Microplastic Ingestion by Zooplankton

Abstract

Small plastic detritus, termed 'microplastics', are a widespread and ubiquitous contaminant of marine ecosystems across the globe. Ingestion of microplastics by marine biota, including mussels, worms, fish and seabirds, has been widely reported, but despite their vital ecological role in marine food-webs, the impact of microplastics on zooplankton remains under-researched. Here, we show that microplastics are ingested by, and may impact upon, zooplankton. We used bio-imaging techniques to document ingestion, egestion and adherence of microplastics in a range of zooplankton common to the northeast Atlantic, and employed feeding rate studies to determine the impact of plastic detritus on algal ingestion rates in copepods. Using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy we identified that thirteen zooplankton taxa had the capacity to ingest 1.7 - 30.6 µm polystyrene beads, with uptake varying by taxa, life-stage and bead-size. Post-ingestion, copepods egested faecal pellets laden with microplastics. We further observed microplastics adhered to the external carapace and appendages of exposed zooplankton. Exposure of the copepod Centropages typicus to natural assemblages of algae with and without microplastics showed that 7.3 µm microplastics (>4000 ml-1) significantly decreased algal feeding. Our findings imply that marine microplastic debris can negatively impact upon zooplankton function and health.

Full text

Microplastic Ingestion by Zooplankton
Matthew Cole,
†,∥,
*Pennie Lindeque,
†
Elaine Fileman,
†
Claudia Halsband,
‡
Rhys Goodhead,
§
Julian Moger,
§
and Tamara S. Galloway
∥
†
Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, United Kingdom
‡
Akvaplan-niva AS, FRAM −High North Research Centre for Climate and the Environment, N-9296 Tromsø, Norway
§
College of Engineering, Mathematics and Physical Sciences: Physics, Physics Building, University of Exeter, Stocker Road, Exeter
EX4 4QL, United Kingdom
∥
College of Life and Environmental Sciences: Biosciences, Geoffrey Pope Building, University of Exeter, Stocker Road, Exeter EX4
4QD, United Kingdom
ABSTRACT: Small plastic detritus, termed “microplastics”,
are a widespread and ubiquitous contaminant of marine
ecosystems across the globe. Ingestion of microplastics by
marine biota, including mussels, worms, fish, and seabirds, has
been widely reported, but despite their vital ecological role in
marine food-webs, the impact of microplastics on zooplankton
remains under-researched. Here, we show that microplastics
are ingested by, and may impact upon, zooplankton. We used
bioimaging techniques to document ingestion, egestion, and
adherence of microplastics in a range of zooplankton common
to the northeast Atlantic, and employed feeding rate studies to
determine the impact of plastic detritus on algal ingestion rates
in copepods. Using fluorescence and coherent anti-Stokes
Raman scattering (CARS) microscopy we identified that
thirteen zooplankton taxa had the capacity to ingest 1.7−30.6 μm polystyrene beads, with uptake varying by taxa, life-stage and
bead-size. Post-ingestion, copepods egested faecal pellets laden with microplastics. We further observed microplastics adhered to
the external carapace and appendages of exposed zooplankton. Exposure of the copepod Centropages typicus to natural
assemblages of algae with and without microplastics showed that 7.3 μm microplastics (>4000 mL−1) significantly decreased algal
feeding. Our findings imply that marine microplastic debris can negatively impact upon zooplankton function and health.
1. INTRODUCTION
It has been estimated that up to 10% of plastics produced
globally enters our oceans, so it is of little surprise that plastic
debris is now a pervasive and resilient pollutant of the marine
environment.
1,2
Larger plastic debris, such as monofilament
line, plastic strapping, and plastic bags, can entangle, garrotte,
drown, or be eaten by an array of marine wildlife.
3
There is
compelling evidence that microplasticssmall plastic <5 mm
in diameteralso negatively impact upon marine biota.
4
Microplastics consist of synthetic polymer products manufac-
tured to be of a small size, such as exfoliates in cosmetics,
5
and
those items derived from the fragmentation of larger plastic
debris, for example polyester fibers from fabrics,
6
polyethylene
fragments from plastic bags
7
and polystyrene particles from
buoys and floats.
8
Typically, high-density plastics (e.g.,
polyvinyl chlorides, polyester) settle out of the water column,
whereas low-density plastics (e.g., polyethylene, polystyrene)
remain buoyant, although freshwater inputs, storms, and
biofilm formation may result in vertical mixing.
9,10
Floating
plastic debris is susceptible to local and ocean currents resulting
in higher-than-average waterborne microplastic concentrations
in areas of confluence.
11
Microplastics are of environmental concern as their small size
makes them available to a wide range of marine biota.
12
Microplastic ingestion has been demonstrated in marine
organisms, including amphipods, lugworms, and barnacles,
4
mussels,
13
decapod crustaceans,
14
seabirds,
15
and fish.
16,17
Ingested microplastics might obstruct feeding appendages,
aggregate, and block the alimentary canal, limit the food intake
of an organism or be translocated into the circulatory
system.
13,14
Further, microplastics may introduce toxicants to
the organism: first, additives incorporated into a plastic during
manufacture to improve its properties (e.g., phthalates for
malleability and polybrominated diphenyl ethers (PDE) for
heat resistance) might leach out of weathered plastic debris;
18,19
second, the large surface area to volume ratio and hydrophobic
properties of microplastics leave them susceptible to the
accumulation of hydrophobic organic contaminants (HOCs)
which could dissociate post-ingestion.
20
Received: February 12, 2013
Revised: May 16, 2013
Accepted: May 21, 2013
Article
pubs.acs.org/est
© XXXX American Chemical Society Adx.doi.org/10.1021/es400663f |Environ. Sci. Technol. XXXX, XXX, XXX−XXX

The extent to which microplastics are ingested and can
impact upon zooplankton is uncertain. Zooplankton have a vital
ecological role in marine ecosystems, both as primary
consumers in the marine food web, and in the case of
meroplankton, consisting of the juvenile life stage of numerous
commercially important species. The widespread presence of
small plastic debris in the water column makes interactions
between zooplankton and microplastics highly likely; indeed,
both small plastic debris and zooplankton >333 μm in diameter
have been recurrently sampled together in sea surface trawls
and by continuous plankton recorders.
4,11,21,22
Zooplankton
display a range of feeding modes, which vary by life-stage,
species and prey availability.
23
Zooplankton can use a
combination of chemo- and mechano-receptors to select prey,
and their ability to preferentially feed on one species of algae
over other algae, plastic beads or detritus has been
demonstrated.
24−26
Laboratory experiments, in which latex
beads were used to model algal ingestion, have shown that
zooplankton have the potential to ingest small plastics.
26−28
Uptake of these small plastics likely results from indiscriminate
feeding modes (e.g., filter-feeding), by which prey with
equivalent spherical diameters (ESD) <100 μm are non-
selectively fed upon.
23,29
Due to the complexities of sampling and extracting
microplastics from the marine environment, existing studies
have largely focused on detritus >333 μm.
1,30
However, there is
evidence of very small microplastics (<100 μm) both in the
benthos and water column. Sampling of shoreline, estuarine
and harbor sediments has shown the presence of ∼20 μm
diameter fibrous polymers,
4,6,31
and microplastic fibers,
granules, films, and polystyrene spheres ranging in size from
38 μm to 1 mm.
32
In the water column, sampling with a 80 μm
mesh in Swedish coastal waters captured 100 000 times greater
concentrations of microplastics than when using a 450 μm
mesh, with a maximal concentration of 102 000 microplastics
per m3sampled near a polyethylene production facility.
33
Sampling of microplastics in this size range is exceptional, as
such there is currently insufficient data to determine realistic
environmental concentrations of these particles.
Here, we investigate the ingestion of minute microplastics,
≤31 μm diameter, by a range of zooplankton species, and
examine their impact on zooplankton function and feeding. To
explore the hypothesis that zooplankton are capable of
ingesting microplastics, 15 zooplankton taxarepresentative
of abundant mesozooplankton in northeast Atlantic coastal
systemswere exposed to polystyrene spheres in the size range
7.3−30.6 μm suspended in natural seawater, then analyzed
using fluorescence microscopy. Using the copepod Temora
longicornis,weexploredwhere0.4−3.8 μmmicroplastics
accumulate, both internally and externally, using a novel
bioimaging technique: coherent anti-Stokes Raman scattering
(CARS) microscopy. Finally, to test the hypothesis that
microplastics negatively impact upon zooplankton feeding, we
exposed the copepod Centropages typicus to natural assemblages
of algae and polystyrene beads, using fluorometry and flow
cytometry to quantify algal ingestion.
2. MATERIALS AND METHODS
2.1. Zooplankton Sampling. Zooplankton sampling was
conducted between November 2011 and October 2012 at
Station L4 (50°15′N, 4°13′W), a coastal site located in the
western English Channel 12 km south of Plymouth, UK.
34,35
A
200 μm mesh was used to collect zooplankton via horizontal
surface tows and vertical hauls. Collected zooplankton were
held in 2 L of seawater within a coolbox, and transported to
controlled-temperature facilities at Plymouth Marine Labo-
ratory (Plymouth, UK). For all experimental procedures, we
maintained the zooplankton at ambient sea-surface temper-
atures (ranging 10−17 °C depending on sampling date).
Specimens were hand-selected under a dissecting microscope
within two hours of sampling, and then collectively held in 2 L
of filtered seawater (0.22 μm Millipore filter) for 24 h to allow
full gut depuration. In all, fourteen mesozooplankton taxa (size:
0.2−20 mm), representative of the most commonly occurring
zooplankton in the western English Channel and covering a
range of life-stages and life-strategies, in addition to cultured
Oxyrrhis marina, a heterotrophic dinoflagellate (size: 15−30
μm), were selected for microplastic ingestion studies (Table 1).
2.2. Natural Seawater Preparation. For the algal
ingestion studies, natural seawater (5 L) was collected from
the sea surface at station L4, passed through a 200 μm mesh
into a polycarbonate carboy and returned to the laboratory
within 2 h. The seawater was further screened with a 100 μm
mesh to ensure the removal of any grazing micrometazoans
then stored in the dark for 24 h at ambient sea-surface
temperature to maintain the natural communities of algae at
normal concentrations. Prior to experimental work, the
seawater was mixed thoroughly by gentle inversion of the
water in the carboy.
2.3. Microplastics. Exposures used commercial polystyrene
spheres (SPHERO Spherotech). With global production rates
of 10.6 million tons in 2001, polystyrene is the fourth most
commonly produced polymer in the world and its presence as a
constituent of marine debris is commonly reported.
30,36
The
bead sizes used in each experiment (0.4−30.6 μm) were
selected to be comparable with the prey size range of the
zooplankton exposed.
23,37
2.4. Microplastic Ingestion by Zooplankton. To
ascertain whether zooplankton ingest microplastics we
conducted exposures using fluorescent polystyrene beads, and
used microscopy to assess uptake. Microplastic suspensions
were made up by pipetting 20 μL of 7.3, 20.6, or 30.6 μm
diameter fluorescently labeled (yellow fluorescence: 400−500
nm excitation, 450−550 nm emission) polystyrene spheres into
glass vials containing 20 mL of filtered seawater (0.1% v/v:
3000 beads mL−1(7.3 μm); 2240 beads mL−1(20.6 μm); 635
beads mL−1(30.6 μm)), then mixed through repeated
inversion. With larger zooplankton (e.g., copepods, decapod
larvae, chaetognaths), individual specimens were added directly
to the vial (n=≥6 per exposure), and fitted to a rotating
plankton wheel (<5 rpm) for 24 h. For smaller zooplankton or
those with low survivability in the laboratory (e.g., bivalve
larvae, gelatinous holoplankton, O. marina), individual speci-
mens were exposed to microplastic suspensions in Petridishes
(n=≥6 per exposure) at ambient sea temperature for 1 h (with
the exception of bivalve larvae which were exposed for 24 h
using this method). Post-exposure, zooplankton were washed
with filtered seawater and transferred to Eppendorf tubes
containing 1 mL of 4% formalin . Ingestion was ascertained by
viewing specimens at ×40−400 magnification with an Olympus
IMT2 inverted light microscope with fluorescence to determine
the presence of polystyrene beads (fluorescing yellow-green)
within the alimentary canal or body cavity of the zooplankton.
To better understand the interactions between zooplankton
and microplastics, both live and preserved copepods and select
zooplankton specimens were viewed under the microscope for
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varying lengths of time to observe the feeding process,
ingestion, gut passage, and egestion of polystyrene beads.
2.5. Interactions between Microplastics and Cope-
pods. To explore the internal distribution and external
adherence of microplastics in zooplankton, we first exposed
the copepod Temora longicornis to polystyrene beads and then
employed CARS microscopy (see below) to visualize their
uptake. Microplastic suspensions were formulated by adding 12
μL of 0.4, 1.7, or 3.8 μm diameter non-labeled polystyrene
spheres to 24 mL of filtered seawater (0.05% v/v: 1 ×106beads
mL−1(0.4 μm), 380 ×103beads mL−1(1.7 μm), and 40 ×103
beads mL−1(3.8 μm)), which were mixed through inversion
and sonication. Individual T. longicornis (n=≥6 per exposure)
were added to each vial, rotated at <5 rpm at ambient sea
temperature for 24 h. Post-exposure, specimens were poured
onto a 200 μm mesh suspended in filtered seawater (to prevent
damage to the copepods), washed gently, preserved in 4%
formalin and then transferred to the bioimaging suite at the
University of Exeter (Exeter, UK).
2.6. Coherent Anti-Stokes Raman Scattering (CARS)
Microscopy. CARS microscopy is a novel microscopy
technique that provides label-free contrast, based on vibrational
spectroscopy
38
which has exceptional capability for locating
polymer particles within biological tissues with subcellular
precision.
39,40
CARS imaging was performed using a custom-
built microscopy system based on a commercial confocal laser-
scanning microscope and a synchronized dual-wavelength
picosecond laser source. Laser excitation was provided by an
optical parametric oscillator (OPO) (Levante Emerald, APE,
Berlin) pumped with a frequency doubled Nd:vandium
picosecond oscillator (High-Q Laser Production GmbH).
The pump laser generated a 6 ps, 76 MHz pulse train at 532
nm with adjustable output power up to 10 W. The OPO
produced collinear signal and idler beams with perfect temporal
overlap and provided continuous tuning over a range of
wavelengths. The signal beam was used as the pump, ranging
from 670 to 980 nm and fundamental of Nd:vandium (1064
nm) used as the Stokes beam. The maximum combined output
power of the pump and Stokes was approximately 1 W, which
was attenuated to reduce the power at the sample to between
15 and 30 mW. To improve the transmission of the near-IR
excitation through the commercial microscope (IX71 and
FV300, Olympus UK) the galvanometer mirrors were replaced
with silver mirrors and the tube lens was replaced with a MgF2
coated lens. The collinear pump and Stokes beams were
directed onto the scanning confocal dichroic which was
replaced by a silver mirror with high reflectivity throughout
the visible and NIR (21010, Chroma Technologies, Bellows
Falls, VT ). The forward-CARS signal was collected by the air
condenser, transmitted by the dichroic mirror and directed
onto a red-sensitive photomultiplier tube (R3896, Hamamatsu
Photonic UK). The epi-CARS signal was collected using the
objective lens and separated from the pump and Stokes beams
by a long-wave pass dichroic mirror (z850rdc-xr, Chroma
Technologies) and directed onto a second R3896 photo-
multiplier tube at the rear microscope port. The CARS signal
was isolated at each photodetector using a single band-pass
filters centered at the anti-Stokes wavelengths. Imaging was
performed using either a 60×water immersion, or 20×air
objective (UPlanS Apo, Olympus UK).
2.7. Impact of Microplastics on Copepod Feeding. To
determine whether microplastics negatively impact upon a
copepod’s ability to ingest natural prey, we exposed the
copepod Centropages typicus to natural assemblages of algae
with and without microplastics, and compared algal ingestion
Table 1. Capacity for a Range of Zooplankton to Ingest
Microplastics, Demonstrated Using Fluorescent
Microscopy
a
organism taxonomy microplastic
ESD (μm)
exposure
duration
(h) ingestion
(Y/P/N?)
Holoplankton (Copepods)
Acartia clausi Copepoda
(Calanoida) 7.3 24 yes
Acartia clausi Copepoda
(Calanoida) 20.6 24 no
Acartia clausi Copepoda
(Calanoida) 30.6 24 partial
Calanus
helgolandicus
Copepoda
(Calanoida) 7.3 24 yes
Calanus
helgolandicus
Copepoda
(Calanoida) 20.6 24 yes
Calanus
helgolandicus
(juv.)
Copepoda
(Calanoida) 20.6 24 yes
Calanus
helgolandicus
Copepoda
(Calanoida) 30.6 24 partial
Centropages
typicus
Copepoda
(Calanoida) 7.3 24 yes
Centropages
typicus
Copepoda
(Calanoida) 20.6 24 yes
Centropages
typicus
Copepoda
(Calanoida) 30.6 24 yes
Temora
longicornis
Copepoda
(Calanoida) 7.3 24 yes
Temora
longicornis
Copepoda
(Calanoida) 20.6 24 yes
Temora
longicornis
Copepoda
(Calanoida) 30.6 24 yes
Holoplankton (Other)
Doliolidae Tunicata 7.3 1 yes
Euphausiidae Euphausiacea 20.6 24 yes
Parasagitta sp. Chaetognatha 20.6 1 no
Parasagitta sp. Chaetognatha 30.6 24 no
Obelia sp. Cnidaria
(Hydrozoa) 20.6 1 partial
Siphonophorae Cnidaria
(Hydrozoa) 20.6 1 no
Meroplankton
Bivalvia (larvae) Mollusca 7.3 24 yes
Brachyura
(megalopa) Decapoda 20.6 24 yes
Brachyura
(zoea) Decapoda 20.6 24 no
Caridea (larvae) Decapoda 20.6 24 yes
Paguridae
(larvae) Decapoda 20.6 24 partial
Porcellanidae
(zoea) Decapoda 30.6 24 partial
Microzooplankton
Oxyrrhis marina Dinoflagellata 7.3 1 yes
a
Microplastic uptake is based upon the number of individuals in a
treatment (n=≥6) that contained beads in their alimentary canals or
body cavity following 1 or 24 h exposures to either 7.3, 20.6, or 30.6
μmfluorescent polystyrene beads. ESD = equivalent spherical
diameter. Scoring system: yes (>50%); partial (<50%); no (0%).
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rates between treatments. In our initial experiment, designed to
identify the size of microplastic that would have the greatest
impact on C. typicus feeding, we exposed individual C. typicus
specimens (n=≥6 per exposure) to 23 mL of natural seawater
containing 0 or 23 μL of 7.3 or 20.6 μmfluorescent polystyrene
beads (0.1% v/v), rotated at <5 rpm for 24 h. To quantify algal
concentrations within the natural seawater pre- and post-
exposure, we vacuum filtered the exposure media through a
glass fiber filter, and then transferred the filter to 7 mL of
acetone, held at 4 °C in the dark for 24 h. The chlorophyll
levels within the acetone solution were measured using a
Turner fluorometer. Since 7.3 μm microplastics had the most
notable impact on C. typicus feeding, we conducted a further
experiment to establish a dose−response relationship between
microplastic concentration and food uptake. Microplastic
suspensions consisted of 0, 2.5, 5, 10, or 20 μL additions of
Figure 1. Microplastics of different sizes can be ingested, egested and adhere to a range of zooplankton, as visualized using fluorescence microscopy:
(i) the copepod Centropages typicus containing 7.3 μm polystyrene (PS) beads (dorsal view); (ii) the copepod Calanus helgolandicus containing 20.6
μm PS beads (lateral view); (iii) a D-stage bivalve larvae containing 7.3 μm PS beads (dorsal view); (iv) a Brachyuran (decapod) larvae (zoea stage)
containing 20.6 μm PS beads (lateral view); (v) a Porcellanid (decapod) larvae, containing 30.6 μm PS beads (lateral view); (vi) 30.6 μm PS beads
in the posterior-gut of the copepod Temora longicornis during egestion, (vii) 1.4 μm PS beads trapped between the filamental hairs of the furca of C.
typicus; (viii) a T. longicornis faecal pellet containing 30.6 μm PS beads; (ix) proportion of copepods (Acartia clausi,Calanus helgolandicus,Centropages
typicus, and Temora longicornis) with microplastics in their guts following 24 h of exposure to 7.4, 20.6, and 30.6 μm polystyrene beads. *denotes
statistically significant (P≤0.05) lower consumption of larger beads compared with that of 7.3 μm beads. Scale bar (gray line): 100 μm.
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7.3 μmfluorescent polystyrene beads in 23 mL of natural
seawater. A 1.8 mL aliquot of natural seawater was taken from
all vials at T0and fixed with 40 μL of 50% glutaraldehyde (4%
final concentration), inverted for 2 min, refrigerated at 4 °C for
30 min and subsequently snap-frozen in liquid nitrogen and
stored in a −80 °C freezer prior to analysis using analytical flow
cytometry. Individual C. typicus (n=≥6 per exposure) were
added to experimental vials, while controls (with no copepod)
were set up to determine natural growth or decline of algae
over the exposure period. The vials were incubated on a
rotating plankton wheel (5 rpm) for 24 h in the dark. Post-
exposure (T24), a further 1.8 mL aliquot was fixed (as with T0).
Flow cytometric analysis was carried out on thawed natural
seawater samples using a BD Accuri C6 flow cytometer.
41
Particle abundance data was subsequently used to calculate the
ingestion rates of algae by C. typicus.
42
2.8. Statistical Analysis. Data was analyzed using Micro-
soft Excel. Student’sttests were used to compare experimental
data with controls, with significant difference attributed where P
≤0.05. Regression analysis was used to analyze the correlation
between algal ingestion rates and microplastic concentration.
3. RESULTS
3.1. Microplastic Ingestion by Zooplankton. The
majority of zooplankton (13 of 15) exposed to polystyrene
beads (7.3−30.6 μm) demonstrated the capacity to ingest
microplastics (Table 1). Organisms exhibiting uptake included
copepods (Figure 1i, ii), bivalve larvae (Figure 1iii) and
decapod larvae (Figure 1iv, v). Only two specimens
chaetognaths (Parasagitta sp.) and siphonophorae (Cnida-
ria)showed no evidence of ingestion. All four species of
copepods examined demonstrated some affinity for ingesting
microplastics, with Centropages typicus and Temora longicornis
able to consume 7.3, 20.6, and 30.6 μm polystyrene beads
(Figure 1ix). The other copepods showed evidence of size-
based selectivity: Acartia clausi ingested 7.3 μm beads but
ingested significantly fewer 20.6 and 30.6 μm beads, and
Calanus helgolandicus showed significantly less affinity for 30.6
μm beads than for 7.3 μm beads. The decapod Brachyurans
demonstrated variability in microplastic ingestion depending
upon life-stage: brachyuran zoea showed no affinity for 20.6 μm
beads, while the more developed brachyuran megalopa readily
ingested such beads. Obelia sp., Paguridae larvae and
Porcellinidae (zoea) exhibited individual variability in their
ability to ingest polystyrene beads, with less than half the
Figure 2. Coherent anti-Stokes Raman scattering (CARS) microscopy: (i) Spontaneous [black] and stimulated [grey] peaks for polystyrene beads,
Raman shifts of 2845 cm−1(C−H) and 3050 cm−1(aromatic C−H) were used to visualize the polystyrene; (ii) 3.4 μm microplastics accumulated in
the alimentary canal [ac] of the copepod Temora longicornis (yellow dots); beads further adhered to the exterior of the copepod’s urosome [u], furca
[f] and posterior swimming legs [sl] (blue dots); (ii) 3.4 μm microplastics (red dots) adhered to the external surface of the posterior swimming legs
of T. longicornis. Scale bar [gray line]: 50 μm.
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exposed specimens in a cohort showing evidence of micro-
plastic uptake.
Live observations of copepods, euphausids, and doliolids
found microplastics were ingested via filter-feeding. In
copepods and euphausids, this process relied upon the rapid
movement of the swimming legs and external appendages,
which generated a feeding current that indiscriminately drew
surrounding beads toward the organism. With doliolids, we
observed the microplastics being drawn through the anterior
siphon into their body cavity, where the polystyrene beads were
entrapped and drawn toward the gut. Oxyrrhis marina, a single
celled heterotrophic dinoflagellate, demonstrated a more direct
method of ingestion, locating particles with their flagella and
then engulfing the polystyrene beads. Post-ingestion, copepods
typically aggregated beads within the anterior midgut, shifted
them to the posterior midgut via peristaltic action (Figure 1i, ii)
and egested them within densely packed faecal pellets (Figure
1vi, viii). Typically, microplastic-laden faecal pellets were
egested within hours. In the absence of food, individual
microplastic beads could remain in the intestinal tract of C.
helgolandicus for up to 7 days (data not shown). During
observations of both live and preserved zooplankton specimens,
including copepods, decapod larvae and euphausids, micro-
plastics often adhered to the specimens’external surfaces. In
copepods that died during the exposure period, polystyrene
beads would coat the carapace in vast numbers; similarly, beads
were observed to cling to the shed carapace of a molting C.
helgolandicus copepodite. In live specimens, microplastics were
found to concentrate between the external appendages of
copepods, including the swimming legs, feeding apparatus,
antennae, and furca (Figure 1vii).
3.2. Interactions between Microplastics and Cope-
pods. CARS microscopy used a blend of transmitted light to
capture the structure of the copepod, and Raman shifts of 2845
cm−1(C−H) and 3050 cm−1(aromatic C−H) to visualize the
polystyrene (Figure 2i). Temora longicornis ingested both 1.7
and 3.8 μm polystyrene beads; use of Z-stackingin which 2D
images at incremental focal plains are layered together to form a
Figure 3. Exposure to increasing concentrations of microplastics in the copepod Centropages typicus (n=≥5). Treatments comprise seawater
containing natural assemblages of algae [A] with 4000 [B], 7000 [C], 11 000 [D], and 25 000 [E] 7.3 μm polystyrene beads per mL. *denotes
statistically significant (P≤0.05) lower ingestion rates (cells individual−1hour−1) than in controls. Graphs show ingestion rates of (i) Synechococcus
sp.; (ii) Picoeukaroytes; (iii) all algae present; (iv) plot comparing positive C. typicus algal ingestion rates at differing microplastics concentrations−
logarithmic regression: R2= 0.70 (P≤0.05).
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3D imageconfirmed that microplastics clumping in the
posterior midgut were, indeed, internalized (Figure 2ii; yellow
dots), but sufficient resolution to identify microplastic trans-
location was not possible. CARS imaging confirmed that
microplastics adhere to the external appendages of the
zooplankton: polystyrene beads (0.4−3.8 μm) accumulated
between the filamental hairs on appendages, including the furca
(Figure 2iii; blue dots), rear swimming legs (Figure 2iii; red
dots) and antennules, and between the segments of the
carapace, particularly around the urosome and swimming legs.
3.3. Impact of Microplastics on Copepod Feeding.
Using chlorophyll concentration as a proxy for algal abundance,
we identified that 7.3 μm microplastics had a significant impact
on algal ingestion by the copepod Centropages typicus (data not
shown) and identified a significant dose−response relationship
between ingestion rates and the concentration of 7.3 μm
polystyrene beads. Exposed to seawatercontaining natural
assemblages of algaeC. typicus ingested ∼12 Synechococcus sp.
ind−1h−1(Figure 3i) and ∼24 picoeukaryotes ind−1h−1(Figure
3ii). These ingestion rates decreased when additionally exposed
to ∼4000 microplastics mL−1; this decrease was statistically
significant at concentrations of ≥7000 microplastics mL−1(t
test: P≤0.05). When considering all of the <20 μm ESD algal
groups identified using flow cytometry −Synechococcus sp.,
picoeukaryotes, nanoeukaryotes, and cryptophytes,in
combination (hereafter referred to as “total algae”), C. typicus
presented total algal ingestion rates of ∼34 algae ind−1h−1in
the absence of microplastics. Total algal ingestion rates for C.
typicus were significantly reduced with the addition of ≥4000
microplastics mL−1(ttest: P≤0.05; Figure 3iii). Furthermore,
we identified a strong, logarithmic relationship (R2= 0.70, P≤
0.05) between the ingestion rate of total algae and microplastic
concentration (Figure 3iv).
4. DISCUSSION
Our results show that a range of zooplankton common to the
northeast Atlantic can ingest microplastics (1.4−30.6 μm
diameter), with capacity for uptake varying between species,
life-stage, and microplastic size. Microplastics were indiscrim-
inately ingested via filter-feeding and later egested in faecal
pellets, typically within a matter of hours. Microplastics
accumulated on the external surface of dead zooplankton, and
were found trapped between the external appendages of live
copepods. We visualized 1.7 and 3.8 μm polystyrene beads
clustered within the alimentary canal and aggregated between
the setae and joints of external appendages. Lastly, we
demonstrated that the presence of 7.3 μm polystyrene beads
could significantly reduce the algal ingestion rate of the
copepod Centropages typicus, in a dose−response relationship.
We demonstrated that 13 zooplankton taxaincluding
holoplankton, meroplankton, and microzooplanktonhave
the capacity to ingest polystyrene beads in the absence of
natural food. All four copepod species showed uptake of
microplastics, with varying degrees of selectivity: T. longicornis
and C. typicus ingested 7.3, 20.6, and 30.6 μm beads, whereas A.
clausi and C. helgolandicus fed on 7.3 μm beads but less
frequently ingested larger beads. Using CARS microscopy, we
further identified that T. longicornis could ingest 1.7 and 3.8 μm
microplastics; however, we found no evidence of 0.4 μm beads
being ingested. Brachyuran larvae only ingested 20.6 μm
polystyrene beads as megalopa (postzoea larvae), with no
uptake observed when in the earlier zoea stage. Microplastics
were also ingested by the filter-feeding euphausids and
doliolids, and Oxyrrhis marina, a heterotrophic dinoflagellete
that ingests motile or immotile prey through engulfment via a
non-permanent cytosome.
43
These findings corroborate the
results of several previous studies, which documented the
uptake of <100 μm microplastics by Acartia tonsa,
28
Calanus
pacificus adults, copepodites and nauplii,
26,44,45
Oxyrrhis
marina,
46
ciliates,
47,48
echinoderm larvae
27
and salps.
49
We did not observe microplastic uptake in Parasagitta sp.
(chaetognaths) following 1 or 24 hour exposures to 30.6 μm
beads, or siphonophorae (Cnidaria) exposed to 20.6 μm
plastics, possibly as a result of handling stress, or more likely
because these zooplankton are raptorial predators and feed
actively, so were not enticed to capture the immotile
microplastics.
37
Furthermore, only 10−50% of Obelia sp.,
Paguridae larvae and Porcellinidae (zoea) specimens presented
with polystyrene beads in their intestinal tracts post-exposure.
As we also observed size-selective ingestion in A. clausi and C.
helgolandicus, it is important to consider how microplastics may
impact on different zooplankton feeding strategies. Zooplank-
ton use both mechanoreception (i.e., detection of pressure
disturbances within the water) and chemoreception (i.e.,
detection of infochemicals emitted by algal cells) to sense
prey.
29,37
As such, the clean immotile beads used in our algal-
free experiments are less likely to be detected by exposed
zooplankton, although it is possible that aged microplastics, that
have developed biofilms during their residence within the
marine environment,
10
may generate a chemosensory response;
this effect was observed in the copepod Eurytemora aff inis
which more readily ingested beads spiked with bacteria than
when offered beads alone.
50
While some copepods will
continuously filter-feed regardless of prey availability, others
(e.g., C. pacificus,A. tonsa) can limit their movement and filter-
feed at reduced rates to conserve energy when faced with low
food concentrations.
51,52
The presence of algae promotes
greater uptake of microplastics in the filter-feeding copepods
Calanus pacificus
26
and Eucalanus pileatus CV copepodites;
53
notably, A. clausi only ingests 16 μm polystyrene beads in the
presence of algae.
24
Some zooplankton can ingest or reject prey
upon capture, depending on surface characteristics and charge
of the particle, both echinoderm larvae and the copepods A.
clausi and E. pileatus can reject plastic beads that coalesced
within their mouthparts.
27,53,54
The presence of microplastics
may also alter the behavior of zooplankton, limiting their
capacity to feed; in Acartia tonsa copepodites, contact with 45
μm plastic beads caused the organisms to “jump”, limiting time
dedicated to feeding bouts and reducing their clearance rates by
60%.
55
Post-ingestion, polystyrene beads were observed to coalesce
within the midgut of copepods prior to egestion. While gut-
retention times of these microplastics were typically similar to
natural food items (i.e., egestion occurred within hours), a
follow-up experiment found some Calanus helgolandicus
individuals retained microplastics for up to 7 days. Micro-
plastics found in the marine environment include fibers,
granules, and fragments manufactured from a range of
polymers;
30
if such irregularly shaped and fibrous microplastics
were ingested, they may become entangled within the intestinal
tract, potentially resulting in a nonbiodegradable gut-blockage
and greater gut-retention times. Plastic fibers entangle within
the intestinal tracts of Nephrops in this manner,
14
wheras
fish
16,17
and seabird dissections
15
have demonstrated that
marine wildlife can retain a range of plastic detritus within their
stomachs near-indefinitely. Prolonged gut-retention times of
Environmental Science & Technology Article
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plastics and gut-blockages in zooplankton may limit the ability
of these organisms to ingest and digest food, and may pose a
toxic risk. During manufacture, a suite of additives (e.g.,
plasticisers, flame-retardants, antimicrobials) are added to
plastics, and large surface area to volume ratio and hydrophobic
properties of microplastics make them particularly susceptible
to the adherence of waterborne contaminants (e.g., PCBs,
DDT, and PAHs).
19
The leaching of additives and disassoci-
ation of toxic contaminants post-ingestion has been modeled in
polychaete worms
56
and demonstrated in streaked shear-
waters.
57
In zooplankton, as with other marine biota, these
contaminants might be considered endocrine-disruptors,
carcinogenic, or toxic, with repercussions for growth, sexual
development, fecundity, morbidity, and mortality.
58,59
Of
further concern is trophic-transfer: microplastics (and con-
taminants released from microplastics) within lower-trophic,
keystone organisms such as zooplankton may result in the
trophic-transfer of these contaminants up the food-chain, with
the potential for bioaccumulation and therefore adverse health
consequences in higher trophic organisms.
Copepods that died during exposures, and shed molts of
copepodites, were coated in microplasticspresumably
because of hydrophobic- or static-attractions between the
negatively charged polystyrene (average zeta potential: −41.8
mV) and organic materiala process that acts to concentrate
microplastics from the surrounding seawater. Our observations
of microplastic-laden faecal pellets egested by copepods
provided no indication that passage through the alimentary
canal had any discernible impact on the microplastics.
However, plastics may alter the density and structural integrity
of faecal pellets with potential repercussions on vertical carbon
flux.
60
During our studies, we also found microplastics were
becoming trapped between the external appendages and
carapace segments of live copepods. We found that very
small microplastics (0.4−3.8 μm) became lodged between the
filamental hairs and setae of the antennules, furca, and the
swimming legs.
29,61
As these appendages have key roles in
copepod function and behavior, this may have repercussions for
locomotion, ingestion, mating, and mechanoreception, that
may limit their ability to detect prey, feed, reproduce, and evade
predators.
We found that the presence of 7.3 μm beads significantly
reduced the amount of algae eaten by the copepod Centropages
typicus, whereas 20.6 μm beads showed no discernible impact
on algal consumption. This suggests C. typicus can preferentially
feed upon algae over 20.6 μm beads (but could not differentiate
between the algae and 7.3 μm beads), or, that only the smaller
beads impact on copepod feeding (i.e., 7.3 μm beads are small
enough to become entrapped between external appendages or
be recurrently ingested). A similar finding has been observed
with Acartia clausi and Calanus pacificus nauplii, which
selectively fed upon small algae while avoiding larger beads,
but could not discriminate between algae and beads of a similar
size.
24,45,54
We found that a concentration of 4000 beads mL−1
was enough to result in significantly reduced algal ingestion
rates. This relationship reached saturation at concentrations of
>5000 beads mL−1. Two previous studies have found similar
results, where the ingestion rates of the copepod A. clausi
24
and
C. pacificus
45
were significantly reduced by the presence of
beads of a similar size to the algae. A reduction in algal feeding
may have severe consequences for copepods, as limited energy
intake, in particular with species that have minimal lipid
reserves (e.g., Centropages,Acartia), could result in decreased
fecundity and growth, or increased mortality.
24,62
We do not
yet know whether 5000 particles mL−1can be considered an
environmentally relevant concentration for microplastics <10
μm in size. Perpetual fragmentation of plastic litter, coupled
with the increasing popularity of household products
containing microscopic plastic exfoliates,
5
suggests marine
plastic debris is becoming, on average, smaller over time.
63
However, due to the complexities of sampling and extraction,
and in the absence of unified sampling methodologies,
microplastics are still considered to be an under-researched
fraction of marine litter, with no consistent data relating to
plastic detritus <333 μm in diameter.
1,30,64
Further, we must
consider that microplastics made of polymers other than
polystyrene, potentially laden with chemical additives or
adhered contaminants, could result in different interactions
with zooplankton with variable impacts on function.
Our findings confirm that ingestion of marine microplastic
debris by zooplankton in the ocean is feasible. Potential impacts
include reduced function and health of the individual, trophic-
transfer of contaminants to predators, and the egestion of faecal
pellets containing microplastics. Better knowledge of the extent
of microplastic contamination of oceans waters is now a
research imperative.
■AUTHOR INFORMATION
Corresponding Author
*Phone: +44 (0)1752 633165; fax: +44 (0)1752 633101; e-
mail: mcol@pml.ac.uk.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
M.C. is supported by a NERC PhD studentship (Grant
I528034). We thank the crews of the Plymouth Quest and MBA
Sepia for the collection of zooplankton and seawater, Rachel
Harmer and Andrea McEvoy for zooplankton identification and
Glen Tarran for assistance with flow cytometry.
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