Ecotoxicology and Environmental Safety 234 (2022) 113394

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Ecotoxicology and Environmental Safety

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

Shrimp and microplastics: A case study with the Atlantic ditch shrimp
Palaemon varians
Reinhard Saborowski *, Špela Korez , Sarah Riesbeck 1, Mara Weidung 2, Ulf Bickmeyer ,
Lars Gutow
Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany

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

Edited by Professor Bing Yan Many invertebrate species inhabit coastal areas where loads of plastic debris and microplastics are high. In the
current case study, we exemplarily illustrate the principal processes taking place in the Atlantic ditch shrimp,
Keywords: Palaemon varians, upon ingestion of microplastics. In the laboratory, shrimp readily ingested fluorescent poly­
Crustacea styrene microbeads of 0.1–9.9 µm, which could be tracked within the widely translucent body. Ingested food
Ingestion
items as well as micro-particles cumulate in the stomach where they are macerated and mixed with digestive
Egestion
enzymes. Inside the stomach, ingested particles are segregated by size by a complex fine-meshed filter system.
Pyloric filter
Oxidative stress Liquids and some of the smallest particles (0.1 µm) pass the filter and enter the midgut gland where resorption of
Superoxide dismutase nutrients as well as synthesis and release of digestive enzymes take place. Large particles and most of the small
particles are egested with the feces through the hindgut. Small particles, which enter the midgut gland, may
interact with the epithelial cells and induce oxidative stress, as indicated by elevated activities of superoxide
dismutase and cellular markers of reactive oxygen species. The shrimp indiscriminately ingest microparticles but
possess efficient mechanisms to protect their organs from overloading with microplastics and other indigestible
particles. These include an efficient sorting mechanism within the stomach and the protection of the midgut
gland by the pyloric filter. Formation of detrimental radical oxygen species is counteracted by the induction of
enzymatic antioxidants.

1. Introduction organisms, and taken up intentionally or by accident (Bern, 1990).

Additionally, microplastics can be transferred across trophic levels via
The number of studies demonstrating the uptake of microplastic consumption of contaminated prey (Farrell and Nelson, 2013).
particles by various marine organisms increased continuously during Crustaceans inhabiting coastal areas or estuaries are particularly
recent years (e.g. Auta et al., 2017; Bour et al., 2018; de Sá et al., 2018; subjected to anthropogenic pollution, including microplastics (Setälä
López-Martínez et al., 2020). Some of them report accumulation of et al., 2016; Sindermann, 2005; Vikas and Dwarakish, 2015). Their
microplastics on the gills of fishes, mollusks, and crustaceans. However, feeding mode as scavengers, deposit feeders, suspension feeders, or
no clear transfer of particles through gill epithelia has been reported yet predators make them vulnerable to microplastic ingestion. Whether
(Watts et al., 2014, 2016). The principal way of microplastic uptake by ingested microplastics have adverse effects on the health of consumers
marine organisms remains via ingestion. Microplastics have been found with different feeding modes depends on the fate of the particles within
in the gastrointestinal tract of numerous aquatic species including fishes the organism. Microplastics may simply pass the digestive tract and
(Lusher et al., 2013; Nadal et al., 2016), fish larvae (Steer et al., 2017), leave the gut along with the fecal material (Ha¨mer et al., 2014; Cole
zooplankton (Desforges et al., 2015), tropical corals (Hall et al., 2015), et al., 2016; Rodríguez-Torres et al., 2020) whereas larger particles (>
and even deep-sea organisms (Taylor et al., 2016). The tiny synthetic 100 µm) may be regurgitated through the esophagus (Saborowski et al.,
particles may easily be mistaken as natural food, such as small plankton 2019). However, very small microplastics in the nanometer size range

* Corresponding author.
E-mail addresses: Reinhard.Saborowski@awi.de (R. Saborowski), Spela.Korez@awi.de (Š. Korez), sarah.riesbeck@ufz.de (S. Riesbeck), mara.weidung@hotmail.
com (M. Weidung), Ulf.Bickmeyer@awi.de (U. Bickmeyer), Lars.Gutow@awi.de (L. Gutow).
1
Present address: Helmholtz Centre for Environmental Research, Permoserstraße 15, 04318 Leipzig, Germany.
2
Present address: Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Wuhanstraße 6, 47051 Duisburg, Germany.

https://doi.org/10.1016/j.ecoenv.2022.113394
Received 13 October 2021; Received in revised form 2 March 2022; Accepted 5 March 2022
Available online 11 March 2022
0147-6513/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
R. Saborowski et al. Ecotoxicology and Environmental Safety 234 (2022) 113394

may be resorbed by the epithelia of digestive organs and translocated to in ultrapure water to eliminate potentially adverse additives and pre­
adjacent tissues (Browne et al., 2008). servatives from the commercial microbead suspension (see supporting
The cellular effects of microplastics are diverse (Sun et al., 2021 and information).
literature cited therein). Ingestion of high-density polyethylene (HDPE)
particles by mussels (Mytilus edulis) induced inflammatory responses and 2.3. Microscopic investigations
lysosomal membrane destabilization in the cells of the intestine (von
Moos et al., 2012). Similarly, polystyrene microparticles of 5 µm accu­ To illustrate the uptake of microbeads and their distribution in the
mulated in the liver of zebrafish (Danio rerio) and caused inflammation digestive tract of P. varians, the shrimp were exposed to high concen­
and cellular oxidative stress through the formation of reactive oxygen trations of fluorescent particles of different sizes (0.1, 2.1, or 9.9 µm,
species (ROS) (Lu et al., 2016). Oxidative stress, in turn, can have Exp. 1 in Table S1). For each particle type, a 50-mL reaction tube was
adverse effects on essential cellular compounds such as proteins, lipids, filled with 35 mL of particle suspension in brackish water (15 PSU). Two
and DNA (Turrens, 2003). In vital cells with functional cellular redox shrimp (30–35 mm length) were placed in the tube for 24 h on a rotator
systems, balanced equilibria are established between ROS and mixer at low rotation speed (10 rpm) to keep the particles in suspension.
ROS-scavenging molecules to protect the cells from oxidative damage After the incubation, the shrimp were photographed under a Nikon SMZ
(Ray et al., 2012). 25 epifluorescence stereo microscope at the respective filter setting to
The first line of defense against ROS in e.g. epithelial cells is the visualize the distribution of the particles within the digestive tract of the
superoxide scavenging enzyme superoxide dismutase (SOD), which widely translucent animal (Saborowski et al., 2019).
converts the superoxide ion into hydrogen peroxide. In response to The distribution of the smallest particles (0.1 µm) within the diges­
increasing superoxide levels, SOD activities are upregulated (Landis and tive organs, the cellular structure of the midgut gland, and the presence
Tower, 2005). Accordingly, elevated enzyme activities can serve as an of reactive oxygen species within the cells was visualized by confocal
indicator for oxidative stress. The increase of ROS and antioxidant laser scanning microscopy (Leica TCS SP5II, Leica Microsystems CMS
enzyme activity in response to micro- and nanoplastic uptake has been GmbH, Wetzlar, Germany). The microscope was equipped with an argon
demonstrated in copepods, rotifers, and bivalves (Jeong et al., 2016, laser (excitation: 488 nm).
2017; Paul-Pont et al., 2016; Ribeiro et al., 2017). Particles are taken up Shrimp were incubated in a suspension of 0.1-µm particles as
into the cells presumably via endocytosis (von Moos et al., 2012; Vogt, described above. After 24 h, the shrimp were sedated on ice, the midgut
2019). However, the cellular mechanisms causing oxidative stress by gland was dissected and stained with Syto-13 (Molecular Probes, D-
microplastics need deeper investigation. 23107, 5 mmol⋅L− 1 solution in DMSO). The dissected organs were
In the current work, we address the effects and reactions in the incubated at room temperature for 30 min in darkness in 15 mmol⋅L− 1
Atlantic ditch shrimp Palaemon varians upon microplastic exposure. This Na-HEPES and 0.5 mmol⋅L− 1 glucose in seawater (pH 8.3) with 10
article is conceived as a case study with a crustacean species, providing μmol⋅L− 1 Syto-13 (Korez et al., 2020).
an overview of the principal processes from the uptake of particles to the Midgut glands of P. varians, which were incubated in a suspension of
internal processing, and the cellular effects they may cause. Our 0.1-µm particles, were also stained with carboxy-2′ ,7′ -difluoro­
approach comprises feeding of shrimp with differently sized fluorescent fluorescein diacetate (CH2DFFDA, Molecular Probes, C-13293, 2
microbeads. Their dispersion within the digestive tract and biochemical mmol⋅L− 1 solution in ethanol) to detect reactive oxygen species (ROS)
effects are discussed in view of the anatomy, functional morphology, (Rivera-Ingraham et al., 2013). The staining solution was composed of
and cytology of the digestive organs. 15 mmol⋅L− 1 Na-HEPES and 0.5 mmol⋅L− 1 glucose in 250 mmol⋅L-1
NaCl, containing 20 µmol⋅L− 1 of CH2DFFDA. The samples were incu­
2. Material and methods bated for 30 min in the dark at room temperature. Prior to imaging, the
samples were washed three times in incubation buffer.
2.1. Shrimp The small particles (0.1 µm) emitted in the red spectrum (645–660
nm). The cell nuclei and ROS emitted in the green spectrum (500–520
Atlantic ditch shrimp (Palaemon varians, see also supporting infor­ nm and 517–527 nm, respectively). Photographs were taken with a 40x
mation) were purchased from an aquaristic supplier (Mrutzek Meeres- optical objective and a resolution of 512 × 512 pixels.
Aquaristik, Ritterhude, Germany). The shrimp were transferred to the Scanning electron microscopy (SEM) was applied to illustrate the
laboratories of the Alfred Wegener Institute, Helmholtz Centre for Polar ultrastructure of the stomach and the midgut gland of P. varians, and to
and Marine Research, where they were maintained in 10-L aquaria with verify the size and shape of the particles used. Stomachs and midgut
diluted seawater (salinity 15 PSU). The water was exchanged every glands of the shrimp were carefully dissected on ice and the filter press
other day. Maintenance and feeding were carried out in a temperature- in the proventriculus was exposed. The organs were then dehydrated in
controlled room at 15 ◦ C and a 12/12 h light/dark cycle. The shrimp an ethanol series: 2 × 15 min in 50% ethanol, 2 × 15 min in 70%
were fed every other day with plant-based fish food (NovoVert, JBL, ethanol, 2 × 15 min in 90% ethanol, 2 × 15 min in 96% ethanol, 30 min
Germany) and once a week with freshly hatched Artemia nauplii (Great in an ethanol-hexamethyldisilazane (HDMS) solution (ratio 1:1) and,
Salt Lake Artemia Cysts, Sanders, USA). The water quality was finally, 60 min in pure HDMS-solution (Korez et al., 2020). After
controlled regularly. Levels of nitrite, nitrate and ammonium were air-drying for 24 h, the samples were mounted on SEM stubs with
monitored and held below critical concentrations by regular exchanges double-sided carbon tape. The microplastic particles were suspended in
of the seawater. Only shrimp in the intermolt stage were used for further demineralized water. Droplets of the differently sized particles were first
investigations. dried on a plastic weighing pan. Then the microplastic particles were
sprinkled over the stubs with double-sided carbon tape. The stubs were
2.2. Microbeads sputter coated with gold-palladium. The samples were inspected and
photographed under the SEM (FEI, Quanta FEG 200).
The microbeads used were Thermo Scientific™ Fluoro-Max fluores­
cent polystyrene (PS) microspheres. These particles are uniform, easy to 2.4. Biochemical investigations
observe within organisms, and easy to follow microscopically when
translocated between organs. The types of microbeads used varied in 2.4.1. Exposure
terms of size and fluorescent properties and were applied at different Twenty-four hours prior to incubation with microplastics, the shrimp
concentrations (Table S1) and according to the experimental question as were fed with plant-based fish food (NovoVert, JBL, Germany). During
described in the following paragraphs. Stock suspensions were prepared the incubation, the animals received no food but only microplastic

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particles at the given concentrations (Exp. 2 in Table S1). 500-mL glass

flasks were filled with 200 mL of filtered seawater, diluted to a salinity of
15 PSU. A specific amount of the particle stock suspension was added to
the flasks to obtain the desired particle concentration. Five randomly
selected shrimp were transferred from the aquaria into each of four
replicate flasks. The flasks were covered with parafilm to avoid evapo­
ration and contamination. Each flask was equipped with an aeration
tube, which was pierced through the parafilm. The aeration suspended
the microbeads in the water. The incubation times were 2, 4, 8, 24, and
48 h. Two additional incubations (6 replicates each) served as negative
controls using the same setup as described above. In the first negative
control, the shrimp received food but no particles. Shrimp were first fed
at the start of the incubation and then 24 h later, with 15 mg plant-based
fish food per 500-mL glass flask. In the second negative control the
shrimp received no particles and no food.

2.4.2. Sample preparation

The shrimp were sedated on ice. The body length of the shrimp was
measured, and the midgut gland was dissected in a Petri dish placed on a
cooling pad. The midgut gland was transferred into pre-weighed 1.5-mL
reaction tubes, weighed, and immediately frozen in liquid nitrogen.
Thereafter, the samples were stored at - 80 ◦ C. Untreated animals (0 h of
incubation) were randomly taken from the aquaria at the beginning of
the feeding trials and processed as described above. The frozen tissues
were extracted immediately before the enzyme activities were
measured. The individual midgut glands were slowly thawed on ice and
homogenized with a conical micro-pestle in 50 μL of ultrapure water.
After centrifugation (10 min, 14,000g, 4 ◦ C), the supernatants were
transferred to new 1.5-mL reaction tubes. Five μL of the extract were
diluted 1:20 in SOD homogenization buffer. The SOD homogenization
buffer (pH 7.6) was prepared with 100 mL Tris-HCl (20 mmol⋅L− 1) and
50 mL EDTA (1 mmol⋅L− 1) in distilled water (modified after Livingstone
et al., 1992). The buffer was filtered (0.2 µm) and stored at 4 ◦ C. Finally,
the diluted extracts were vortexed and referred to as ‘samples’ in the
following. The remains of the extracts were stored at - 80 ◦ C. Every step Fig. 1. Palaemon varians with ingested fluorogenic microbeads of 9.9 µm
was performed on ice. (green), 2.1 µm (blue), and 0.1 µm (red) in diameter. a) Appearance of
microbeads in the stomach and in the midgut gland. Merged images of trans­
mission and electronically intensified fluorescence spectra. b) Lateral view of
2.4.3. Enzyme assays
the shrimp and schematic illustration of the stomach and the midgut gland and
Superoxide dismutase (SOD) was assayed after Livingstone et al.
gut with green (9.9 µm), blue (2.1 µm), and red (0.1 µm) microbeads indicated.
(1992), with slight modifications and adaptations for shrimp midgut c) Excreted fecal string with 9.9 µm microbeads. Merged transmission and
gland tissue samples (for details see the Supporting Information). fluorescence images.

2.5. Statistical analyses apparently dispersed into the midgut gland, which extends posteriorly
from the stomach (Fig. 1b). Excreted feces contained the fluorescent
The temporal variation of SOD activity in the midgut gland of microbeads (Fig. 1c). Smaller fluorescent particles within the fecal pel­
P. varians was analyzed for each food type separately using one-factorial lets were not visible from outside due to shielding by the fecal pellet
analyses of variance (ANOVA) and Tukey’s posthoc tests. Prior to the content.
ANOVA, the data were tested for equal variances using Levene’s test.
Solely in the case of the 9.9-µm microbeads, the variances were het­
erogeneous and could not be homogenized by data transformation. 3.2. The stomach and the pyloric filter
Additionally, we compared the overall SOD activity among the treat­
ments by an ANOVA. To account for the heteroscedasticity of the data, The stomach is a complex flexible chitinous capsule. It consists of an
as revealed by Levene´s test, the significance level was adjusted to p = anterior cardiac part and a smaller posterior pyloric part. The pyloric
0.01. stomach contains a fine-meshed filter system (Fig. 2a, b), which fulfils
pivotal functions in the separation of digestible compounds and indi­
3. Results gestible solid particles. The pyloric filter consists of two inner and two
outer valves. The inner valves join dorsally at the so-called filter crest,
The shrimp readily ingested the offered food and the fluorescent forming a ‘W′ -shape in cross section. Each side of the filter is covered by
microparticles. There was no mortality. a field of forklike setae, projecting dorsally to the ventrolateral partition
(Fig. 2c, d). This separates the dorsal passageway for solids and the
3.1. Dispersion of microplastics in the digestive organs ventral passageway for fluids (King and Alexander, 1994). The ven­
trolaterally projecting filter setae are connected by longitudinal ridges.
The largest particles of 9.9 µm (green) were present in the cardiac Between the longitudinal ridges four to six pyloric filter setae are
stomach (Fig. 1a). The 2.1-μm particles (blue) accumulated in the bundled, thus forming transversal channels. The pyloric filter setae are
anterior part of the cardiac stomach and advanced further into the dorsal studded with densely packed setules. The gaps between the setules have
chamber of the pyloric stomach. The smallest particles of 0.1 µm (red) a width of approximately 170 nm. The chyme produced in the stomach

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R. Saborowski et al. Ecotoxicology and Environmental Safety 234 (2022) 113394

Fig. 2. The pyloric filter of Palaemon varians. a,b) Transmitted light microscopic view on the pyloric filter. c,d) Scanning electron microscopic details of the pyloric
filter with longitudinal costae (LC) bearing vertical pyloric filter setae (PFS). Scale bars in a and b: 100 µm, c: 300 µm, d: 50 µm.

is pressed through the pyloric filter. The fluid fraction of the chyme decreased.
enters the midgut gland where nutrients are resorbed while solids are Shrimp feeding on 2.1-µm microbeads showed a significant increase
retained and directed into the gut for egestion. in SOD activity (ANOVA: F4,17 = 4.98; p < 0.01 – Fig. 6b). The SOD
activity increased steadily from 725 ± 60 U⋅g− 1 at the beginning of the
3.3. The midgut gland incubation period to 1337 ± 115 U⋅g− 1 after 24 h of incubation. After
48 h of incubation the SOD activity was slightly lower at 1015
The midgut gland (syn. hepatopancreas) consists of numerous blind ± 93 U⋅g− 1.
ending tubules with a monolayer of cells forming the tubule wall. The Exposure to 9.9-µm microbeads also resulted in an increase in SOD
nuclei of the cells, stained with Syto-13, indicate the arrangement of the activity (ANOVA: F4,17 = 6.21; p < 0.01 – Fig. 6c) with the activity being
tubule with their distal tips and the cellular monolayer surrounding the highest at 1076 ± 44 U⋅g− 1 after 8 h of incubation. The heterogeneous
tubule lumen (Fig. 3). The diameter of the tubules is up to 200 µm. The variances (Levene’s test: F4,17 = 3.54; p = 0.03) increased the proba­
tubules become wider towards the proximal regions of the midgut gland. bility of erroneously accepting the differences in SOD activity as sta­
The scanning electron micrograph shows longitudinally broken tubules tistically significant. However, the variances within the groups were
and membranous fragments of the cell monolayer. The tubule lumen small and the initial increase in SOD activity was steep, suggesting
extents to about 50 µm. Specific cells cannot be identified, but large clearly elevated activities after 8 h of incubation. After 24 h of incuba­
vacuoles of the B-cells are suggested. tion, the SOD activity declined to 765 ± 107 U⋅g− 1 and to 711
Individual microbeads of 0.1 µm could not be resolved within the ± 29 U⋅g− 1 after 48 h of incubation.
midgut gland. However, several hours after feeding on these particles, Feeding on commercial fish food had no effect on the SOD activity of
agglomerations of fluorescent particles appeared adjacent to the midgut the shrimp (ANOVA: F4,17 = 0.34; p = 0.85 – Fig. 6d). The SOD activities
gland tissue (Fig. 4). Their shape was cylindrical with a length of up to varied only little between the incubation times from 726 ± 90 U⋅g− 1 at
500 µm and a width of up to 160 µm. the beginning of the incubation period and 855 ± 60 U⋅g− 1 after 24 h of
Incubation of midgut gland tissue with CH2DFFDA revealed distinct incubation. Similarly, the control without food and without particles
green fluorescent spots in the cells of the midgut gland tubules (Fig. 5). showed no significant variation in SOD activity (ANOVA: F4,17 = 1.65;
The lumen of the tubule showed a weak and diffuse fluorescence. p = 0.21 – Fig. 6e). The activities varied from 768 ± 74 U⋅g− 1 at the
beginning of the incubation, increased towards 1030 ± 83 U⋅g− 1 after
24 h, and decreased again to 944 ± 15 U⋅g− 1 at the end of the
3.4. SOD-activity
incubation.
No differences in the overall SOD activity were detected among the
The SOD activity increased significantly after exposure to 0.1-µm
treatments (F4,105 = 2.75; p = 0.03) when the significance level was
microbeads (ANOVA: F4,17 = 4.17; p = 0.02 – Fig. 6a). At the beginning
adjusted to p = 0.01 to account for the heteroscedasticity of the data.
of the incubation period, the initial average (± SEM) SOD activity was
518 ± 140 U⋅g− 1. After 4 h the activity increased to 953 ± 155 U⋅g− 1
and reached the maximum of 1120 ± 79 U⋅g− 1 after 8 h. After 24 h
(1045 ± 120 U⋅g− 1) and 48 h (1003 ± 95 U⋅g− 1) the activities slightly

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Fig. 3. Midgut gland tubules of Palaemon varians: fluorescent image of a) Syto-13 dyed cell nuclei (green) and b) fluorescent image merged with brightfield image of
midgut gland. c) Scanning electron micrograph (SEM) of longitudinally broken midgut gland tubule showing the tubule lumen (TL), the cell monolayer (ML), the
distal tip of the tubule (DT), and vacuoles (V) of B cells. No microplastics were administered. All scale bars represent 100 µm.

4. Discussion in the water column and 22–702 items per kg dry sediment (Table S2).
Small items in the size range < 100 µm dominated the microplastic
Microplastics have no nutritional value, except a more or less samples in the coastal areas of the North Sea (Lorenz et al., 2019). The
evolved biofilm on the surface (Michels et al., 2018). Nevertheless, size range of the collected particles depends on the mesh size of the
numerous reports showed that a huge range of marine invertebrates, sieves, which was 20 µm in the study by Lorenz et al. (2019). Smaller
including crustaceans, mollusks, chaetognaths, tunicates, and cnidar­ particles mostly passed the filter, while particles < 100 µm as well as
ians do ingest microplastic particles in the µm size range accidentally or fragments thereof are in the size range of potential food items and
intentionally (Cole et al., 2013), among them mysid shrimp and caridean resemble the administered 9.9-µm particles in our experiment.
shrimp (Setälä et al., 2014; Devriese et al., 2015; Korez et al., 2020). Investigations on the microplastic burden of wild individuals of
However, the effects of microplastic ingestion are only marginally un­ P. varians are lacking. However, studies on mussels and brown shrimp
derstood and seem to differ between consumers, potentially because of from the same area showed up to 6.9 plastic items per gram (Table S3).
morphological and physiological variations among species. Moreover, previous laboratory studies showed that P. varians readily
ingest microplastic beads (9.9 µm) and fibers of up to 200 µm (Sabo­
rowski et al., 2019). The present observations confirm the uptake of
4.1. Exposure and uptake of particles microplastics of different size by P. varians. Moreover, they indicate that
microplastics are taken up by the shrimp in misperception of food,
The Atlantic ditch shrimp, Palaemon varians, lives in brackish la­ rather than accidently along with the food as shown for the uptake of
goons, estuaries, and salt marshes from Scandinavia to the Mediterra­ sand grains by the brown shrimp Crangon crangon (Schmidt et al., 2021).
nean, where it faces anthropogenic pollution. Microplastic In the current study, no food was offered simultaneously with the
concentrations in its area of distribution range from 0 to 70 items per m3

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through carboxylate surface modifications.

This distribution of the particles inside the shrimp can be explained
by the anatomy and function of the digestive tract. The digestive tract of
crustaceans is subdivided into foregut, midgut, and hindgut. The foregut
is composed of the esophagus and the stomach. The midgut consists of
the midgut gland (hepatopancreas) and the anterior part of the gut. The
hindgut terminates in the anus. The stomach and the midgut gland are
connected via a short-paired duct. The foregut, including the stomach,
and the hindgut are of ectodermal origin and are protected against
mechanical and chemical damage by a chitinous layer. Simultaneously,
this chitinous layer prevents passive or active translocation of particles
into the surrounding tissue. Nutrients are made available for metabolism
only when the macerated and predigested chyme passes from the
stomach into the midgut gland, which is the principal organ of nutrient
resorption and digestive enzyme synthesis.
The midgut, including the midgut gland, is of endodermal origin,
thus lacking a protective chitinous layer (Ceccaldi, 1989; Sousa and
Petriella, 2006). Once entering the midgut gland tubules, ingested
microplastics may interact with the epithelial cells of the midgut gland,
which are basically resorption cells (R-cells), fibrillary cells (F-cells),
and blister-like cells (B-cells). The R-cells facilitate nutrient resorption
and nutrient storage (Ceccaldi, 1989). Similarly, B-cells may be involved
in the translocation of microplastics. They contain a distinctive vacuole,
which grows while the cell matures. The vacuoles are believed to collect
substances and molecules, which ought to be degraded and resorbed or
egested, i.e. denatured enzymes or cellular waste (Vogt, 2019; Štrus
et al., 2019). The epithelium of the midgut gland is the most vulnerable
site of the digestive system where microplastics can translocate into the
tissue to induce cytotoxic responses.
In other taxa, such as the bivalve Mytilus edulis, microplastics are
ingested through the esophagus as well. The particles were taken up into
Fig. 4. Confocal laser scanning micrographs showing two aspects (a, b) of Syto- the stomach and transported into the digestive gland. There, they
13 stained cell nuclei (green) and agglomerations of microparticles (0.1 µm, accumulated in the lysosomal system and caused a strong inflammatory
red) adjacent to the midgut gland tissue. Merged images of transmission, green,
response demonstrated by the formation of granulocytomas and lyso­
and red spectra.
somal membrane destabilization (von Moos et al., 2012). Microplastic
ingestion was reported for a variety of species with different focus on the
microplastics. uptake and the cellular effects of particles. Recent reviews summarize
Administration of differently sized fluorescent microbeads indicates the current knowledge (e.g. Wang et al., 2019; Coyle et al., 2020; Prinz
that the largest particles (9.9 µm) were retained in the stomach and and Korez, 2020).
subsequently egested via the gut, whereas smaller particles (2.1 and
0.1 µm) apparently translocated into the surrounding tissue. Leaching of
4.2. Effects and cellular responses
the fluorescent dye from the microbeads, as observed by Schür et al.
(2019) did not occur in our study. This is probably due to the different
Many studies have shown adverse effects of ingested micro- and
nature of the particles. The particles used in our study are internally
nanoplastics on invertebrates including reduced feeding rates, energy
dyed while the particles used by Schür et al. (2019) were surface-dyed
reserves, fitness, and fecundity, as well as immune suppression (e.g.

Fig. 5. Detection of reactive oxygen species with the fluorescent probe CH2DFFDA in midgut gland tissue of Palaemon varians. a) Upon oxidation, CH2DFFDA is
converted into the fluorescent derivate difluorofluorescein (DFF). b) Transmission photograph and c) merged fluorescence and transmission images. Arrows indicate
spots of DFF derived fluorescence. Scale bars represent 100 µm.

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Fig. 6. Activities of superoxide dismutase in the midgut glands of Palaemon varians exposed to microbeads (a, b, c) and two controls; d) plant based commercial food,
and e) treatment where both food and particles were absent. Different letters indicate statistically significant differences between groups (p < 0.05, n = 4).

Besseling et al., 2013; Cole et al., 2013; Wright et al., 2013; Li et al., Polystyrene particles induce oxidative stress in rotifers and activate by
2016; Auta et al., 2017). However, in experiments the load of micro­ phosphorylation two factors of the mitogen-activated protein kinases
plastics often exceeds environmentally detected concentrations (Cun­ (MAPKs) pathway, JNK and p38 (Jeong et al., 2016).
ningham and Sigwart, 2019). Thus, adverse effects may be attributed to In the present investigation, SOD-activities increased rapidly already
a high exposure, potentially inducing false satiation and malnutrition. after few hours of exposition of the shrimp in microplastic suspension.
Moreover, in some polymers the cellular response is induced by the These findings indicate a significant production of reactive oxygen
plastic additives or leachates rather than by the microparticles them­ species, as confirmed by the ROS marker CH2DFFDA. SOD-activity as
selves (Zimmermann et al., 2020). To minimize the risk of such misin­ immediate cellular reaction responds rapidly to an imbalance of the
terpretation, we diluted and washed the particles with pure water and oxidative state (Timofeyev et al., 2006). These results are in agreement
removed additives as well as possible. with recent studies by Jeong et al. (2016, 2017) who showed in rotifers
Other studies report little or no effects of microplastics on in­ and copepods, that small polystyrene particles (0.05 µm) entailed higher
vertebrates, such as e.g. sea urchin larvae (Kaposi et al., 2014), marine ROS production and higher antioxidant activities than larger particles.
isopods (Ha¨mer et al., 2014), amphipods (Bruck and Ford, 2018), and This indicates that antioxidant enzymes are suitable cellular markers of
decapods (Carreras-Colom et al., 2018). It appears reasonable that ef­ oxidative stress occurring after ingestion of microplastics and that
fects of microplastics differ between species. For example, species, smaller particles, especially nanoparticles, cause stronger cytotoxic ef­
which commonly take up considerable quantities of indigestible parti­ fects than larger ones (Jiang et al., 2008; Lee et al., 2013; Jeong et al.,
cles with their food, such as diatom frustules, plant fibers, or sand grains, 2016). Upon ingestion and translocation of these particles into the
may be better prepared to cope with ingested microplastics (Saborowski digestive organs, the cells may identify them as pathogens and react by
et al., 2019; Korez et al., 2020; Schmidt et al., 2021). generating destructive superoxide ions (O2-) through e.g.
Studies addressing the cytotoxic potential of particles in in­ NADPH-oxidase activation (Inada et al., 2012; Brandes et al., 2014). In
vertebrates are sparse. Incorporation and translocation of microplastics this way, microplastic particles might have an indirect hazardous effect
into cells of the digestive tract of the blue mussel (Mytilus edulis), were on cells by activating their endogenous defense mechanisms. Upon
accompanied by encapsulation of particles and the formation of eosin­ chronic exposure, this may entail damage of cellular compounds such as
ophil granulocytomas, an inflammatory response, known to occur after membranes and DNA. Additionally, chronic activation of endogenous
parasite infestation (Lowe and Moore, 1979; von Moos et al., 2012). defense mechanism may deplete resources needed for other

7
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physiological processes, such as growth and reproduction, potentially 5. Conclusions

leading to fitness reduction.
The largest particles (9.9 µm) also caused increases in SOD activity Aquatic invertebrates are in many ways affected by microplastics.
although they are assumed not to enter the midgut gland. Possibly, these The Atlantic ditch shrimp, P. varians, readily and indiscriminately ingest
particles were at least partly fragmented by the action of the gastric mill. microplastics. The conceptual model (Fig. 7) illustrates the fate of
The grinding of the food may generate very small particles or even microplastics and other indigestible material upon ingestion in shrimp.
nanoparticles, which are not visible by light microscopy and able to Large items, such as microplastic fibers or bivalve shell fragments or
enter the midgut gland. This process has been shown in Antarctic krill, polychaete bristles derived from food, remain in the stomach and may
langoustine, and freshwater amphipods (Dawson et al., 2018; Cau et al., be discarded through the esophagus by regurgitation. Smaller particles,
2020; Mateos-Cárdenas et al., 2020). such as the experimentally administered 9.9-µm beads, are passed from
The overall SOD activity did not vary among the treatments although the stomach into the gut for defecation. The fine-meshed pyloric filter
the temporal development of the SOD activities differed fundamentally prevents the passage of these particles into the midgut gland, which is
between the particle treatments and the control treatments. The initial the principal organ of nutrient resorption. The smallest particles
increase and the subsequent decline in SOD activity resulted in a (0.1 µm), however, can pass the pyloric filter and advance into the
considerable variation of the overall SOD activity in the particle treat­ midgut gland, where they cause oxidative stress as indicated by the
ments (but not in the control treatments), which prevented the detection occurrence of radical oxygen species in epithelial tissue and the rapid
of statistically significant differences in SOD activity among the increase of the anti-oxidative enzyme superoxide dismutase. Accord­
treatments. ingly, P. varians shows efficient mechanical and biochemical protection
mechanisms to reduce the uptake of indigestible material and to coun­
4.3. Protection against microplastics teract ROS formation.

Ideally, the first line of protection should be the selective refusal of Funding
particles as discussed for bivalves in view of microplastic uptake (Ward
et al., 2020). Since selective refusal is not the case in P. varians, the This study received no external funding.
shrimp have to rely on internal selective mechanisms. Beads of 9.9 µm
diameter passed the digestive tract and were egested with fecal material, CRediT authorship contribution statement
although some of them might have been fragmented. Fibrous micro­
plastics first remained in the stomach but are regurgitated after a few R. Saborowski: Conceptualization, Supervision, Data curation,
hours through the esophagus (Saborowski et al., 2019). Accordingly, the Writing − original draft, review & editing. Špela Korez: Investigation,
stomach and its various morphological features are crucial for the pro­ Methodology, Data collection and curation, Writing − review & editing.
cessing of the ingested food and the allocation of nutrients and indi­ Sarah Riesbeck: Investigation, Methodology, Data collection. Mara
gestible items. Weidung: Investigation, Methodology, Data collection. Ulf Bickmeyer:
The fine-meshed chitinous filter system at the base of the pyloric Methodology, Writing − review & editing. Lars Gutow: Conceptuali­
stomach functions as a selective barrier to prevent the passage of larger zation, Supervision, Writing − review & editing.
particles into the midgut gland. A paired duct connects the pyloric
stomach with the midgut gland and another duct leading into the gut Declaration of Competing Interest
functions as the pyloric passage for solids. Indigestible particles, which
are not regurgitated but are too big to pass the pyloric filter, are directly The authors declare that they have no known competing financial
passed into the hindgut for excretion. The pyloric filters of crustaceans interests or personal relationships that could have appeared to influence
with outer and inner valve assembled with setae and setules seem to the work reported in this paper.
have taxon-specific mesh sizes of 100–200 nm (Korez et al., 2020). In
the shrimp Penaeus merguiensis it is assumed that particles smaller than
about 170 nm may pass the filter barrier (King and Alexander, 1994).
In P. varians, particles of 9.9 µm in diameter were too large to pass
the pyloric filter. Similarly, 2.1 µm sized microbeads were retained by
the filter setae, whereas 0.1 µm particles seem to pass the filter and enter
the midgut gland. Nevertheless, a vast number of these particles seems
to be also retained by the pyloric filter. The aggregations that we
observed adjacent to the midgut gland tissue have a similar diameter as
the fecal strings of the shrimp. Therefore, it is likely, that these aggre­
gations were separated from the gut nearby the junction to the pyloric
stomach during the dissection of the midgut gland. The smallest parti­
cles of 0.1 µm diameter were too small to be resolved by light micro­
scopy. However, previous studies showed that indigestible particulate
matter accumulated in the vacuoles of the midgut gland of crayfish and
green tiger shrimp (Loizzi, 1971; Al-Mohanna and Nott, 1986). Shortly
after feeding on food spiked with colloidal gold or thorium dioxide, the Fig. 7. Conceptual model of the fate of microparticles in the digestive tract of
indigestible particles accumulated in the vacuoles of the B-cells. Within the Atlantic ditch shrimp, Palaemon varians. Ingested material (food in the
24 h, the enlarged vacuolar content was released into the lumen of the wider sense) is macerated in the stomach. Large indigestible items like shell
midgut gland tubules and excreted with the feces. In this way, excretion fragments, spines, or fibers are regurgitated through the esophagus. Smaller
indigestible items are retained by the pyloric filter (PF) and directed into the
of the particles through the feces appears likely (Štrus et al., 2019; Vogt,
gut for defecation. The liquid share of the chyme and smallest particles pass the
2019), preventing or reducing the translocation of particles to tissues
pyloric filter and enter the midgut gland tubules. Here, nutrient resorption and
and organs. This mechanism would efficiently protect the cells from interaction with the epithelial cells takes place, potentially causing cellular
detrimental accumulation of indigestible microparticles. stress by reactive oxygen species. Indigestible components of the chyme,
including smallest particles, are pushed back from the midgut gland tubules
into the gut and defecated as well.

8
R. Saborowski et al. Ecotoxicology and Environmental Safety 234 (2022) 113394

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Channel area. Mar. Pollut. Bull. 98, 179–187. https://doi.org/10.1016/j.
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