DOI: 10.5285/0298edcf-93b1-4a6c-9a81-d9c785149b39
title: Lipid mass and free fatty-acid composition of Calanus hyperboreus (CV) exposed to pristine and biofouled microplastics during shipboard incubation experiments (southeastern Greenland, summer 2024)

description: This dataset provides measurements of total lipid mass and free fatty-acid composition of the Arctic copepod Calanus hyperboreus, copepodite stage CV, collected during three independent shipboard microplastic exposure experiments conducted aboard the RRS Sir David Attenborough in July-August 2024 during the KANG-GLAC cruise (SD041). Copepods were exposed to pristine and biofouled nylon-6 microplastics at concentrations 0, 20 and 200 microgram per liter under food availability and starvation conditions. Total lipid mass was quantified gravimetrically and normalized to individual dry weight. Fatty-acid composition was analysed as fatty-acid methyl esters using gas chromatography-mass spectrometry (GC-MS) and expressed as relative abundance (percent of total detected fatty acids). 

Funding was provided by:
- Natural Environment Research Council (NERC) IAPETUS2 Doctoral Training Partnership (NE/S007431/1)
- UK Research and Innovation (UKRI) Future Leaders Fellowship project CUPIDO (MR/T020962/1)
- European Research Council (Horizon 2020) project ANTSIE (Grant 864637)
description: Sampling and experiments were conducted between 2024-07-25 and 2024-08-31. Sampling of copepods for Experiment I was performed on 2024-07-24, for Experiment II - on 2024-08-04 (first morning hours), and for Experiment III - on 2024-08-17 and 2024-08-18. The duration of Experiment I was from 2024-07-27 to 2024-08-02, Experiment II - from 2024-08-04 to 2024-08-10, and Experiment III - from 2024-08-19 to 2024-08-25.

Copepods were collected along the southeastern Greenland shelf with a motion-compensated 200 µm Bongo net. Copepods for Experiment I and Experiment II were collected from individual single stations (67.492° N, 33.003° W and 68.107° N, 30.423° W, respectively). For Experiment III, copepods were collected from three stations: two adjacent sites (67.414° N, 32.683° W; 67.411° N, 32.683° W) and a third site about 31 km to the north (67.693° N, 32.577° W).

Immediately after capture, copepods were acclimated for 12-24 h in 1 L beakers with 1.2 µm-filtered Arctic seawater at 7 °C. Only active, undamaged individuals were used. Three shipboard experiments were conducted: Experiment I exposed copepods to pristine microplastics (MP) with food, Experiment II to pristine MP without food, and Experiment III to biofouled MP with food. Each experiment included Control (0 MP), Low MP (20 µg L-1), and High MP (200 µg L-1) treatments. Experiments I and II used three replicate 50 mL Falcon tubes with two copepods per tube. Experiment III used three replicate 50 mL Falcon tubes with one copepod per tube due to limited specimen availability at that time of the season. Fed treatments received a mixed algal diet (Nannochloropsis, Tetraselmis, Pavlova, Isochrysis, Thalassiosira weissflogii; Reefphyto, UK) at 4,600 cells mL-1 every two days. Tubes were mounted horizontally on a vertical rotator (1 rpm, 90° tilt) and opened every two days for feeding (where applicable), and gentle aeration.

Microplastics consisted of polyamide nylon-6 powder (Goodfellow, AM306010). Pristine MP were prepared in a clean, plastic-free laboratory at British Antarctic Survey before cruise commencement via size-fractionation to 5-25 µm, and dilution (0.25 mg in 25 mL 2 µm-filtered Milli-Q water) to produce a 0.01 mg mL-1 stock suspension. Defined volumes of this stock (100 µL and 1 mL) were then pipetted before experiment commencement into 50 mL Falcon tubes and diluted to a full volume with 1.2 µm-filtered Arctic seawater to obtain final MP concentrations of 20 and 200 µg L-1. For biofouled MP, 1 mg of particles was prepared identically, transported on board in pre-cleaned aluminium foil envelope, and incubated in 100 mL Arctic seawater for 27 days at 7 °C with constant mixing. Immediately before the experiments, defined volumes of the continuously mixed suspension were pipetted directly into Falcon tubes and brought to 50 mL to achieve equivalent MP concentrations.

At the end of each experiment, no mortality or behavioural changes were observed. Copepods were rinsed and frozen individually at -80 °C.
At the stage of the lipid extraction, copepods were freeze-dried for 24 h, and two or three individuals from different tubes were pooled per replicate to account for inter-individual variability and to meet FA detection limits (n = 3 per treatment). Copepods were then weighed for dry mass, and spiked with 20 µL of 5alpha(H)-cholestane as the internal standard. Lipids were extracted using dichloromethane:methanol (9:1 v/v), with sonication and 24 h incubation with solvent, evaporated under vacuum at 30 °C, re-dissolved, and dried under nitrogen gas flow before gravimetric quantification (µg lipid mg-1 DW ind-1). Lipids were then methylated with methanol containing 5% HCl at 70 °C for 12 h, extracted with hexane:dichloromethane, dried, and silylated with BSTFA prior to analysis. After that, samples were analysed using GC-MS.

Due to low internal-standard recovery, analyses were based on relative FA abundances (percent total area), retaining only FAs contributing &gt;1 percent in all replicates.


Instrumentation and software:
Samples were analysed using a Thermo Trace 1300 GC-MS equipped with a RESTEK FAMEWAX column (30 m x 0.25 mm x 0.25 µm). A 0.8 µL aliquot was injected in splitless mode. The oven was programmed from 100 °C (3 min) to 230 °C at 2 °C min-1, with a final hold of 32 min, using helium as the carrier gas at 1 mL min-1. Fatty-acid methyl esters (FAMEs) were identified by retention times and mass spectra using the LIPID MAPS® spectral library and confirmed with a commercial FA standard mix (Supelco 37-component FAME mix, CRM47885, Merck) run under identical conditions.
Chromatograms were processed using Thermo Xcalibur Qual Browser software. Fatty-acid identification was supported by comparison with certified reference standards and mass spectral libraries, and nomenclature follows conventions used in the LIPID MAPS® Structure Database.
Data handling, calculation of summary statistics, and preparation of final datasets were conducted in R version 4.3.2.
description: gas chromatography-mass spectrometry (GC-MS)
 LIPID MAPS spectral library
 R version 4.3.2
description: For MP preparation and experimental work, all materials were pre-cleaned three times with Milli-Q water and once with ethanol (2 µm-filtered). All laboratory procedures for lipid extraction were carried out using pre-combusted or solvent-rinsed glassware to minimise contamination. Procedural blanks were included alongside all samples throughout lipid extraction, silylation, and GC-MS analysis to quantify background contamination. Fatty-acid peaks detected in blanks at corresponding retention times were removed from sample chromatograms before further analysis. Biological replication was maintained at the incubation level during experiments. Individuals were then pooled (2-3 specimens) within replicates to ensure sufficient material for lipid extraction and to reduce individual variability, yielding a final replication of n=3 per treatment for each experiment, including the control treatment without MP under identical conditions. No mortality was observed during incubations, and no samples were excluded based on behavioural criteria. Lipid mass was normalised to dry weight to account for size differences among individuals. Fatty-acid composition is reported as relative abundance (% of total detected fatty acids). Only fatty acids contributing more than 1% of total fatty-acid abundance across all samples were retained to reduce analytical noise. No missing values are present, and no data interpolation or imputation was applied.