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Microplastics of different characteristics are incorporated into the larval cases of the freshwater caddisfly Lepidostoma basale

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Microplastics of different characteristics are incorporated into the larval cases of the freshwater caddisfly Lepidostoma basale Sonja M. Ehlers*, Werner Manz, Jochen H. E. Koop ABSTRACT: Plastic pollution is present in aquatic systems worldwide. While numerous studies investigate microplastic interactions with marine organisms, microplastic effects on freshwater organisms, especially insects, are rarely studied. Moreover, microplastic studies mainly focus on microplastic dietary uptake. The presence of microplastics in animal constructions, as commonly known from macroplastics incorporated into birds' nests, is largely unknown. So far, microplastics have only been observed in the tubes of a marine polychaete species. In freshwater systems, common caddisfly (Trichoptera) larvae build cases by using larval silk and mineral grains from benthic sediments that are at the same time known as microplastic sinks. Therefore, we examined caddisfly cases for microplastic presence. We collected caddisfly cases in the field, disintegrated them using hydrogen peroxide, and determined microplastic polymer type through micro-Fourier-transform infrared spectroscopy (µFTIR). We found primary and secondary microplastics of different shapes, colors, sizes and chemical compositions (e.g., polypropylene, polyethylene, polyvinyl chloride). Therefore, this is the first study to show that microplastics are present in the biological construction of a freshwater organism. Larval stages are usually more vulnerable than adult individuals, and microplastics can transport persistent organic pollutants and emit toxic leachates. In the caddisfly larval case, those substances are in close proximity to the sensitive larval body which may be harmful for the larva and may eventually impede its development. Furthermore, we discuss the potential of caddisfly larval cases to act as microplastic bioindicators in freshwater habitats.
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AQUATIC BIOLOGY
Aquat Biol
Vol. 28: 67–77, 2019
https://doi.org/10.3354/ab00711 Published July 11
1. INTRODUCTION
Freshwater ecosystems worldwide, including
streams and rivers, are polluted with microplastic
debris (Eerkes-Medrano et al. 2015). Such micro -
plastics (plastic particles <5 mm, Moore 2008) vary in
shape (fragment, film, pellet [spherical], foam and
fiber; Free et al. 2014), color and chemical compo -
sition, and are classified as primary and secondary
microplastics (Cole et al. 2011). Primary microplastics
such as spherical microbeads are manufactured to be
of a very small size. These spheres have multiple
appli cations, e.g. as exfoliating agents in cosmetics,
and may reach the environment because waste water
treatment plants are often inefficient in removing
them (Rochman et al. 2015). In contrast, secondary
micro plastics, such as fragments, films and fibers, are
created when larger plastic debris becomes (e.g.
mechanically or photolytically) degraded into smaller
plastic pieces (Cole et al. 2011). Plastic items from
land-based sources can be transported to streams
and rivers by wind, rain drainage, waste water and
improper disposal of plastic waste (Duis & Coors
2016). The industrial mass production of plastics
© The authors 2019. Open Access under Creative Commons by
Attribution Licence. Use, distribution and reproduction are un -
restricted. Authors and original publication must be credited.
Publisher: Inter-Research · www.int-res.com
*Corresponding author: sehlers@uni-koblenz.de
Microplastics of different characteristics are
incorporated into the larval cases of the
freshwater caddisfly Lepidostoma basale
Sonja M. Ehlers1,2,*, Werner Manz2, Jochen H. E. Koop1
1Department of Animal Ecology, Federal Institute of Hydrology, 56068 Koblenz, Germany
2Institute for Integrated Natural Sciences, University of Koblenz-Landau, 56070 Koblenz, Germany
ABSTRACT: Plastic pollution is present in aquatic systems worldwide. While numerous studies
have investigated microplastic interactions with marine organisms, microplastic effects on fresh-
water organisms, especially insects, have been rarely studied. Previous studies have mainly focused
on dietary uptake of microplastics, but the presence of microplastics in animal constructions is
largely unknown. To date, microplastics have only been observed in the tubes of a marine poly-
chaete species. In freshwater systems, common caddisfly (Trichoptera) larvae build cases by using
larval silk and mineral grains from benthic sediments, which are known microplastic sinks. There-
fore, we examined caddisfly cases for microplastic presence. We collected caddisfly Lepidostoma
basale cases in the field, disintegrated them using hydrogen peroxide, and determined microplastic
polymer type through micro-Fourier-transform infrared spectroscopy. We found primary and sec-
ondary microplastics of different shapes, colors, sizes and chemical compositions (e.g. poly propylene,
polyethylene, polyvinyl chloride). Thus, this is the first study to show that microplastics are present
in the biological construction of a freshwater organism. Larval stages are usually more vulnerable
than adult individuals, and microplastics can transport persistent organic pollutants and emit toxic
leachates. In the caddisfly larval case, those substances are in close proximity to the sensitive larval
body, which may be harmful for the larva and may eventually impede its development. We discuss
the potential of caddisfly larval cases to act as microplastic bioindicators in freshwater habitats.
KEY WORDS: Synthetic polymers · Freshwater insects · Trichoptera · Case construction · Stream
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Aquat Biol 28: 67–77, 2019
started in the 1940s (Cole et al. 2011), and microplas-
tics were reported in the oceans as early as the 1970s
(Carpenter et al. 1972, Carpenter & Smith 1972).
Since then, numerous studies have examined the
impact of microplastics on marine organisms (Erren
et al. 2013, Wright et al. 2013, Egbeocha et al. 2018),
including vertebrates (Germanov et al. 2018) and
invertebrates (Cole et al. 2013, Kaposi et al. 2014).
Rivers are important sources of marine microplastics,
as they transport plastic debris from inland waters to
the ocean (Lebreton et al. 2017). Despite this impor-
tant pathway, freshwater microplastics have been
considerably less studied than microplastics in the
marine environment (Eerkes-Medrano et al. 2015).
Regarding the interactions of microplastics with
freshwater organisms, recent studies have mainly
focused on microplastic ingestion by vertebrates, e.g.
fish (Horton et al. 2018, McNeish et al. 2018), and
invertebrates, such as mollusks (Su et al. 2018) and
crustaceans (Weber et al. 2018), and on the resulting
physiological effects (Ding et al. 2018, Redondo-
Hasselerharm et al. 2018). Despite the great abun-
dance and ecological importance of aquatic insects
(Suter & Cormier 2015), microplastic studies on those
organisms are just emerging and largely focus on
micro plastic dietary uptake (Kim et al. 2018, Nel et
al. 2018, Windsor et al. 2019).
Besides microplastic ingestion, microplastics may
be incorporated into the structures built by animals.
For instance, the marine tube-building polychaete
Gun na rea gaimardi (Quatrefages, 1848) incorporates
microplastics into its housing (Nel & Froneman 2018),
and thereby fixes microplastic particles in a biologi-
cal construction. Similar biological structures in fresh-
water habitats are larval cases that several epi benthic
caddisfly (Trichoptera) species build. It has recently
been suggested that microplastics may be fixed in
caddisfly larval cases, but no analysis such as micro-
Fourier-transform infrared (µFTIR) spectro scopy has
been performed to verify that the particles which
were observed in caddisfly larval cases were micro-
plastics and that they were not, for example, parts of
ceramics or cardboard (Tibbetts et al. 2018). Hence,
to investigate if freshwater microplastics may be in -
corporated into caddisfly larval cases, we analyzed
whether microplastics are present in those cases and
which characteristics (shape, polymer type, color,
size) such microplastics possess.
After hatching from the eggs, the larvae of many
caddisfly species start building cases to protect them-
selves from predators (Boyero et al. 2006) and from
environmental influences (Zamora Muñoz & Svens-
son 1996). As the larvae grow, new material is added
to the anterior end of the case and secured with silk
filaments produced by the larvae (Stewart & Wang
2010). For case building, caddisfly larvae actively
collect different biotic (e.g. leaves; Sheath et al. 1995,
Moretti et al. 2009) and abiotic (e.g. sediment grains;
Gaino et al. 2002, Okano et al. 2012) materials, and
some species use both material types (Hansell 1972).
Different caddisfly species differ in their preferences
for certain case-building materials (Hanna 1961).
Recently, a few potential sentinel species for micro -
plastic pollution in freshwater ecosystems have been
suggested, including tadpoles (Hu et al. 2018) and
Asian clams (Su et al. 2018), but none has been estab-
lished so far. Therefore, we discuss the potential role
of caddisfly larval cases as bioindicators for fresh-
water microplastics which might facilitate microplas-
tic assessment in streams and rivers.
Given that a marine polychaete species incorpo-
rates microplastics into its tube (Nel & Froneman
2018), and freshwater sediments are a sink for micro -
plas tics (Castañeda et al. 2014), we hypothesized that
microplastics would be fixed in the larval cases of the
caddisfly Lepidostoma basale, which uses sediment
grains for case building (Skuja 2010). We tested our
hypothesis through an observational field study.
2. MATERIALS AND METHODS
2.1. Study site
We conducted our study in the Saynbach stream
(50.438399°N, 7.5732430°E), located in the Schloss-
park Sayn, a public park in the town of Bendorf
(Rhineland-Palatinate, Germany). This stream origi-
nates in Himburg (Rhineland-Palatinate) and flows
directly into the Rhine River. The stream has a length
of 43.7 km, a width of 3 to 4 m, a depth of 6 to 40 cm
and a catchment area of 219 km2(Beckmann et al.
2005). Downstream of our study site in the Schloss-
park Sayn, the Saynbach passes the Bendorf waste-
water treatment plant before leading into the Rhine.
The abiotic conditions of the Saynbach are regu-
larly measured by the Rhineland-Palatinate State
Office for the Environment (Landesamt für Umwelt)
at a nearby location (50.429° N, 7.565°E). On 10 April
2018 at 15:40 h Central European Summer Time (1 d
before we collected caddisfly larvae; see Section 2.2),
water temperature was 13.6°C, turbidity was 8 for-
mazin nephelometric units, pH was 8.4, conductivity
(at 20°C) was 264 µS cm−1, and oxygen concentration
was 10.9 mg l−1. The Saynbach sediment at the study
site consisted of sand and fine gravel.
68
Ehlers et al.: Microplastics in caddisfly larval cases
2.2. Caddisfly larvae collection and identification
On 11 April 2018, we manually collected case-
bearing caddisfly larvae in individual glass vials
(Wheaton, DWK Life Sciences) that contained 70%
ethanol. All larvae were collected at random and
had intact cases. To avoid airborne microplastic con-
tamination of the larvae, we wore cotton clothes.
Furthermore, the vials used for sampling were previ-
ously rinsed with ultrapure water.
At the lab, we identified 29 of the 30 caddisfly
larvae as Lepidostoma basale (Kolenati, 1848) under a
bin ocular microscope according to morphological
species keys (Wallace et al. 1990, Waringer & Graf
2011). This caddisfly (former synonym: Lasiocephala
ba sa lis) occurs in streams and rivers across Europe
(Moretti et al. 1981, Beisel et al. 1998, Hoffmann 2000,
Chadd & Extence 2004, Bonada et al. 2008, Skuja
2010, Verdonschot et al. 2010). It lives on woody de-
bris, shows facultative xylophagy (Hoffmann & Hering
2000) and feeds on biofilms (Schulte et al. 2003).
L. basale larvae can feed on invertebrates (Schulte et
al. 2003) and leaf litter (Hoffmann 2000). Annual
L. basale development proceeds through 5 larval
stages and 1 pupal stage (Verdonschot et al. 2010). In
all larval stages (i.e. instars), L. basale constructs min-
eral cases (Skuja 2010); in the fifth-instar larva, the
case has a maximum length of ca. 1.5 cm (Hoffmann
2000). In L. basale, individuals of different larval stages
(and thereby of different sizes) can be found simul tan -
eously at the same location (Verdonschot et al. 2010).
We identified the remaining caddisfly larva as
either Sericostoma personatum (Spence in Kirby &
Spence, 1826) or Sericostoma flavicorne Schneider,
1845, whose taxonomic differentiation is not fully
understood (Waringer 1987, Malicky 2005), but has
recently been clarified to some extent by genetical
analyses (Weigand et al. 2017). S. personatum larvae
are detritivore-shredders that feed on leaves (Friberg
& Jacobsen 1999). As we only found 1 individual of
this species, we present information on microplastics
in its larval case as an additional observation but
focus on the larval cases of L. basale.
2.3. Preparation of caddisfly larval cases
We measured caddisfly larval case length along the
convex case side using a digital microscope (VHX-
2000, Keyence). We then removed the larvae from
their cases using metal forceps. To remove any parti-
cles that adhered to the case surface but were not
fixed in the case matrix, we carefully rinsed all cases
in ultrapure water and put them into individual glass
Petri dishes. Next, we dried the cases at 40°C for 4 h
in a drying cabinet and immediately measured their
dry weight using an analytical balance (XS205 Dual-
Range Analytical Balance, Mettler Toledo).
To prevent microplastic contamination, we thor-
oughly cleaned all lab surfaces and glassware us ing
70% ethanol and ultrapure water before starting lab-
work. Furthermore, to prevent microplastic cross-
contamination between the caddisfly larval cases, we
carefully rinsed our forceps between samples. Finally,
to prevent airborne microplastic contamination, we
immediately covered all Petri dishes containing cad-
disfly larval cases with aluminum foil.
2.4. Caddisfly larval case oxidation and
density separation
For case disintegration, we transferred all rinsed
caddisfly larval cases into individual glass beakers in
which we submerged each case in 20 ml of a hydro-
gen peroxide solution (34.5−36.5% H2O2) to disinte-
grate them and remove any organic substances pres-
ent. All beakers were then covered with parafilm and
left on a shaking table at 150 rpm for 7 d until the
cases were completely disintegrated. Nuelle et al.
(2014) previously showed that such H2O2solutions
can successfully reduce organics in sediment sample
matrices similar to caddisfly case matrices. Occasion-
ally, stirring the samples with a glass rod can help in
case disintegration. We ran blanks parallel to the
case disintegration and scanned them for microplas-
tics, but did not detect any parafilm or microplastic
particles in the blanks. However, we found natural
fibers in the blanks which we identified using µFTIR.
After case disintegration, we separated the result-
ing minerals from the microplastics through density
separation. To do so, we transferred each sample to a
glass separation funnel to which we added potassium
formate (99%) until the solution was saturated at
about 1.6 g ml−1, a similar approach to that used to
separate microplastics from sediment grains in sedi-
ment samples (Zhang et al. 2016). Again, we ran
blanks with the density separation to exclude micro-
plastic contamination from, for example, the lid of the
container in which the potassium formate was stored.
After ap proximately 3 h, the mineral grains had set-
tled to the bottom of the funnel. We then drained the
grains and filtered the supernatant onto aluminum
oxide filters (Ano disc filter; pore size 0.2 µm; diame-
ter 47 mm, What man) using a pressure filtration unit
(model 16249, Sartorius). We chose these filters to en -
69
Aquat Biol 28: 67–77, 2019
able µFTIR measurements in transmission mode dur-
ing further microplastic analyses (Löder et al. 2015).
We placed the filters in small aluminum bowls, cov-
ered them with aluminum foil and placed them in a
drying cabinet (50°C) for 2 d. We then visually in -
spected each filter for microplastics using a digital
microscope (VHX-2000, Keyence) and identified all
microplastics by their unnatural color and shape
(Hidalgo-Ruz et al. 2012). The shapes for microplastic
particle classification were sphere, film (thin and
small layer), fragment (part of a larger plastic item)
and fiber (Su et al. 2016, 2018). We recorded the oc -
currence of microplastics and measured their dimen-
sions (maximum length of fragments, films and
fibers, and the diameter of the spheres).
2.5. µFTIR analyses of microplastics found in
caddisfly larval cases
We manually analyzed all microplastics found in the
L. basale cases using a Hyperion 2000 FTIR micro-
scope equipped with a mercury-cadmium telluride
detector (Bruker) in a wavenumber range of 4000−
600 cm−1 with 32 co-added scans and a spectral reso-
lution of 4 cm−1. The software used was OPUS 7.5, and
we compared the obtained spectra with the Bruker
database. As in previous studies, only particles with a
hit quality of over 700 were considered as microplas-
tics (Bergmann et al. 2017). We analyzed the micro-
plastics using attenuated total reflectance (µATR)
with a germanium crystal and an ATR 20× objective.
For some particles that were difficult to analyze, we
used µATR and transmission mode (with a 15× infra -
red objective). For measurements in transmission
mode, the blank aluminum oxide filter was used for
background measurements. Measuring the blank fil-
ter material as a background in transmission meas-
urements is a common procedure for µFTIR measure-
ments in microplastics research (Löder et al. 2015).
2.6. Sediment and water samples
We manually collected 5 sediment samples within
meters of the caddisfly collection site using yellow
containers. As in previous studies, sediment was sam -
pled to a depth of ca. 10 cm and as little surface water
as possible was collected along with the sediment
(Tibbetts et al. 2018). At the laboratory, we freeze-
dried the wet sediment samples, a common pro cedure
in microplastic research (Matsuguma et al. 2017), and
took subsamples with a dry weight of 15.64 ± 0.17 g
(mean ± SE, n = 5 sediment subsamples, range: 15.16−
16.15 g). We then transferred each subsample to an
individual glass beaker which we covered with alu-
minum foil to avoid airborne microplastic contamina-
tion of our samples. To facilitate the sediment ana -
lysis, we digested organic matter using 20 ml 10 M
KOH and 20 ml H2O2(34.5−36.5%) on a shaking
table. The beakers were covered with parafilm, and
controls were run in parallel to the digestion. KOH
was then neutralized with formic acid, as aluminum
oxide membrane filters are sensitive to alkaline con-
ditions. Afterwards, sediment grains were separated
from microplastics using density separation with
potassium formate. Again, blanks were run during di-
gestion and density separation. Moreover, we took 4
water samples (again using yellow containers) with a
volume of 467.5 ± 11.09 ml (mean ± SE, n = 4 water
samples, range: 450− 500 ml) each close to the caddis-
fly collection site. In the laboratory, the water samples
were transferred to glass beakers and freeze-dried.
Afterwards, we digested organic materials inside the
suspended sediment fraction using 10 ml 10 M KOH
and 20 ml H2O2(34.5−36.5%), neutralized the KOH
with formic acid and performed density separation as
well as filtration onto aluminum oxide membrane fil-
ters. Again, no microplastic particles were found in
the blanks. After transferring the samples onto alu-
minum oxide membrane filters, sediment and water
microplastic loads were analyzed under the digital
microscope as well as under the µFTIR. Again, µFTIR
measurements were conducted using µATR, and only
particles with a hit quality greater than 700 were con-
sidered as microplastics. Furthermore, special care
was taken to avoid contact between the germanium
crystal and sand grains to prevent any damage to the
crystal. No yellow particles that might have come
from the containers for sediment and water sampling
were found inside the samples.
3. RESULTS
3.1. Microplastics in caddisfly cases
In Lepidostoma basale, the average larval case
length was 0.85 ± 0.04 cm (mean ± SE, n = 29 cases,
range: 0.65−1.40 cm) and average larval case dry
weight was 6.40 ± 0.63 mg (mean ± SE). The Sericos-
toma personatum/flavicorne larval case had a length
of 1.26 cm and a dry weight of 34.07 mg.
Out of the 29 L. basale larvae, 17 individuals (59%)
had microplastics of different shapes fixed into their
cases (Fig. 1). The average microplastic load per indi-
70
Ehlers et al.: Microplastics in caddisfly larval cases
vidual was 1.14 ± 0.28 microplastics per caddisfly lar-
val case (mean ± SE, n = 29 caddisfly larval cases,
range: 0−6 microplastics per individual case). The
average microplastic load was 0.36 ± 0.09 microplas-
tics mg−1 case dry weight (mean ± SE, n = 17 caddis-
fly larval cases, range: 0.07 − 1.44 microplastics mg−1
case dry weight). In the S. personatum/flavicorne
case we detected 2 polyamide (PA) microplastics.
µFTIR spectroscopy revealed that microplastics
in the L. basale cases consisted of the polymers poly -
pro py lene (PP), PA, acrylonitrile-butadiene-styrene
(ABS), a blend of PA and ABS, polyacrylamide
(PAM), thermoplastic polyurethane (TPU), vinyl ester
resin (VE), polyvinyl chloride (PVC), polyethylene
(PE) and polyester (Fig. 2). The microplastic particles
incorporated into the caddisfly cases also showed a
wide spectrum of colors (Fig. 3).
Furthermore, we found rayon, a cellulose-based,
semi-synthetic material, in the cases. It is debated as
to whether rayon should be counted as microplastic,
considering its highly processed structure and poten-
tial environmental effects (Song et al. 2015). In our
study, rayon was rare, so we did not consider it as a
microplastic.
The microplastic fragments had an average length
of 236.09 ± 33.44 µm (mean ± SE, n = 11 microplastic
fragments, range: 40−375 µm). The films had an
average length of 98.92 ± 18.57 µm (n = 12 films,
range: 27−191 µm). The plastic spheres had an aver-
age diameter of 91.20 ± 25.88 µm (n = 5 spheres,
range: 29−163 µm) and the microplastic fibers had an
average length of 1872.40 ± 1434.03 µm (n = 5 fibers,
range: 295−7601 µm).
71
Fig. 1. Relative amount (in %) of different microplastic shapes
(sphere, fiber, fragment, film) in Lepidostoma basale cases
(n = 17), sediment (n = 5 samples) and water (n = 4 samples)
Fig. 2. Relative amount (%) of microplastic polymer types
(PP: polypropylene; TPU: thermoplastic polyurethane; PAM:
poly acrylamide; PA: polyamide; ABS: acrylonitrile-butadi-
ene-styrene; PVC: polyvinyl chloride; VE: vinyl ester resin;
PE: poly ethylene) of microplastic fragments, films, spheres
and fibers found in Lepidostoma basale caddisfly cases
Fig. 3. Relative number (in %) of differently colored micro-
plastics found in Lepidostoma basale cases (n = 17), sedi-
ment (n = 5 samples) and water (n = 4 samples). Transp.:
transparent
Aquat Biol 28: 67–77, 2019
3.2. Analyses of sediment and water samples
The sediment subsamples had a microplastic load
of 0.09 ± 0.05 microplastics g−1 (mean ± SE, n = 5 sedi-
ment subsamples, range: 0−0.255 microplastics g−1).
Most of the sediment microplastics were fibers
(Fig. 1). According to µFTIR measurements, sediment
microplastics consisted of polyethylene, acryl and
polyester. The 4 water samples contained a micro-
plastic load of 0.003 ± 0.001 microplastics ml−1 (n = 4
water samples, range: 0.002−0.007 microplastics ml−1).
Among these microplastics, we found urea form alde -
hyde resin and silicone rubber.
4. DISCUSSION
In our study, we found primary (spheres) as well as
secondary (fibers, films, fragments) microplastics in -
corporated into the larval cases of Lepidostoma basa -
le. The spheres accounted for 15.2% of the micro-
plastics found and resemble those that are commonly
used in cosmetics (Tanaka & Takada 2016). In con-
trast, secondary microplastics, such as the fragments,
films and fibers in the caddisfly cases, are created
when larger plastic debris becomes degraded into
smaller plastic pieces (Cole et al. 2011). The L. basale
individuals that we analyzed had primary and sec-
ondary microplastics of different shapes, polymer
types, sizes and colors fixed in their cases (see Figs. A1
& A2 in the Appendix). Those results were verified by
µFTIR analysis, a crucial step in microplastic analysis,
as up to 70% of particles found in the environment can
visually be misidentified as microplastics (Hidalgo-
Ruz et al. 2012). Hence, we showed that freshwater
microplastics of different characteristics are incorpo-
rated into L. basale larval cases.
As caddisfly larval cases contain material available
in the water body the larva inhabits, the microplas-
tics found inside the L. basale case matrix in this
study must be a subset of the microplastics sus-
pended in the Saynbach. Thus, caddisfly case analy-
sis may provide information on the diversity of micro-
plastics present in a caddisfly’s habitat. Inside the
caddisfly larval case matrix, the microplastic parti-
cles are fixed with silk, and mineral cases show a par-
ticularly high stability (Otto & Svensson 1980). As L.
basale mineral cases contain a low fraction of organic
material and lower amounts of sand grains than sed-
iment samples, a filter resulting from the caddisfly
case processing is easier to visually analyze than a
filter with a sediment microplastic fraction. The latter
is often characterized by not fully digested organic
residues and small sand grains which could not fully
be removed by density separation (Mathalon & Hill
2014). For instance, we found a small yellow PP
sphere with a diameter of 43 µm and a small orange
PP film with a length of 73 µm in the caddisfly cases
(see Fig. A1F) which might have been easily over-
looked in visual sediment analysis due to their simi-
larity to sediment sand grains.
The disintegration of the cases enabled us to ana-
lyze all microplastics present in the caddisfly cases.
Some microplastics which might have been part of
the inner case wall or were previously covered by
sediment grains inside the case matrix (see Fig. A3 in
the Appendix) would have been overlooked without
dissolving the cases. Also, the usage of the digital
microscope al lowed us to record microplastic par ticles
which are normally not visible to the naked eye.
Nuelle et al. (2014) showed thata7dlongtreatment
with 35% H2O2successfully digests organic materials
but might lead to color and size loss in some polymer
types. The strong colors of the microplastics that we
found in the caddisfly cases (see Fig. A1) suggest that
there was no bleaching of microplastics due to the
usage of H2O2, perhaps aided by microplastic protec-
tion inside the caddisfly case matrix during digestion.
The cases exhibited microplastics with a range of
chemical compositions (PE, PP, TPU, PA, PAM, VE,
PVC, ABS, polyester). Low-density microplastics such
as ABS (density 1.02−1.07 g cm−3) can float in the wa-
ter column (McCormick et al. 2016), whereas high-
density microplastics such as PVC (density ca. 1.20−
1.45 g cm−3, Avio et al. 2017) are denser than fresh-
water and accumulate in sediments (Wright et al.
2013). In the caddisfly cases, we found not only plas-
tics with high densities such as PVC, but also micro-
plastics with low density such as ABS, PE (density
0.93− 0.98 g cm−3, Avio et al. 2017) and PP (density
0.89− 0.91 g cm−3, Avio et al. 2017). Hence, it is likely
that factors such as turbulence (McCormick et al.
2016) or an increase in microplastic density caused by
a biofilm cover (Lagarde et al. 2016, Rummel et al.
2017) led to low-density microplastic sedimentation
and made it available for caddisfly larvae. Similarly,
we found PE in the sediment samples. The fact that
the sediment samples showed higher microplastic
loads than the water samples corroborates that the
microplastics in the sampled caddisfly cases stem
from the stream sediments. However, as water flows
through the caddisfly case (Williams et al. 1987), it
cannot be ruled out that some microplastics inside the
caddisfly case may come from the water column. The
fact that the microplastic load in the caddisfly cases
was higher than in the sediment and water samples
72
Ehlers et al.: Microplastics in caddisfly larval cases
indicates that caddisfly cases may act as microplastic
sinks in freshwater habitats.
The µFTIR analysis revealed that a high proportion
of the microplastics were made of PP and that many
spheres consisted of PE, which are the most com-
monly used plastics (Zhang et al. 2016). They serve
as material for different single-use items such as
plastic bags, bottle caps and drinking straws which
are used in high quantities and regularly accumulate
in the environment after disposal (Andrady 2011).
Our results suggest that the plastic types which are
most frequently used, and are therefore probably the
most prevalent in the environment, are incorporated
into caddisfly cases. This finding implies that caddis-
fly cases may potentially be used as quantitative
bioindicators for microplastics. However, future stud-
ies should test the potential of caddisfly larval cases
as quantitative freshwater microplastic bioindicators
by comparing microplastic loads in caddisfly cases
from streams differing in plastic pollution. Further-
more, future studies could examine if microplastic
loads in caddisfly larval cases may change according
to seasonal flow rates. For instance, amounts of
microplastics ingested by chironomid larvae show
the same seasonal fluctuations as sediment micro-
plastic loads (Nel et al. 2018).
In our study, the only polymers which we found in
the caddisfly cases as well as in the sediment samples
were PE and polyester, suggesting that L. basale
cases may have the potential to act as qualitative
microplastic bioindicators. The reason why we did
not find more similarities between microplastics in
caddisfly cases and in the sediment and water sam-
ples may be the limited number of specimens from
just 1 species which was analyzed in our study. Dif-
ferent caddisfly species use different case building
materials and may therefore more likely represent
the variety of microplastics present in their habitats
when analyzed together.
In our study, all sediment and water microplastic
shapes were reflected in the L. basale cases. How-
ever, microplastic spheres were only found in the
caddisfly cases and not in the sediment and water
samples. An explanation for this may be that spheres
were present in the sediment and water samples, but
could not be detected as they were covered by
residues which are characteristic for filters from sedi -
ment and water samples. In the caddisfly cases, the
spheres were as small as 29 µm and were yellow or
transparent and colorless, which may further restrict
the chances of finding them on filters covered by
residues. Therefore, analyzing caddisfly cases might
help identify microplastics which are present in an
aquatic habitat but cannot easily be detected in sedi-
ment and water samples.
The disintegration of caddisfly cases used in our
study does not require extensive amounts of chemi-
cals and sieving and is therefore less costly and less
time consuming than sediment analyses (Klein et al.
2015). Hence, screening of caddisfly cases might be a
cost- and time-effective first step in assessing if
microplastics of different characteristics are present
in a freshwater habitat.
As the case-building larvae of some caddisfly spe-
cies occur in brackish and marine environments
(Haage 1968, Mouro et al. 2016), future studies
should investigate if their cases contain microplastics
and may thereby provide information on microplastic
characteristics in marine habitats. The fact that a
marine polychaete species (Nel & Froneman 2018)
and freshwater caddisfly larvae incorporate micro-
plastics inside their tube-like structures may indicate
a general microplastic fixation process present across
different ecosystems. Other freshwater organisms
such as dipterans also construct tube-like structures
(Brennan & McLachlan 1979), and in terrestrial sys-
tems, termites use soil particles for building mounds
which have sizes similar to the microplastics found in
our study (Jouquet et al. 2002). Furthermore, bag-
worm moth larvae construct self-enclosing bags
using organic and inorganic materials (Rhainds et al.
2009). These biological constructions could be ana-
lyzed for microplastics in future studies and poten-
tially provide information on ecosystem-wide pro-
cesses of microplastic fixation.
Future research should also examine the effects of
microplastics in insect constructions as indirect stres-
sors. As plastics often contain harmful additives (Her-
mabessiere et al. 2017), might increase the risk of
cancer (Erren et al. 2013) and may transport patho-
genic bacteria (McCormick et al. 2014), microplastics
in caddisfly cases may affect caddisfly larvae. For in -
stance, plastic leachates harm brown mussel larvae
(Gandara e Silva et al. 2016), and PVC (which was ob -
served in the caddisfly cases) leads to increased mor-
tality in barnacle larvae (Li et al. 2016). We conclude
that caddisfly larval cases may have the potential to
be used as freshwater microplastic bioindicators.
However, future studies should analyze micro plastic
contents of cases from different caddisfly species and
from habitats differing in plastic pollution levels.
Acknowledgements. We thank Georg Reifferscheid for pro-
viding the pressure filtration unit, and Friederike Stock and
Niklas Arendt (Department of Biochemistry and Ecotoxicol-
ogy, Federal Institute of Hydrology, BfG) for lab assistance,
Barbara Anderer and Esther Behring (Department of Animal
73
Aquat Biol 28: 67–77, 2019
Ecology, BfG) for caddisfly identification, Esther Behring for
field assistance and Bettina Salinus (Department of Animal
Ecology, BfG) for lab assistance. We also thank 4 anonymous
reviewers for their constructive comments on the original
manuscript. This research did not receive any specific grant
from funding agencies in the public, commercial or not-for-
profit sectors.
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Fig. A1. Examples of microplastics found in Lepidostoma basale cases. (A) Blue polypropylene (PP) fragment, (B) yellow acry-
lonitrile-butadiene-styrene (ABS) film, (C) transparent polyethylene (PE) sphere, (D) black PP fiber, (E) red thermo plastic
polyurethane (TPU) film, (F) orange PP film
Appendix. Examples of microplastics, µFTIR spectra, and a picture of the analyzed caddisfly species.
Ehlers et al.: Microplastics in caddisfly larval cases 77
Fig. A2. Spectra of microplastics (red spectra) measured with micro-Fourier-
transform infrared spectroscopy (µFTIR) in attenuated total reflectance (µATR)
mode. The blue spectra are reference spectra from the Bruker spectra database.
PE: polyethylene; PA: polyamide; PP: polypropylene
Fig. A3. Case-bearing Lepidostoma basale larva that had
microplastics fixed in its case which are not visible to the
naked eye
Editorial responsibility: Victor Benno Meyer-Rochow,
Oulu, Finland
Submitted: January 24, 2019; Accepted: April 18, 2019
Proofs received from author(s): July 6, 2019
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... Hermit crabs have also been documented using litter as mobile homes instead of shells (Lavers and Bond, 2017). Similarly, case building caddisfly have been found to incorporate microplastics and other artificial particles into their cases (Ehlers et al., 2019), which could affect the physical properties of their cases (Ehlers et al., 2020). ...
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... As aerial insectivores, the preferred prey of tree swallows are caddisflies, dragonflies, damselflies and mayflies that develop in freshwater systems before emerging as adults. Microplastics and particles are found in these and other macroinvertebrates (e.g., Ehlers et al., 2019;Windsor et al., 2019;Simmerman and Coleman Wasik, 2020;Garcia et al., 2021). Since tree swallows are prey for other predators, and trophic transfer of microplastics is known to occur between avian species (Hammer et al., 2016), it is conceivable that tree swallows may be a source of microplastics when consumed by higher trophic predators. ...
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