Microplastic characterisation

A microplastic feeding stock suspension was made from commercially available (Cospheric LLC CA, USA—UVPMS-BG-1.025) fluorescent green PE microbeads (27–32-µm diameter, 1.030 or 1.026 g cm−3). The beads were confirmed to be PE by Fourier transform infrared (FTIR) spectroscopy using a PerkinElmer FTIR spectrometer (Supplementary Fig. 4). The bead size range was selected to closely conform to the size range of the algal food, simultaneously offered to the krill (see below). Density was selected to be close to neutrally buoyant in 0 °C seawater. The physical properties of the microbeads were characterised using images of beads subsampled from the feeding stock (see Sample analysis section below).

Exposure design

Mixed sex Antarctic krill were collected from the Southern Ocean (66.33 S, 59.34 E) in the Austral summer of 2014/2015. Krill were maintained in the Marine Research Facilities at the Australian Antarctic Division, Tasmania according to previously established methods until use in experiments49. The exposure design followed previously described methods50,51. Adult krill (n = 65, wet weight: 0.556 ± 0.117 mg, length: 41.1 ± 3.7 mm) were acclimatised for 24 h prior to the start of experiments in 5-L glass beakers. Krill were randomly selected for use in the experiment from apparently healthy free swimming schooling adults. Krill were collected into buckets by repeatedly dipping a small net into the same region of the tank as the krill schooled anticlockwise. Buckets contained 15 krill; these were randomly distributed amongst beakers, so each beaker contained five adult krill in 4 L seawater. Five krill per 4 L is the maximum density krill can be maintained under experimental conditions. Block randomisation was applied to distribute krill amongst treatments, and the investigator was not blinded to the treatments. The sex of individuals was not determined in the experiment. Seawater temperatures were maintained at 0 °C (±0.5) and beakers were kept in total darkness throughout the experiment but were exposed to a small amount of red light from a headlamp worn by handlers during the water changes. Exposure seawater was collected from Bruny Island, Tasmania, and filtered to 0.2 µm. Filtered seawater was pre-chilled to 0 °C (±0.5) before krill were added. The dietary exposure suspension was prepared daily from stock using fluorescent plastic microbeads with concentrated instant non-viable algae Thalassiosira weissflogii (Reed Mariculture Inc, CA, USA). The size range for T. weissflogii cells was 5–20 µm according to the manufacturer. Although this is slightly smaller than the microplastics beads, Antarctic krill can feed efficiently on particles >2 µm up to whole zooplankton (~3 mm). Dietary exposure suspensions were made up as a portion of the krill’s dietary requirements under laboratory conditions, 100% algae equates to 0.00798 mg T. weissflogii (dry weight) per beaker. Harvested krill were euthanized in liquid nitrogen or formalin. The seawater physiochemical parameters for the two experiments are outlined in Supplementary Tables 1–2. Mortality for all experiments is given in Supplementary Note 1.

Particle size experiment

Four-day feeding and egestion experiments were carried out on 45 Antarctic krill. Nominal daily exposure suspensions were made up to 20 or 80% microplastics by weight, which equated to approximately 29 or 116 beads mL−1 (402 or 1606 µg L−1). Control krill were fed 100% algae and all treatments were carried out in triplicate. Krill were transferred daily to exposure suspension and allowed to feed for 4 h, before being transferred with a stainless steel dip net to a clean beaker for 20 h. Before transfer, krill were flushed with cold fresh filtered seawater to remove plastics that may adhere to the exoskeleton. Upon transfer to the exposure suspension, krill were observed to be feeding almost immediately. Faecal pellets were collected after 24 h exposure on days 1 and 4. An extra sample point was added on day 4 to assess fine scale temporal variation after repeated exposure, thus faecal pellets were collected at 4 and 24 h on day 4 (refer to Supplementary Fig. 2). All beakers of krill were harvested for particle size and tissue localisation analysis after 96 h. Three krill from each beaker were randomly selected for particle size analysis (n = 18 krill total). As the beads were fragmented after ingestion, the total bead ingestion rates could not be calculated from stomach content or egested material.

Tissue localisation experiment

To investigate tissue localisation of ingested plastic, two krill from each beaker were randomly selected, fixed in formalin, and used for histological cryo-section (20 μm) analysis. Slides were stained with H&E or remained unstained. Slides were examined using an Olympus BX60 fluorescence microscope or Zeiss-780 Laser Scanning Confocal microscope with a fluorescent filter of 488-nm excitation and 526-nm emission.

In addition, to investigate if krill could fragment plastics <1 µm and the possibility of fragments entering the digestive gland, five krill were exposed to 100% plastic diet (approx. 2063 µg L−1 or 149 beads mL−1) for 24 h, with no water changes. These five krill were all used for tissue localisation analysis.

Egestion experiment

To examine particle sizes egested over an extended period, 15 krill divided into three beakers were exposed for 10 days to a 20% diet (approx. 401 µg L−1) of plastic following the same basic design as the Particle Size Experiment. After 10 days, the diet was switched to 100% algae for five days. Faecal pellets were collected at 4 and 24 h every day of the five day egestion period. Faecal material was pooled per beaker per 24 h resulting in 15 samples.

Sample analysis

Body burden analysis was carried out using an enzyme digestion followed by visual identification of ingested microplastics under a fluorescent microscope. Krill were flushed with Milli-Q water, blotted dry, weighed (to 3 d.p), and heated to 65 °C in a water bath, after which the exoskeleton was removed. Krill were then homogenised using a glass rod, and digested using proteinase K adapted from Cole et al.52, which was previously shown to have negligible effects on PS bead integrity. Digestion efficacy was not optimal as hard chitinous structures often remained after digestion. Digested krill were filtered under vacuum onto Millipore gridded 0.45 µm filters and air dried overnight. Filters were fixed between glass coverslips and analysed for microplastics using a Zeiss-780 Laser Scanning Confocal microscope with a fluorescent filter with a Plan-Apochromat 10 × /0.45 M27 lens, with a numerical aperture of 0.45. Microplastic fragments were imaged in five randomly selected squares (6.97 × 6.98 mm; total area of 2.4 cm2) on the filter paper, which accounted for 25% of the total filtered area. Images were verified by eye, and compared to controls to examine for undigested chitinous material with autofluorescence. Of the 165 images taken, 2 images were excluded on the basis of chitinous material with autofluorescence (See Fig. 2. panel A for example of excluded image). These were too large to be mistaken as a microplastic beads and were clearly distinguishable as mandibles. The diameter (major axis when particles were fitted to an ellipse) of each particle within each image was measured using imaging software (FIJI GPL v2)53. A minimum threshold was applied to the fluorescence intensity of each image to ensure only beads were counted by the imaging software. Thresholds were set to a minimum of 65 and maximum of 255 which allowed background material, including undigested exoskeleton (except for mandibles), algal cells and the filter paper, to be excluded without interference to the analysis. Size exclusions were applied to particles which had a diameter >50 µm, on the basis these were two or more beads too close together for the imaging software to distinguish individual beads and accurately measure size. See Supplementary Note 1 and Supplementary Figs. 5–6 for further information.

Bead fragmentation

To test that the sample analysis procedures were not responsible for fragmenting the beads, procedural blanks were carried out in a pilot study and throughout the experiment. Procedural blanks consisted of krill and beads or just beads. Krill blanks consisted of seven krill taken from a pilot study. The krill were digested as per the method described in the Sample Analysis section, except the krill were not homogenised. After digestion, exoskeleton remained intact but the tissue was completely digested, krill were vortexed and the stomach was opened to liberate any remaining beads. The sample was then filtered and imaged as per the method described in the Sample Analysis section. Bead blanks consisted of beads in the absence of krill and were not homogenised. Beads were added to buffer and enzyme, then digested, filtered and imaged as per methods outlined in the Sample Analysis section. Bead blanks were examined after enzyme digestion with FTIR spectroscopy (Supplementary Fig. 7), but ingested beads and fragments were unable to be detected on the cellulose filters with FTIR due to the low concentration and/or small size of the particle.

Statistical analysis

Two-sample Kolmogorov–Smirnov tests (two-tailed, α ≤ 0.05) were used to compare the particle size distribution from the stock microbeads to the size distribution of plastics isolated from the digested krill, and from the particles isolated from the faecal pellets. The proportion of whole beads compared to fragments in digested krill and in faecal pellets was compared between doses using Chi-squared analysis (two tailed, α ≤ 0.05). For all proportion tests (Chi-squared and linear regression), beads with a diameter ≥25 µm were classified as whole beads, beads <25 µm were considered fragments. This cut off was selected by eye using the standard distribution of the stock beads. Kolmogorov–Smirnov tests (two tailed, α ≤ 0.05) were used to test for normality. The data were log 10 transformed and comparison between fragment size, sample time and plastic dose in the faecal pellets was determined with a two-way ANOVA (two tailed, α ≤ 0.05). Multiple linear regression was used to examine relationships between the length and weight of the krill and their ability to fragment plastics. Means are expressed as mean ± standard deviation (S.D.) unless otherwise stated.

Data availability

Data and image macro coding are available from the corresponding author on request.