Marine Pollution Bulletin 194 (2023) 115403

Contents lists available at ScienceDirect

Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Baseline

Fate of microplastic captured in the marine demosponge

Halichondria panicea
Peter Funch a, *, Rachael A. Kealy b, c, Josephine Goldstein a, b, c, Jonathan R. Brewer c,
Vita Solovyeva c, d, Hans Ulrik Riisgård b
a
Department of Biology, Genetics, Ecology, and Evolution, Aarhus University, Denmark
b
Marine Biological Research Centre, Department of Biology, University of Southern Denmark, Denmark
c
Danish Molecular Biomedical Imaging Center (DaMBIC), University of Southern Denmark, Denmark
d
Department of Physics, Carl von Ossietzky University of Oldenburg, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: Microplastic particles are widespread pollutants in the sea and filter-feeding sponges have recently been sug­
Microplastic particles gested as useful monitoring organisms. However, the fate of microplastic particles in sponges is poorly under­
Live-cell imaging stood, yet crucial for interpreting monitoring data. The present study aims to help develop sponges as more
Sandwich culture
useful monitoring organisms for microplastic in the sea. Here, we describe the fate of inedible (2 and 10 μm)
Sponge explant
SEM
plastic beads compared to that of edible bacteria and algal cells captured in the marine demosponge Halichondria
Monitoring organism panicea. Small Cyanobium bacillare cells entered the choanocyte chambers and were phagocytized by choano­
cytes, while larger Rhodomonas salina cells were captured in incurrent canals and phagocytized in the mesohyl.
Small 2 μm-beads were captured by choanocytes and subsequently expelled into the excurrent canals after 58 ±
34 min. Larger 10 μm-beads were captured in the incurrent canals and transferred to the mesohyl, where
amoeboid cells moved them across the mesohyl before they were expelled into the excurrent canal after 95 ± 36
min. SEM observations further indicated engulfment of plastic beads on the outer sponge surface. This insight
provides useful information on how sponges, in general, treat microplastic particles of various sizes. It helps us
understand actual measured sizes and concentrations of microplastic particles in sponges in relation to those in
the ambient water.

1. Introduction flagellated cells (choanocytes), which in the leuconoid-type sponge are

Microplastic particles are widespread pollutants in the marine
environment and increasing concentrations of these man-made pollut­
ants need to be monitored (Saliu et al., 2022; Fallon and Freeman, 2021;
Girard et al., 2021; Modica et al., 2020). Traditional sieving techniques
have failed to assess the microplastic particulate fraction <200 μm
adequately (Lindeque et al., 2020) and therefore filter-feeding sponges,
which process large quantities of water with high retention efficiency of
very small particles, have been suggested to possess a potential as
monitoring organisms for microplastics (Celis-Hernández et al., 2021;
Girard et al., 2021). However, interpretation of monitoring data on
microplastics in sponges is reliant on knowledge of capture and fate of
these particles in the sponges, as it appears from the present study.
Sponges are simple multicellular filter-feeders that generate water
flow through an aquiferous system by means of water-pumping
arranged in choanocyte chambers (CCs) (Larsen and Riisgård, 1994;
Fig. 2 therein). The CCs pump water from the inhalant to the exhalant
canals, which are lined by a 2 to 3 μm thick single cell layer of endo­
pinacocytes (Bergquist, 1978). Suspended particles, predominantly
phytoplankton (3 to 30 μm) and free-living bacteria (0.5 to 2 μm) along
with microplastic particles can enter the inhalant canals through small 5
to 50 μm porous openings (ostia) in the outer sponge surface (Lüskow
et al., 2019; Riisgård and Larsen, 2010; Reiswig, 1975). Larger particles
(5 to 50 μm) are captured in the inhalant canals and phagocytosed by the
endopinacoderm of inhalant canals, whereas smaller particles (<5 μm)
enter into the CCs through openings (prosopyles) to be subsequently
captured by the collar filter of the choanocytes with a microvilli sieve
size around 0.1 μm and ingested by the choanocytes (Turon et al., 1997;
Larsen and Riisgård, 1994; Imsiecke, 1993; Weissenfels, 1992; Reiswig,
1975, 1971; Schmidt, 1970). The micro-filtered water is pumped out of

* Corresponding author.
E-mail address: funch@bio.au.dk (P. Funch).

https://doi.org/10.1016/j.marpolbul.2023.115403
Received 16 May 2023; Received in revised form 2 August 2023; Accepted 6 August 2023
Available online 14 August 2023
0025-326X/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
P. Funch et al.
the CCs into an exhalant canal through an apopyle opening. The filtered
water, along with expelled waste material, leaves the sponge with the
exhalant jet through an exhalant opening (osculum) (Riisgård and
Larsen, 2022; Goldstein et al., 2019; Kealy et al., 2019; Wolfrath and
Barthel, 1989).
Motile cells (phagocytes) in the mesohyl between inhalant and
exhalant canals are involved in processing and transport of particles
(Leys et al., 2009; Leys and Eerkes-Medrano, 2006; Imsiecke, 1993;
Reiswig, 1971; Schmidt, 1970). The sequence of ingestion, digestion,
and defecation into the excurrent canal has been described in a fresh­
water sponge by Imsiecke (1993, Fig. 12 therein). However, it has been
shown that uptake may also occur via phagocytosis of particles captured
on the outer sponge surface which is lined with exopinacocytes (Gaino
et al., 1994; Willenz and Van de Vyver, 1982). Even though sponges lack
conventional nerves and muscles, overloading with particles may trigger
oscular and body contractions during which the water flow through the
aquiferous system is reduced and eventually shut down (Goldstein et al.,
2020, 2019; Kealy et al., 2019).
Sponges can filter large volumes of water, up to six times their body
volume per minute, while efficiently retaining particles as small as 0.1
μm (Riisgård and Larsen, 2022; Ludeman et al., 2017; Riisgård et al.,
2016). Therefore, sponges have been considered to be suitable organ­
isms for monitoring microplastic pollution in aquatic environments
(Giametti and Finelli, 2022; Girard et al., 2021; Modica et al., 2020).
However, the fate of microplastic particles taken up by sponges is not
well understood. It is important to determine whether these particles
accumulate in the sponge over time or are efficiently expelled. The
present study aims to describe the fate of inedible microplastic particles
(2 and 10 μm-beads) in comparison to the fate of edible bacteria and
algal cells of similar sizes in the marine demosponge Halichondria pan­
icea. The sponge captures edible and inedible particles in the same way.
However, they are suggested to be treated differently, resulting in the
digestion of the organic particles and the expulsion of plastic particles
after a certain time. Using both small and larger ‘standard’ microplastic
particles may provide useful information about size-dependent expul­
sion times. Such insight is suggested to help understand how sponges, in
general, treat microplastic particles. In this way, the present study may
help to understand the actual sizes and concentrations of microplastic
particles measured in sponges in relation to those found in the ambient
water. Thus, the overall objective of the study is to contribute to the
development of using sponges as more useful monitoring organisms for
microplastics in the sea.
2. Materials and methods
Capture and transport of small and large organic particles (bacteria
and algal cells) and inorganic particles (2 and 10 μm plastic beads) were
studied in sponge sandwich cultures by means of microscope time-lapse
observations. In this way, it was possible to observe the fate of the
various captured particles, providing information on the digestion time
of edible organic particles versus the expulsion time of inedible plastic
particles. Further, scanning electron microscopy (SEM) was used to
visualize the capture sites and uptake of the various types of particles
within single-osculum sponge explants. Epifluorescence microscopy
with UV-extinction was used to observe edible (untreated) and inedible
fluorescent melamine resin microbeads.
2.1. Sponge cultures and particle types
Specimens of the demosponge Halichondria panicea were collected at
different sampling sites in the inlet of Kerteminde Fjord, Denmark
(55◦ 26′59″N, 10◦ 39′40″E) between June and December 2019. To prepare
explants, sponge cuttings were placed on glass slides and transferred to
flow-through aquaria with aerated bio-filtered seawater (15 to 25 psu,
10 to 15 ◦ C). After attaching to the glass slides, the sponges regenerated
into actively pumping specimens with a single osculum within one to
2
Marine Pollution Bulletin 194 (2023) 115403
two weeks (Kealy et al., 2019; Kumala et al., 2017). Following the same
explant technique as described above, we prepared sponge sandwich
cultures to facilitate regeneration of thin sponge explants in the narrow
space between two glass slides (Wyeth et al., 1996). For this purpose,
sponge cuttings were mounted on ibidi μ-dishes (Ø 35 μm, ibiTreat
polymer coverslip bottom) with attached coverslips. The space between
the two coverslips was around 100 to 200 μm. Explants and sandwich
cultures were continuously fed with Rhodomonas salina cells by means of
a dosing pump (~5000 cells mL− 1; corresponding to ~6 μg Chl a L− 1, cf.
Riisgård et al., 2013). Specimens of cultured sponges were randomly
picked from the cultivation tanks and maintained in filtered (38 μm)
seawater under constant mixing for two to three days prior to the
experiments.
Four different particle-seawater (20 psu) stock solutions were pre­
pared to explore particle transport in Halichondria panicea. Two stock
solutions of edible particles of two sizes were prepared from cell cultures
of Cyanobium bacillare and Rhodomonas salina (cell dimensions: 2.6 ±
0.2 μm × 2.1 ± 0.2 μm and 10.1 ± 0.7 μm × 6.2 ± 0.4 μm, respectively).
Two other stock solutions of inedible particles were prepared using
melamine resin based microparticles (microbeads) of corresponding
diameters (2.0 ± 0.2 μm, FITC-, i.e., fluorescein-5-isothiocyanate-
marked, and 10.3 ± 0.8 μm, rhodamine B-marked, respectively;
Fig. 1). The concentration of particles in each seawater stock solution
(R. salina: 1.0 ± 0.3 × 106 cells mL− 1, C. bacillare: 3.5 ± 1.5 × 107 cells
mL− 1, 10 μm-beads: 4.3 ± 0.3 × 105 particles mL− 1, 2 μm-beads: 5.4 ±
3.0 × 107 particles mL− 1) was determined by manual counts (100-fold
dilution; n = 10 replicates on 0.2 μm polycarbonate filters) using epi­
fluorescence microscopy with UV-excitation (Leica Leitz Laborlux S).
2.2. Live-cell imaging of sponge sandwich cultures
Sponge sandwich preparations in ibidi μ-dishes (3 mL) with filtered
seawater (15 ◦ C, 20 psu) were exposed to edible cells (Cyanobium
bacillare and Rhodomonas salina) or inedible particles (2 and 10 μm
beads) at final concentrations resembling trigger levels for contractile
responses and studied using combined transmitted brightfield and epi­
fluorescence microscopy (Nikon Eclipse Ti-E with integrated Andor Zyla
sCMOS camera, LED-illumination; Nikon FITC filter cube with λem =
465–495 nm and λem = 515–555 nm for Cyanobium bacillare, Cy5-
(cyanine-5-) 4040C Semrock Brightline filter cube for 2 μm-beads with
λex = 628/40 nm and λem = 692/40 nm and Nikon TRITC (tetrame­
thylrhodamine-isothiocyanate) filter cube for R. salina and 10 μm-beads
with λex = 540/25 nm and λem = 605/55 nm). Image stacks at a step size
of 1–2 μm (to achieve a total optical slice thickness of 20 μm) were ac­
quired using time-lapse microscopy (interval: 1 min, duration: 1–2 h)
with a Nikon Plan Apo 20× NA 0.75 air or a 60× NA 1.4 oil objective,
respectively, for live-cell imaging of sandwich cultures. Using imaging
software ImageJ (version 1.53c), we tracked the transport of particles
through the sponge to measure the time of edible particles to be
phagocytized versus time of plastic beads to be expelled into the
excurrent canal and out of the sponge with the exhalant jet through the
osculum.
2.3. Scanning electron microscopy (SEM)
After acclimatization in (15 mL) sterile filtered (0.2 μm) seawater
(20 psu, 15 ◦ C) for 3 h, single-osculum explants were exposed to edible
cells (Cyanobium bacillare and Rhodomonas salina) or inedible particles
(2 μm- and 10 μm-beads; Fig. 1) at final concentrations resembling
trigger levels for contractile responses for 1 h (n = 3 per treatment).
Sponges were fixed with 2 % OsO4 in 0.1 M cacodylate buffer overnight
(~18 h), rinsed (3×) with sterile filtered seawater and transferred to a
seawater solution with 1 % glutaraldehyde in 4 % cacodylate buffer (pH
= 7.4) for 1 h on ice. After another rinsing step with sterile filtered
seawater and storage with a remnant of secondary fixative at 4 ◦ C
overnight, sponge explants were dehydrated in a graded ethanol series
P. Funch et al. Marine Pollution Bulletin 194 (2023) 115403

Fig. 1. Examined particle types. Scanning electron micrographs (left panel) and epifluorescence signals (right panel) of edible and inedible particles of two size
groups: (A, B) Cyanobium bacillare (cell dimensions: 2.6 ± 0.2 μm × 2.1 ± 0.2 μm), (C, D) 2 μm-melamine resin based microbeads (FITC-marked), (E, F) Rhodomonas
salina (cell dimensions: 10.1 ± 0.7 μm × 6.2 ± 0.4 μm) and (G, H) 10 μm-melamine resin based microbeads (rhodamine B-marked).

up to 70 % (30 min per 10 % step) and divided in two parts with a razor
blade before further stepwise transfer (75, 80, 96 %) to 100 % ethanol.
For cryofracture, each fixed specimen was placed in a sealed Parafilm
sleeve filled with 100 % ethanol and placed in liquid nitrogen until the
ethanol had solidified. The sleeve with the specimen was then placed on
a precooled brass piece, held in place with a precooled haemostat and
freeze fractured with a precooled razor blade hit by a rubber mallet. The
fractured specimen was transferred to 100 % ethanol at room temper­
ature, and the Parafilm was removed. Each fractured specimen was
critical point dried with CO2 (EMS850), mounted on aluminium stubs
with double adhesive carbon tape, sputter coated with a 6 nm thick layer
of platinum (LEICA EM SCD 500; 5 min) or a 45 nm thick layer of gold
(Edwards S150B; 3 min), respectively. The fractured specimens were
examined using a FEI NOVA NanoSEM 600 scanning electron micro­
scope equipped with an ETD detector at 2–3 or 5 kV and the position of
the four different types of particles in different components of the
aquiferous system was documented.

3
P. Funch et al. Marine Pollution Bulletin 194 (2023) 115403

Fig. 2. Halichondria panicea. Schematic illustrations of different pathways for particle (p) capture and transport in the aquiferous system of sponge sandwich cultures,
based on time-lapse observations. A) Cyanobium bacillare, B) 2 μm-bead, C) Rhodomonas salina and D) 10 μm-bead. Arrows indicate the direction of water flow,
phagocytosis of edible cells (A, C) is marked by x. in-c: incurrent canal, pro: prosopyle, cc: choanocyte chamber, c: choanocyte, ap: apopyle, enp: endopinacoderm, m:
mesohyl, ex-c: excurrent canal, sp.: spicule. Scale bars: 20 μm.

4
P. Funch et al.
3. Results
It was observed that Cyanobium bacillare cells entered the choanocyte
chambers (CCs) of Halichondria panicea sandwich cultures through one
of the four prosopyles and were phagocytized by choanocytes within 41
± 21 min (n = 6) (Fig. 2). Small 2 μm-beads were also captured by
choanocytes in the CCs, but were subsequently (via the mesohyl, see
Discussion) expelled into the excurrent canals, typically after 58 ± 34
min (n = 6) (Fig. 2). Rhodomonas salina cells were captured in the
incurrent canals, transferred to the mesohyl, where amoeboid cells
moved them to a position near to the CCs to be disintegrated and
phagocytized within 58 ± 25 min (n = 6) (Fig. 2). Larger 10 μm-beads
were also captured in the incurrent canals and transferred to the mes­
ohyl where amoeboid cells moved them across the mesohyl before they
were expelled into the excurrent canal after 95 ± 36 min (n = 6) (Fig. 2).
The amoeboid cells involved in particle transport in the mesohyl were
spindle-shaped and had a prominent nucleus, long cytoplasmic pro­
jections, an average cell length of 17.1 ± 6.8 μm, a cell diameter of 5.6
± 1.5 μm and a nucleus diameter of 2.6 ± 0.6 μm (n = 6). Motility of
amoeboid cells in the mesohyl was observed in all sandwich cultures and
the speed of the cells (about 1 μm min− 1) was unaffected by sponge body
5
Marine Pollution Bulletin 194 (2023) 115403
contractions observed during the study. Endopinacocytes lining the in-
and excurrent canals of sandwich cultures were slightly longer (23.6 ±
6.1 μm) than mesohyl cells and distinctively flattened (3.1 ± 0.6 μm).
These and the above observations were supported by SEM as it appears
from the following section.
The position of different particle types in cryofractured Halichondria
panicea explants was determined using scanning electron microscopy
(SEM). Here, Cyanobium bacillare cells were observed near ostia and
inside CCs (Fig. 3A, C, E). In the latter case, some cells were fixed in the
process of being phagocytized by choanocytes. Small 2 μm-beads had
been phagocytized by exopinacocytes on the outer sponge surface
(Fig. 3B), in contact with amoeboid cells of the mesohyl (Fig. 3D), and in
endopinacocytes lining the excurrent canals (Fig. 3F). Larger Rhodo­
monas salina cells were seen on the outer sponge surface on exopina­
cocytes (Fig. 4A, C), and in the incurrent canals where they were often
entangled in mucus-like strands (Fig. 4E). Large 10 μm-beads were
observed engulfed by exopinacocytes on the outer sponge surface
(Fig. 4B), in endopinacocytes in the mesohyl (Fig. 4D), and engulfed in
endopinacocytes lining the excurrent canals (Fig. 4F).
Fig. 3. Halichondria panicea. Scanning electron mi­
croscopy of cryofractured sponge explants after
exposure to small edible cells (Cyanobium bacillare;
left panel) and inedible particles (2 μm-beads; right
panel). Small edible particles (A, C, E; arrows) in an
ostium (A), inside a choanocyte chamber (C) and
engulfed by a choanocyte (E). Small inedible particles
(B, D, F) which have been phagocytized by exo- (B)
and endopinacocytes (F), in contact with a mesohyl
cell (D) and on the cell surface of an endopinacocyte
lining an excurrent canal (F). p: particle, exp: exopi­
nacocyte, os: ostium, enp: endopinacocyte, c:
choanocyte, ap: apopyle, m: mesohyl cell, fl: flagel­
lum, mv: microvilli, sp.: spicule.
P. Funch et al.
4. Discussion
The present study supports previous observations of selective feeding
on particles of variable size and nutritional quality in sponges (Turon
et al., 1997; Imsiecke, 1993; Reiswig, 1971). Thus, bacteria and other
small particles (<5 μm) are captured by the microvilli collar sieves of the
flagellar water-pumping choanocytes in the choanocyte chambers
(Weissenfels, 1992). Here, the choanocytes phagocytose the captured
particles, which are transferred to the mesohyl where they are digested
and the remnants finally expelled into the exhalant canals (Imsiecke,
1993), typically after <1 h (n = 6) (Fig. 2). Larger (>5 μm) particles are
captured in the incurrent canals and phagocytosed by endopinacocytes
to be subsequently transferred to a phagocyte in the mesohyl and
digested (Imsiecke, 1993, Fig. 12 therein). The observed translocation of
inedible 2 μm- and 10 μm-beads across the mesohyl of Halichondria
panicea mediated by amoeboid cells that apparently “surround and
push” the particles towards the exhalant canal to be expelled (Fig. 2)
agrees with previous studies on removal of incompletely digested
organic material (Imsiecke, 1993; Wolfrath and Barthel, 1989; Reiswig,
1971). Thus, Wolfrath and Barthel (1989) found that H. panicea ingested
algal cells and plastic microspheres in the same manner, but when the
6
Marine Pollution Bulletin 194 (2023) 115403
Fig. 4. Halichondria panicea. Scanning electron mi­
croscopy of cryofractured sponge explants after
exposure to large edible cells (Rhodomonas salina; left
panel) and inedible particles (10 μm-beads; right
panel). Large edible particles (A, C, E) on the cell
surface of exopinacocytes (A, C) and near the endo­
pinacocytes lining an incurrent canal (E); the particles
are trapped in mucus-like strands. Large inedible
particles (B, D, F) engulfed by an exopinacocyte (B),
in the mesohyl (D), engulfed by an endopinacocyte (F;
arrow) and in excurrent canals (D, F). p: particle, exp:
exopinacocyte, os: ostium, sp.: spicule, enp: endopi­
nacocyte, pro: prosopyle, m: mesohyl cell, ap:
apopyle.
particles had been phagocytized their transport was different: digestible
algae were packed in fecal pellets while plastic beads were “more
rapidly” passed through the sponge and egested as single particles. In a
study by Leys and Eerkes-Medrano (2006) on feeding in the calcareous
sponge Sycon coactum, individuals were offered suspensions of 0.1, 0.5
and 1.0 μm latex beads, and already 5 to 10 min after being fed it was
observed that the choanocytes were filled with phagosomes containing
beads, which indicated that latex-bead uptake occurred within a few
minutes after particles entering the sponge, and sponges fixed 1 h after
feeding still contained beads, but not after 6 h.
The observation of algal cells trapped in mucus-like strands in the
incurrent canals (Fig. 4E) is not immediately understandable to us.
However, Langenbruch and Weissenfels (1987) observed “web-like
structures” in the incurrent canals, and Imsiecke (1993) observed “a
delicate cytoplasmic bridge” connecting two choanocyte chambers
forming a “filtration element” on which algal cells were caught in the
incurrent canal. Further, Langenbruch and Weissenfels (1987) suggested
that the net-like structures may “retain large particles and prevent the
prosopyles of the choanocyte chambers from becoming clogged.”
Engulfment of particles captured on the outer sponge surface (Fig. 4)
shows that particles can also be taken up by exopinacocytes as described
P. Funch et al.
by Willenz and Van de Vyver (1982) in a freshwater sponge and by
Gaino et al. (1994) in two Antarctic sponges. Thus, microplastic particles
may be associated with the outer epithelia (ectosome) of sponges as
suggested by Girard et al. (2021) who found that “microparticulate
pollutants” are incorporated “either in skeletal fibers or the ectosome”.
The present study shows that sponges capture both edible and inedible
particles in the same way but subsequently treat them differently,
resulting in digestion of the organic particles (bacteria and algal cells)
and efficient expulsion of microplastic beads. The use of both small and
larger ‘standard’ microplastic particles has provided useful information
about size-dependent expulsion times, which is important for under­
standing how sponges, in general, treat ingested microplastic particles
(MP). However, the fate of more ‘natural’ MP, such as fibers and frag­
ments of various sizes and chemical compositions (cf. Hidalgo-Ruz et al.,
2012), was not studied. Nonetheless, we are aware that the coated
pristine microbeads used in the present study may not entirely represent
aged MP in nature. Nevertheless, we have determined that the expulsion
time of larger MP is likely to be longer than for smaller MP. Furthermore,
the concentration of MP can be expected to be higher in freshly collected
sponges than in the surrounding water because it reflects a steady-state
concentration resulting from the simultaneous intake and expulsion of
MP. This insight may render sponges more usable as monitoring or­
ganisms. Here, it should be remembered that the steady-state concen­
trations of various sizes of MP are different. Thus, smaller MP can be
expected to be present in sponges in lower concentrations that larger
MP, even though they may be present at similar concentrations in the
ambient water. Such insights, to be examined in future studies, may help
to further develop sponges as more useful monitoring organisms for MP
in the sea. With a volume-specific filtration rate of 6 mL water filtered
per minute per mL sponge volume (mL min− 1 mL− 1) = 6 min− 1 (Riisgård
et al., 2016) and an ambient microplastic concentration of 1 particle
mL− 1, a sponge may ingest (I) (6 × 60=) 360 particles (mL sponge)− 1
h− 1 and if the steady-state particle concentration in the sponge is
measured to be for example G = 200 particles (mL sponge)− 1, the
elimination time of the plastic particles is E = G/I = 0.56 h, implying a
200 times higher concentration in the sponge compared to the ambient
water. However, it has been measured that the concentration of
microplastics in sponges was only about 8 times higher than in the
surrounding seawater (Celis-Hernández et al., 2021) indicating a very
short elimination time. Further, Fallon and Freeman (2021) found that
the occurrence of microplastic particles in sponge samples was relatively
low considering the high level of these particles in the ambient seawater,
and therefore they suggested that sponges may have “some capacity to
egest the particles”, as confirmed by the present study. The fate of
microplastics engulfed by exopinacocytes on the outer sponge surface
has so far not been documented, but it may be put forward as a hy­
pothesis that the mesohyl may also in this case serve as a transit zone for
expelling inedible particles with the exhalant jet. Finally, it should be
emphasized that the steady-state concentration of microplastic particles
measured in a sponge depends on the size distribution of microplastic
particles in the ambient water. In the present study 2 μm-beads were
expelled after <1 h, while 10 μm-beads were typically expelled after <2
h, which indicates that small microplastic particles may generally be
expelled faster than larger particles.
5. Conclusions
Based on epifluorescence microscopy and live-cell imaging of sponge
sandwich cultures exposed to edible cells and inedible particles, com­
bined with SEM of sponge explants, it has been observed that uptake of
inedible plastic beads within a sponge is like that of edible cells. How­
ever, the living cells are digested whereas the plastic particles are effi­
ciently expelled into the exhalant canals and carried out of the sponge
with the exhalant jet. The present information about size-dependent
expulsion times is important for understanding how sponges, in gen­
eral, treat microplastic particles (MP). The expulsion time of larger MP is
7
Marine Pollution Bulletin 194 (2023) 115403
longer than of smaller MP, and the concentration of MP is likely to be
higher in freshly collected sponges than in the surrounding water. Such
insight makes sponges more useful as monitoring organisms.
CRediT authorship contribution statement
Peter Funch: Conceptualization, Investigation, Data curation,
Writing – review & editing, Funding acquisition. Rachael A. Kealy:
Conceptualization, Investigation, Data curation. Josephine Goldstein:
Conceptualization, Visualization, Investigation, Data curation, Writing –
review & editing. Jonathan R. Brewer: Investigation, Data curation,
Funding acquisition. Vita Solovyeva: Investigation, Data curation.
Hans Ulrik Riisgård: Conceptualization, Data curation, Writing –
original draft, Writing – review & editing, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This work was supported by the Independent Research Fund
Denmark [grant number 8021-00392B] and VILLUM FONDEN [grant
number 40834]. The authors acknowledge the support for image
acquisition at the Danish Molecular Biomedical Imaging Center (DaM­
BIC, University of Southern Denmark), supported by the Novo Nordisk
Foundation (NNF) [grant agreement number NNF18SA0032928]. We
thank Ramon Liebrechts at the Interdisciplinary Nanoscience Center
(iNANO), Aarhus University, Denmark, for technical support with the
scanning electron microscope and Lars Kumala for advice on the prep­
aration of sandwich cultures.
References
Bergquist, P.R., 1978. Sponges. University of California Press, Berkeley and Los Angeles
(268 pp.).
Celis-Hernández, O., Ávila, E., Ward, R.D., Rodríguez-Santiago, M.A., Aguirre-T’ellez, J.
A., 2021. Microplastic distribution in urban vs pristine mangroves: using marine
sponges as bioindicators of environmental pollution. Environ. Pollut. 284, 117391
https://doi.org/10.1016/j.envpol.2021.117391.
Fallon, B.R., Freeman, C.J., 2021. Plastics in Porifera: the occurrence of potential
microplastics in marine sponges and seawater from Bocas del Toro, Panamá. PeerJ 9,
e11638. https://doi.org/10.7717/peerj.11638.
Gaino, E., Bavestrello, G., Cattaneo-Vietti, R., Sarà, M., 1994. Scanning electron
microscope evidence for diatom uptake by two Antarctic sponges. Polar Biol. 14,
55–58. https://doi.org/10.1007/BF00240273.
Giametti, S.D., Finelli, C.M., 2022. Detection of plastic-associated compounds in marine
sponges. Mar. Pollut. Bull. 175, 113141 https://doi.org/10.1016/j.
marpolbul.2021.113141.
Girard, E.B., Fuchs, A., Kaliwoda, M., Lasut, M., Ploetz, E., Schmahl, W.W., Wörheide, G.,
2021. Sponges as bioindicators for microparticle pollutants? Environ. Pollut. 268,
115851 https://doi.org/10.1016/j.envpol.2020.115851.
Goldstein, J., Riisgård, H.U., Larsen, P.S., 2019. Exhalant jet speed of single-osculum
explants of the demosponge Halichondria panicea and basic properties of the sponge-
pump. J. Exp. Mar. Biol. Ecol. 511, 82–90. https://doi.org/10.1016/j.
jembe.2018.11.009.
Goldstein, J., Bisbo, N., Funch, P., Riisgård, H.U., 2020. Contraction-expansion and the
effects on the aquiferous system in the demosponge Halichondria panicea. Front. Mar.
Sci. 7, 113. https://doi.org/10.3389/fmars.2020.00113.
Hidalgo-Ruz, V., Gutow, L., Thompson, R.C., Thiel, M., 2012. Microplastics in the marine
environment: a review of the methods used for identification and quantification.
Environ. Sci. Technol. 46, 3060–3075. https://doi.org/10.1021/es2031505.
Imsiecke, G., 1993. Ingestion, digestion, and egestion in Spongilla lacustris (Porifera,
Spongillidae) after pulse feeding with Chlamydomonas reinhardtii (Volvocales).
Zoomorphology 113, 233–244. https://doi.org/10.1007/BF00403314.
Kealy, R.A., Busk, T., Goldstein, J., Larsen, P.S., Riisgård, H.U., 2019. Hydrodynamic
characteristics of aquiferous modules in the demosponge Halichondria panicea. Mar.
Biol. Res. 15, 531–540. https://doi.org/10.1080/17451000.2019.1694691.
P. Funch et al.
Kumala, L., Riisgård, H.U., Canfield, D.E., 2017. Osculum dynamics and filtration
activity studied in small single-osculum explants of the demosponge Halichondria
panicea. Mar. Ecol. Prog. Ser. 572, 117–128. https://doi.org/10.3354/meps12155.
Langenbruch, P.-F., Weissenfels, N., 1987. Canal systems and choanocyte chambers in
freshwater sponges (Porifera, Spongillidae). Zoomorphology 107, 11–16. https://
doi.org/10.1007/BF00312124.
Larsen, P.S., Riisgård, H.U., 1994. The sponge pump. J. Theor. Biol. 168, 53–63. https://
doi.org/10.1006/jtbi.1994.1087.
Leys, S.P., Eerkes-Medrano, D.I., 2006. Feeding in a calcareous sponge: particle uptake
by pseudopodia. Biol. Bull. 211, 157–171. https://doi.org/10.2307/4134590.
Leys, S.P., Nichols, S.A., Adams, E.D., 2009. Epithelia and integration in sponges. Integr.
Comp. Biol. 49, 167–177. https://doi.org/10.2307/4134590.
Lindeque, P.K., Cole, M., Coppock, R.L., Lewis, C.N., Miller, R.Z., Watts, A.J.R., Wilson
Mc-Neal, A., Wright, S.L., Galloway, T.S., 2020. Are we underestimating microplastic
abundance in the marine environment? A comparison of microplastic capture with
nets of different mesh-size. Environ. Pollut., 114721 https://doi.org/10.1016/j.
envpol.2020.114721.
Ludeman, D.A., Reidenbach, M.A., Leys, S.P., 2017. The energetic cost of filtration by
demosponges and their behavioural response to ambient currents. J. Exp. Biol. 220,
995–1007. https://doi.org/10.1242/jeb.146076.
Lüskow, F., Riisgård, H.U., Solovyeva, V., Brewer, J.R., 2019. Seasonal changes in
bacteria and phytoplankton biomass control the condition index of the demosponge
Halichondria panicea in temperate Danish waters. Mar. Ecol. Prog. Ser. 608, 119–132.
https://doi.org/10.3354/meps12785.
Modica, L., Lanuza, P., Garcia-Castrillo, G., 2020. Surrounded by microplastic, since
when? Testing the feasibility of exploring past levels of plastic microfibre pollution
using natural history museum collections. Mar. Pollut. Bull. 151, 110846 https://
doi.org/10.1016/j.marpolbul.2019.110846.
Reiswig, H.M., 1971. Particle feeding in natural populations of three marine
demosponges. Biol. Bull. 141, 568–591. https://doi.org/10.2307/1540270.
Reiswig, H.M., 1975. The aquiferous systems of three marine Demospongiae. J. Morphol.
145, 493–502. https://doi.org/10.1002/jmor.1051450407.
Riisgård, H.U., Larsen, P.S., 2010. Particle capture mechanisms in suspension-feeding
invertebrates. Mar. Ecol. Prog. Ser. 418, 255–293. https://doi.org/10.3354/
meps08755.
Marine Pollution Bulletin 194 (2023) 115403
Riisgård, H.U., Larsen, P.S., 2022. Filtration rates and scaling in demosponges. J. Mar.
Sci. Eng. 10, 643. https://doi.org/10.3390/jmse10050643.
Riisgård, H.U., Pleissner, D., Lundgreen, K., Larsen, P.S., 2013. Growth of mussels Mytilus
edulis at algal (Rhodomonas salina) concentrations below and above saturation levels
for reduced filtration rate. Mar. Biol. Res. 9, 1005–1017. https://doi.org/10.1080/
17451000.2012.742549.
Riisgård, H.U., Kumala, L., Charitonidou, K., 2016. Using the F/R-ratio for an evaluation
of the ability of the demosponge Halichondria panicea to nourish solely on
phytoplankton versus free-living bacteria in the sea. Mar. Biol. Res. 12, 907–916.
https://doi.org/10.1080/17451000.2016.1206941.
Saliu, F., Biale, G., Raguso, C., La Nasa, J., Degano, I., Seveso, D., Galli, P., Lasagni, M.,
Modugno, F., 2022. Detection of plastic particles in marine sponges by a combined
infrared micro-spectroscopy and pyrolysis-gas chromatography-mass spectrometry
approach. Sci. Total Environ. 819, 152965 https://doi.org/10.1016/j.
scitotenv.2022.152965.
Schmidt, I., 1970. Phagocytose et pinocytose chez les Spongillidae. Z. vgl. Physiol. 66,
398–420. https://doi.org/10.1007/BF00299939.
Turon, X., Galera, J., Uriz, M.J., 1997. Clearance rates and aquiferous systems in two
sponges with contrasting life-history strategies. J. Exp. Zool. 278, 22–36. https://doi.
org/10.1002/(SICI)1097-010X(19970501)278:1<22::AID-JEZ3>3.0.CO;2-8.
Weissenfels, N., 1992. The filtration apparatus for food collection in freshwater sponges
(Porifera, Spongillidea). Zoomorphology 112, 51–55. https://doi.org/10.1007/
BF01632994.
Willenz, P., Van de Vyver, G., 1982. Endocytosis of latex beads by the exopinacoderm in
the freshwater sponge Ephydatia fluviatilis: an in vitro and in situ study in SEM and
TEM. J. Ultrastruct. Res. 79, 294–306. https://doi.org/10.1016/S0022-5320(82)
90005-3.
Wolfrath, B., Barthel, D., 1989. Production of faecal pellets by the marine sponge
Halichondria panicea Pallas (1799). J. Exp. Mar. Biol. Ecol. 129, 81–94. https://doi.
org/10.1016/0022-0981(89)90064-6.
Wyeth, R.C., Leys, S.P., Mackie, G.O., 1996. Use of sandwich cultures for the study of
feeding in the hexactinellid sponge Rhabdocalyptus dawsoni (Lambe, 1892). Acta
Zool. 77, 227–232. https://doi.org/10.1111/j.1463-6395.1996.tb01266.x.