer (IRMS, Thermo Finnigan GmbH, MAT 253 MS, Germany) connected via a ConFlo IV interface unit to a gas chromatograph combustion periphery (GC-C, Thermo Scientific Trace 1310 GC, Italy; Thermo Scientific, GC Isolink, Germany). The separation column in the GC consisted of a non-polar column coated with 5% phenyl-polysilphenylenesiloxane (BPX5, 60 m, 0.32 mm inner diameter, film thickness of 1μm; SEG Analytical Science, Ringwood, Victoria, Australia). For each run, 2 µl of sample was injected via a PTV injector in splitless mode. The temperature program was as follows: Initial temperature at 50 °C, heat up at 12 °C / min to 120 °C, hold for 17 min, heat up at 3 °C / min to 180 °C, linger for 10 min, heat up at 5 °C / min to 200 °C, hold for 6 min, heat up to 250 °C at 10 °C / min and hold for 7 min. Each sample was injected and measured at least three times using helium (He) as the carrier gas. The standard deviation from three runs was usually &lt; 1.0 ‰ for all 13 AAs.
description: # Bulk Carbon and Amino Acid nitrogen isotope data from Baltic cod (*Gadus
 morhua*) and European flounder (*Platichthys flesus*) muscle tissue
 samples from the western and central Baltic Sea
 [https://doi.org/10.5061/dryad.12jm63z48
 ](https://doi.org/10.5061/dryad.12jm63z48) There are currently four TP
 models available that are suitable to estimate the TP of vertebrates
 (Germain et al., 2013; Nielsen et al., 2015; Bradley et al., 2015; Brault
 et al., 2019). They were tested and partly modified to identify, which TP
 model is most suitable for cod and flounder, respectively. Specifically,
 CSIA data of cod and flounder from the western Baltic Sea were chosen to
 validate and calibrate the TP models based on the assumption that their
 TPs at this reference site should be close to the global mean TP value for
 cod of 4.1 ± 0.2 and for flounder of 3.3 ± 0.2, respectively (Froese and
 Pauly, 2022). The rationale for this is that the TP of cod and flounder
 from the western Baltic Sea should not be largely impacted by any trophic
 lengthening in the mesozooplankton compartment (Loick-Wilde et al., 2019),
 since filamentous, N<sub>2</sub>-fixing cyanobacteria are
 mainly absent in the western Baltic Sea. ## Description of the data and
 file structure The excel file contains all component-specific stable
 isotope data of amino acids measured for cod and flounder as well as their
 injection numbers and standard deviations. In addition, the data are
 colour-coded and divided according to fish species and catch area. The TP
 models tested, their results and the weighted means of the raw amino acid
 data are colour-coded and marked. Missing standard deviation data, caused
 by single injection, are blank. The additional readme sheet within the
 excel sheet contains an explanation of each of the variables in the
 dataset, its measurement units, and -if it concerns a derived variable
 like the different used TP-models - including the literature reference as
 found in the associated manuscript.