The effect of planktivorous fish (juvenile Perca fluviatilis) on the taxonomic diversity of bacterial community colonising microplastic particles

Abstract

Very few studies have investigated the influence of fish on the degradation rate of microplastic particles (MPs). It could be expected that their effect might be not only direct, through digestion and passage through the gut, but also indirect, through the alteration of the density and composition of the bacterial community on the surface of MPs. This study aims to test the short-term effects of fish presence altering the water chemical parameters and the abundance and genetic diversity of the aquatic bacteria colonising MPs, which may contribute to their faster degradation. This was tested in a triplicate experiment, with each replicate lasting 10 days in the presence or absence of spherical MPs, polystyrene (PS) or polyethylene (PE) and the presence or absence of a perch. The animal was separated from the MPs with a plankton net. We determined the chemical parameters of the water, the number of bacteria in the water and on the MPs’ surface, the taxonomic diversity of bacteria in the water and those present on the MPs using 16S rRNA gene (16S rDNA) sequencing, and the alterations of the MPs’ surface using a scanning electron microscope. The exposure to fish increased the concentration of ammonium, nitrates, and orthophosphates and the number of bacteria in the water, which in turn resulted in an increase in the abundance of bacteria and the number of taxa at the family level on the MPs’ surface. The positive effect of fish exudates on the abundance of bacteria on MPs’ surface was greater in the case of PE than in PS. The observed effects did not affect the MPs’ surface in any of the types of plastic during the experiment, but they may play a significant role in MPs decomposition over a longer than applied time period.

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The effect of planktivorous fish (juvenile
Perca fluviatilis) on the taxonomic diversity
of microplastic particles-colonized bacterial
community
M. Pyznar, P. Maszczyk, B. Kiersztyn, K. Dąbrowski, M. L. Zebrowski, J.-S. Lee
& E. Babkiewicz
To cite this article: M. Pyznar, P. Maszczyk, B. Kiersztyn, K. Dąbrowski, M. L. Zebrowski, J.-S.
Lee & E. Babkiewicz (2023) The effect of planktivorous fish (juvenile Perca fluviatilis) on the
taxonomic diversity of microplastic particles-colonized bacterial community, The European
Zoological Journal, 90:1, 414-430, DOI: 10.1080/24750263.2023.2217200
To link to this article: https://doi.org/10.1080/24750263.2023.2217200
© 2023 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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The effect of planktivorous sh (juvenile Perca uviatilis) on the
taxonomic diversity of microplastic particles-colonized bacterial
community
M. PYZNAR
1
, P. MASZCZYK
1*
, B. KIERSZTYN
1
, K. DĄBROWSKI
1
,
M. L. ZEBROWSKI
1
, J.-S. LEE
2
, & E. BABKIEWICZ
1,3
1
Department of Hydrobiology, Faculty of Biology, University of Warsaw, Warsaw, Poland,
2
Department of Biological
Sciences, College of Science, Sungkyunkwan University, Suwon, South Korea, and
3
Biological and Chemical Research
Centre, University of Warsaw, Poland
(Received 7 November 2022; accepted 19 March 2023)
Abstract
Very few studies have investigated the inuence of sh on the degradation rate of microplastic particles (MPs). It could be
expected that their effect might be not only direct, through digestion and passage through the gut, but also indirect, through the
alteration of the density and composition of the bacterial community on the surface of MPs. This study aims to test the short-
term effects of sh presence altering the water chemical parameters and the abundance and genetic diversity of the aquatic
bacteria colonising MPs, which may contribute to their faster degradation. This was tested in a triplicate experiment, with each
replicate lasting 10 days in the presence or absence of spherical MPs, polystyrene (Ps) or polyethylene (Pe) and the presence or
absence of a perch. The animal was separated from the MPs with a plankton net. We determined the chemical parameters of the
water, the number of bacteria in the water and on the MPs’ surface, the taxonomic diversity of bacteria in the water and those
present on the MPs using 16S rRNA gene (16S rDNA) sequencing, and the alterations of the MPs’ surface using a scanning
electron microscope. The exposure to sh increased the concentration of ammonium, nitrates, and orthophosphates and the
number of bacteria in the water, which in turn resulted in an increase in the abundance of bacteria and the number of taxa at the
family level on the MPs’ surface. The positive effect of sh exudates on the abundance of bacteria on MPs’ surface was greater in
the case of Pe than in Ps. The observed effects did not affect the MPs’ surface in any of the types of plastic during the experiment,
but they may play a signicant role in MPs decomposition over a longer than applied time period.
Keywords: Bacteria, biodegradation, sh associated microbiota, sh exudates, freshwater sh, genetic diversity, microplastics
1. Introduction
Since the invention of organic polymers in the early
1900s, annual production of those light, multipur-
pose, insulation, and cheap materials has consis-
tently increased, reaching 368 million tons in 2019
(Plastics Europe, and EPRO 2021). It has long been
known that plastic accumulates in the environment
due to improper waste disposal practices (Kenyon &
Kridler 1969). However, until 1972, the main focus
was on larger plastic items. It was only
E. J. Carpenter who noticed the microplastics
contamination (Carpenter et al. 1972), although
the term itself was coined in an article investigating
traces of plastic fragments and bres in marine habi-
tats, entitled Lost at Sea: Where Is All the Plastic?
(2004), which ignited the microplastics research
boom.
Microplastics are any synthetic solid particles or
polymeric matrices with a regular or irregular shape
and a size ranging from 1 µm to 5 mm (Cole et al.
2011); other operationally dened size fractions are
macroplastics (≥5 mm; Moore 2008; Lechthaler
*Correspondence: P. Maszczyk, Department of Hydrobiology, Faculty of Biology, University of Warsaw, Warsaw, Poland. Email: p.maszczyk@uw.edu.pl
The European Zoological Journal, 2023, 414–430
Vol. 90, No. 1, https://doi.org/10.1080/24750263.2023.2217200
© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been
published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

et al. 2020) and nanoplastics (<1 µm; Klaine et al.
2011; Gigault et al. 2018; Maszczyk et al. 2022a
2022b), of either primary or secondary manufactur-
ing origin (purposely produced in microscopic sizes
or degraded from larger plastic fragments) (Cole
et al. 2011). Polypropylene, polyethylene, polystyr-
ene, polyvinylchloride, and polyethylene terephtha-
late are the most commonly detected microplastic
contaminants in environments around the globe
(e.g. Ng & Obbard 2006; Hwang et al. 2020;
Stanton et al. 2020). Microplastic particles (MPs)
are commonly found in environments ranging from
marine (Cole et al. 2011) and freshwater (Eerkes-
Medrano et al. 2015), through soil ecosystems
(Chae & An 2018), even in hard-to-reach places
such as the Mariana Trench (Peng et al. 2018),
Arctic and Antarctic (Bessa et al. 2019) or in the
sediments of remote mountain lakes (Allen et al.
2019). MPs are also present in food items such as
table salt (Danopoulos et al. 2020) and beer (Shruti
et al. 2020) and were also found in animals (Browne
et al. 2008), including human (Ragusa et al. 2021)
tissues. The earliest traces of microplastics in living
organisms were found in the digestive tissues of
freshwater sh in a museum collection dating back
to the 1950s (Hou et al. 2021). Due to the increas-
ing worldwide production of plastic materials, the
concentrations of MPs in the environment are
increasing, and predictive models show that this
trend will only accelerate in the coming years if
appropriate mitigation measures are not taken (van
Wijnen et al. 2019).
Currently, plastic pollution is receiving world-
wide attention due to emerging discoveries regard-
ing its impact on the environment and human
health. The inuence of microplastic pollution
was conrmed at all integrative levels of biological
organisation, including genetic (Sun et al. 2021),
cellular (Prinz & Špela 2020), physiological
(Franzellitti et al. 2019), organismal (Schrank
et al. 2019), population (Amariei et al. 2021)
and community (Lozano & Rillig 2020;
Zebrowski et al. 2022). The effect may differ
depending on particle degradability (biodegrad-
able or non-biodegradable; Tang et al. 2021;
Zebrowski et al. 2022), size and shape
(Covernton et al. 2019), chemical properties (Xu
et al. 2018), colour (Chen et al. 2020) and con-
centration (Sun et al. 2022). However, the
reported effects in microplastic studies range
from no observed toxicity to observations of
a variety of toxicological endpoints (Booth &
Sřrensen 2020). Furthermore, the concentrations
used in the studies that found the effect usually far
exceed those found in the natural environment
(Lenz et al. 2016; Ockenden et al. 2021).
It has been demonstrated that various living organ-
isms can affect the qualitative and quantitative (crush-
ing MPs into a larger number of smaller fractions)
characteristics of MPs. The presence of organisms
can affect the circulation of MPs in the environment,
including their removal from the water column by
deposition in sediments (Pukos et al. 2023) and trans-
fer within food webs (Elizalde-Velázque et al. 2020).
Biotic degradation of plastics can occur directly: phy-
sically (by biting or chewing (So et al. 2022)) or che-
mically (enzymes during digestive degradation
(Dawson et al. 2018; Cornwall 2021)).
Among the life forms examined in this context,
microorganisms have received the most attention
(Bahl et al. 2020; Matjašič et al. 2021; Venkatesh
et al. 2021). Despite plastic’s hydrophobicity, its
possession of stable covalent bonds and its high
molecular weight, it has been suggested that, due
to evolutionary selective pressure, the more the
environment is exposed to plastic pollution, the
higher its plastic-degrading potential (Yoshida
et al. 2016). Plastic degrading bacteria have been
discovered in habitats ranging from the open ocean
to insect (Danso et al. 2019) and clitellate (Huerta
Lwanga et al. 2018) digestive tracts.
Microbial plastic degradation can be divided into two
categories: direct (a microbe using plastic as a source of
nutrients) or indirect, causing deterioration (alteration
of physical and chemical traits) or degradation (frag-
mentation) of a polymer through exposure to
a microorganism’s metabolic products (Singh &
Sharma 2008). Indirect biotic degradation might as
well occur by organism-associated microbial activity
(Widyananto et al. 2021). However, the indirect type
of biotic degradation is the least studied. Studies on
free-living and animal-associated bacterial plastic degra-
dation are scarce; only one study conrmed the effect of
coral-associated bacteria (Widyananto et al. 2021).
According to a systematic literature review on bio-
degradation studies on synthetic plastics, most of the
bacteria claimed to possess those properties belong to
the phyla Proteobacteria (48.0%), Firmicutes
(37.4%), and Actinobacteriota (9.8%). Other phyla,
although with rare mention of plastic-degrading
strains, are as follows: Acidobacteriota,
Actinobacteriota, Gemmatimonadota, Bacteroidota,
Cyanobacteria, Fusobacteriota, Deinococcota and
Nitrospirota. The most common genera mentioned
were Pseudomonas, Bacillus and Brevibacillus
(Matjašič et al., 2021).
MPs are good substrates for microbial colonisa-
tion due to their low biodegradability, high surface-
area-to-volume ratio, and hydrophobicity (Murphy
Fish presence versus bacteria on microplastics 415

et al. 2020), because of this they provide new micro-
bial niches in aquatic environments (Yang et al.
2020). The colonisation of microplastics is well
documented and occurs within a few days
(Harrison et al. 2014). Apart from outside factors,
like temperature or water ow speed, microplastic
characteristics, such as surface roughness (Kerr &
Cowling 2003) and polymer type (Frère et al. 2018)
inuence the process of biofouling and the composi-
tion and structure of that bacterial community. It is
well established that bacteria growing in biolms are
usually physiologically and taxonomically distinct
from free-living bacteria. Biolms are typically char-
acterised by dense clusters of bacterial cells. These
cells secrete extracellular polymeric substances that
hold the cell aggregates together in a biolm matrix
(Hall-Stoodley et al. 2004).
In the natural environment, MPs come into con-
tact with a variety of bacterial strains, including
bacteria associated with freshwater sh, which have
not been studied in-depth in the context of MPs’
colonisation and degradation potential and their
inuence on the genetic diversity of the MPs’ bac-
terial community. Fishes possess a diverse asso-
ciated bacterial community (Wilson et al. 2007),
which increases the possibility that some of these
bacterial strains could potentially have
a biochemical or physical impact on MPs.
Feeding habits and genotype clearly inuence the
gastrointestinal microbiota of sh, but
Proteobacteria (including Enterobacter, Aeromonas,
Acinetobacter, Vibrio, Pseudomonas, and
Achromobacter) and Firmicutes are the dominant
phyla in all sh species (e.g. Cahill 1990).
Bacteroidota (e.g. Flavobacterium) and
Actinobacteriota (e.g. Corynebacterium and
Micrococcus) are other dominant phyla, except in
carnivorous sh (Cahill 1990; Li et al. 2014).
Other dominant microbiota in the intestine of fresh-
water sh are represented by Fusobacteriota and
Tenericutes (Kashinskaya et al. 2019). This general
pattern was conrmed for the microbiota of the
perch (Perca uviatilis), which is dominated by the
phyla Proteobacteria, Firmicutes and
Fusobacteriota, as well as Tenericutes
(Kashinskaya et al. 2019). As the composition of
the microbial communities associated with the sh
is dominated by the phyla in which bacterial taxa
having the ability to break down MPs were identi-
ed most often (e.g. Proteobacteria, Firmicutes,
Actinobacteriota, Bacteroidota, and
Fusobacteriota), it is very likely that the sh pre-
sence may result in an increase in the contribution
of those taxa on the surface of MPs, which in turn
may result in an increase in the degradation rate of
MPs. The composition of the microbiota of perch at
the genus level varied between different studies,
being dominated either by Myroides,
Prochlorococcus, Anabaenopsis, Cetobacterium and
Fusobacteriaceae (Zha et al., 2018) or Aeromonas
spp. and Pseudomonas spp. (Goldschmidt-Clermont
et al., 2008) or Escherichia spp., Bacillus spp.,
Aerobacter spp., and Proteus spp. (Horsley 1977).
So far, the bacteria used in biodegradation studies
have primarily come from plastic-polluted environ-
ments or animal intestines (Matjašič et al. 2021),
without taking into account the degradation poten-
tial of animal-associated free-living bacteria. Due to
the fact that the presence of aquatic organisms,
including sh, changes both the water chemistry
(e.g. the content of nutrients and pH) and the taxo-
nomic composition of microorganisms, it might
affect the rate of MPs’ degradation. These assump-
tions have yet to be experimentally validated.
Therefore, the aim of our research was to test several
hypotheses concerning the effect of sh presence on
the bacterial community colonising MPs and in
turn, their potential to increase the decomposition
rate of MPs. The rst: the presence of a sh affects
the quantitative (density) and qualitative (taxon-
omy) characteristics of free-living bacteria and bac-
terial communities colonising the MPs’ surface by
changing water chemistry parameters and introdu-
cing new bacterial strains. The second: there is
a notable difference in sh inuence on bacterial
communities attached to the surface of different
types of MPs. The third: the presence of the sh
indirectly (through alterations of the water chemistry
and bacterial composition on MPs particles) inu-
ences the quality features of the MPs’ surface, mea-
sured as the diameter and circumference of the
particles.
2. Materials and methods
2.1. Experimental animals
Juvenile European perch (Perca uviatilis) were col-
lected by seining in Lake Roś, located in the
Masurian Lake District in Poland, a region where
microplastic freshwater contamination has been
conrmed (Kaliszewicz et al. 2020). Planktivorous
sh are abundant in this postglacial, dimictic,
eutrophic lake (Jachner 1991) that is also exposed
to strong anthropogenic pressure. During the day,
juvenile perch, which are planktivorous, migrate
there to the littoral, where they prey mainly on
copepods (Jachner 1991). All captured sh mea-
sured less than 7 cm in length. Before the experi-
ment, the animals were kept for 6 days in an
416 M. Pyznar et al.

aquarium with preconditioned tap water (homoge-
nised and ltered through a 2 µm mesh lter), an air
pump, and an aquarium heater, in order to accli-
mate them to the laboratory environment, and then
randomly selected for appropriate variants. The
experimental procedure was approved by the First
Warsaw Local Ethics Committee for Animal
Experimentation (Permit protocol No. 1350P1/
2022).
2.2. Microplastics
In the experiments, we used two different types of
synthetic, non-biodegradable polymers, with
a density similar to that of natural water (Zhou
et al. 2021), in the form of microspheres. The rst
one was polystyrene (Ps), slightly heavier than
water, which causes its faster sedimentation
(d = 1.05 g × cm
−3
), with a mean diameter of
258.3 µm, catalogue number PS250K (Lab261®,
USA). The second type of plastic was a high-
density polyethylene (Pe), slightly lighter than
water (d = 0.96 g × cm
−3
), which causes oating
close to the surface, with a mean diameter of
275 µm, catalogue number 110616–4 (Cospheric®,
UK). Those two polymers are among the materials
most commonly identied as microplastic pollutants
(Wu et al. 2016).
2.3. Experimental setup
The experimental system for sh was comprised of
eighteen 5 L glass containers placed randomly in
a water bath (L = 220, W = 40, H = 40 cm,
V = 352 L) holding internal submersible water-
heaters (Aquael® Neoheater 150 W) and water
pumps (Aquael® Circulator 500), inside a room,
which allowed to maintain stable temperature
(21 ± 0.2°C) and light conditions. The light inten-
sity reected the summer photoperiod (16 L:8°D,
10.0 ± 0.5 µmol°×°m
−2
× s
−1
, just under the surface
of each of the containers) measured with Li-Cor 189
quantum sensor measuring radiance (Li-Cor
Biosciences®). The potential inuence of external
light sources was eliminated by wrapping the walls
of the water bath with a black, opaque lm. The
water bath was lled with aerated tap water, which
was conditioned 6 days before the experiments (l-
tered through 2 µm mesh lters). The experimental
containers were connected to the 8-channel aeration
pumps (Hailea® ACO-9630) via pipes ending with
glass tubes in order to minimise the contact of
experimental plastic with laboratory plastic. In each
of the 18 containers, a 150 µm net attached to
a U-shaped frame was placed, which separated
MPs from the animal in order to avoid the possibi-
lity of potential MP degradation as a result of pas-
sage through the digestive system. The intentional
placement of the net in control tanks was aimed at
ensuring uniform experimental conditions.
A concentration of 3000 MPs × L
−1
was utilized to
promote adequate surface area for bacterial prolif-
eration. During the experiment, we minimised the
contact of the experimental organisms with plastic
objects other than those used as a factor in the
experiment. For example, all containers, tubes, and
frames that came into contact with water in each
tank were made of glass.
2.4. Experimental procedure
The experiment was conducted in July 2021 at the
Hydrobiological Station in Pilchy, University of
Warsaw, thanks to the authorities of the station.
Eighteen containers were the combination of six
variants replicated simultaneously three times, dur-
ing a 10-day experiment. Each day at 10 a.m., the
sh were fed with frozen bloodworms, and tempera-
ture and oxygenation were measured using an oxy-
gen probe (YSI ProODO). Any leftover food debris
was immediately removed. After 10 days, we col-
lected samples of water and MPs. We examined
the variants with the presence or absence of a sh
and the presence of two types of microplastics. The
experiment included the following variants: a sh
and Ps particles (F-Ps), a sh and Pe particles
(F-Pe), Pe particles only (Pe), Ps particles only
(Ps), sh only (F) and water only (C) as a control.
A randomly selected perch was placed in each con-
tainer with a sh variant (F, F-Pe and F-Ps). The
experimental conditions, i.e. the size and structure,
the amount of water, light exposure, temperature, and
oxygen supply, were the same for each glass container,
representing a single replicate of one of the six variants.
At the end of the experiment, in order to examine
changes in water chemistry caused by the presence of
a sh or possibly by the presence of MPs, concentra-
tions of ammonium nitrogen, nitrates, and orthopho-
sphates were measured in each of the 18 glass
containers. For the analysis of ammonium ions, we
used a method proposed by Holmes et al. 1999. For
the analysis of total nitrogen, we used commercially
certied Merck-Millipore cell tests (Spectroquant®
nitrogen (Total) Cell Test 1.14537) according to the
manufacturer’s instructions, using the Merck
Spectroquant Pharo 300 spectrophotometer. The
orthophosphate concentrations were determined
using the molybdenum method (Nagul et al. 2015)
using a Shimadzu UV-VIS 1201 spectrophotometer.
Fish presence versus bacteria on microplastics 417

For the calculation of bacteria abundance, sam-
ples of MPs and water were conserved with 2%
formalin and processed according to Porter and
Feig (1980). Free-living bacteria and bacteria
attached to MPs were stained with DAPI
(4’,6-diamidino-2-phenylindole, 10 min, temp.
24°C, nal DAPI concentration – 1 µg L
−1
), sus-
pended on the surface of 0.2-µm, black polycar-
bonate membrane lters (Millipore) by ltration
and then examined under Nikon epiuorescence
E450 microscope equipped with Nikon Digital
Camera DXM 1200 F and NIS elements software
(Nikon). In water samples, free-living bacteria
were counted in 10 randomly selected 10 elds
of view. In MPs’ samples, biolm bacteria were
counted on the surface of 10 randomly selected
particles and expressed as the number of bacteria
per µm
2
.
For taxonomic identication of bacteria on MPs
nearly 2000 particles were collected through
a sterilised strainer and immediately frozen for
further DNA isolation. For taxonomic identica-
tion of free-living bacteria 200 ml of water from
the experimental tank was ltered through the
0.2-µm polycarbonate lter (Merck Isopore™
Membrane Filters), and the lter was also placed
in the Eppendorf probe and frozen. The lters were
cut into small pieces prior to DNA isolation. DNA
extraction was performed using the Gene MATRIX
Soil DNA Purication Kit (EURx, Poland) accord-
ing to the manufacturer’s instructions.
In order to examine the surface and determine
any potentially visible surface alterations caused by
the bacteria and biolm presence, we used scanning
electron microscopy (SEM). The images of the par-
ticles were obtained in the Laboratory of Electron
and Confocal Microscopy at the Faculty of Biology,
University of Warsaw. The dried microplastic parti-
cles were mounted on a SEM mounting stub, sput-
ter-coated with gold using the POLARON SC7620
metal sputtering machine by Microtech, and exam-
ined with the use of a LEO 1430VP ZEISS scanning
electron microscope with 400 × magnication.
For the ratio of the changed surface to
unchanged, for the number of visible bacteria on
the surface and for the presence or absence of bio-
lm on SEM photographs, we used ImageJ 1.53 g
software (Schindelin et al. 2012).
2.5. Data analyses
The statistical analysis was performed using the
R platform (v.4.2.0). In all statistical tests, the signi-
cance level was set to α = 0.05. Before selecting an
appropriate test, we checked the assumption for
ANOVA (analysis of variance) by performing the
Shapiro–Wilk normality test and Levene’s test for
homogeneity of variance.
To test the differences in the abiotic parameters
(temperature and oxygen concentration) between all
the experimental treatments, we performed a three-
way mixed ANOVA using the “ezANOVA” function
(the “ez” package v.4.4–0). The Greenhouse–Geisser
correction was applied if the sphericity checked by
Mauchly’s test was violated. The presence of the sh
and MPs (Ps or Pe) were set as between-subject factors,
and time as a within-subject factor. To test the effects of
the presence of a sh and MPs (Ps or Pe) on the
number of bacteria in water, the chemical parameters
of water quality (the concentration of ammonium nitro-
gen, nitrates and orthophosphates) and the effect of the
presence of a sh on the number of bacteria on different
types of MPs and the microplastics’ physical properties
(diameter and circumference), we conducted a two-
way ANOVA using the “aov_car” function (“afex”
package v.1.1–1).
To determine the taxonomic composition of the
bacterial community, Bray–Curtis-based NMDS
(nonparametric multidimensional scaling) was per-
formed on relative abundance of bacteria domain
representatives on the family level.
Planned contrasts for estimated marginal means
(EMMs; the “emmeans” package v.1.7.5) were used
to compare the effect of sh with control separately
for each of the types of microplastics on the chemi-
cal parameters of water quality, on the number of
free-living bacteria, on the number of bacteria on
MPs and on MPs’ physical properties. The “holm”
p-value adjustment was applied to control Type
I error ination as a result of multiple testing.
3. Results
3.1. Microplastics physical properties – diameter and
circumference
The presence of sh did not affect the diameter and
circumference of MPs at the end of experiments
(Table I, Figure 1(a, b)); however, the circumfer-
ence depended on the type of MPs (Table I,
Figure 1(a, b)). The diameter and circumference of
Pe (in relation to Ps) were greater in both the pre-
sence and absence of sh (Table S1, Supplementary
materials, Figure 1(a, b)).
3.2. Experimental conditions
Oxygen concentration was affected by the presence
of the sh (Table II). The effect was apparent in
418 M. Pyznar et al.

both the absence of MPs and in the presence of Pe
(Figure 2). The temperature value and oxygen con-
centration were not affected by the presence of MPs
(Table II, Figure 2).
3.3. Chemical parameters of water quality
Generally, the presence of a sh had a signicant
effect on the concentrations of orthophosphates and
ammonium ions, however there was no signicant
effect of sh on the total nitrogen concentration
(Table II). The presence of MPs did not affect any
of the chemical parameters of water (Table II). The
concentration of orthophosphates was greater in the
presence of sh than in its absence in the treatments
with MPs (Table S2, Figure 2). The concentration
of orthophosphates was also signicantly higher in
the presence of the sh and Pe, compared to the
presence of the sh without Pe (Table S2, Figure 2).
In the presence of the sh, the concentration of
orthophosphates was signicantly higher in the
treatment with Pe than in the treatment with Ps
(Table S2, Figure 2).
The ammonia concentration was higher in the
presence of the sh (both with and without MPs,
independently of their type) compared to the control
(Table S2, Figure 2), and was greater in the pre-
sence of sh than in its absence in both types of MPs
(Table S2, Figure 2). Moreover, the concentration
of ammonia was signicantly higher in the presence
of the sh and Ps, compared to the presence of the
sh without Ps (Table S2, Figure 2). The total
nitrogen concentration was signicantly higher in
the treatment with sh and Ps compared to the
treatment with Ps without sh (Table S2, Figure 2).
3.4. Number of bacteria
The number of free-living bacteria was affected by
the sh presence and by the type of MPs (Table III,
Figure 3(a)). In all variants with the presence of the
Table I. The results of two-way ANOVA to test the effect of the presence of the sh (F) and MPs type on MPs’ physical properties
(diameter and circumference) at the end of experiments. Statistically signicant (α = 0.05) differences are marked with bold (df – degrees
of freedom. SS – Sum of Squares. F – F-statistics, P – p-value).
Parameter Factor; Interaction df SS F P
Microplastics diameter F 1 70 0.512 0.478
MPs type 1 1797 13.186 0.478
F × MPs type 1 38 0.275 < 0.001
Microplastics circumference F 1 858 0.568 0.455
MPs type 1 22,188 14.710 < 0.001
F × MPs type 1 21 0.014 0.906
Figure 1. (a) Selected parameters characterising the surface of the MP: diameter and circumference at the end of the experiments (mean ±
1SD) in all of the treatments. Statistically signicant differences between treatments are denoted with different letters, (b) SEM images of
MPs at the end of the experiments in all of the treatments. Magnication ×400.
Fish presence versus bacteria on microplastics 419

sh (with and without each of the types of MPs) the
number of bacteria was greater compared to the
control (Table S3, Figure 3(a)). In the Pe treat-
ments, the number of bacteria was greater in the
presence than in the absence of sh (Table S3,
Figure 3(a)).
In the case of the number of bacteria on the
MPs’ surface, we observed both the effect of the
sh presence and the type of MPs (Table III,
Figure 3(b)). The number of bacteria on the
MPs’ surface was greater in the presence than in
the absence of sh and at the surface of Ps than Pe
particles (Table. S3, Figure 3(b)). The analysis of
the planned contrasts revealed that the effect of the
sh was signicant in each of the plastic types, and
the difference between MP types was signicant in
both the presence and absence of sh (Table S3,
Figure 3(b)).
3.5. Taxonomic composition of bacterial community
The most abundant bacterial taxa at the phylum
level in all samples were Proteobacteria, followed
by Bacteroidota and Verrucomicrobiota. In free-
living bacterial communities, greater contribution
of Verrucomicrobiota and Actinobacteriota was
apparent in the sh than in no-sh media. The
most apparent difference between free-living
bacteria and bacteria attached to MPs particles was
the lower contribution of Bacteroidota and
Proteobacteria and the greater contribution of
Bdellovibrionota in the latter than in the former.
Moreover, Actinobacteriota were present in water
samples in the sh treatments but not on the surface
of particles, neither in sh nor in no-sh treatments.
Also, Cyanobacteria was present only in free-living
bacteria fraction in several treatments (Pe, F-Pe and
Ps), but not on the surface of MPs (Figure 4(a, b)).
On the MPs’ surface, greater contributions of
Verrucomicrobiota and Bacteroidota and a lower con-
tribution of Proteobacteria were in the sh medium in
relation to the no sh medium. On the surface of Ps
particles in the sh medium, the signicant phylum was
Fusobacteriota, and on the surface of Pe particles in the
sh medium, the signicant phyla were Myxococcota
and Acidobacteriota (Figure 4).
The number of taxa at the family level of the free-
living bacteria was lower in the sh medium, and
greater in the presence of MPs (in both sh and no
sh medium) in relation to the control (Figure 5(a)).
The number of taxa at the family level of bacteria on
MPs’ surfaces was greater in the presence than in
the absence of sh in both types of MPs (Figure 5
(b)). In the absence of sh, the number of taxa on
MPs was lower compared to the number of taxa of
free-living bacteria. In the presence of sh, this rela-
tion was reversed.
Table II. Results of the three-way mixed ANOVA to test the effect of all experimental treatments (F – presence of the sh, type of MPs
and Ti – time) on the abiotic parameters (temperature and oxygen concentrations) and the results of the two-way ANOVA to test the
effect of the presence of the sh and type of MPs on the chemical parameters of water quality (the concentrations of orthophosphates,
ammonium ions, and total nitrogen). Statistically signicant (α = 0.05) differences are marked with bold (df – degrees of freedom, F –
F-statistics, P – p-value).
Parameter Type of parameter Factor; Interaction df F P
Abiotic
parameters
Temperature F 1 0.010 0.919
MPs type 2 0.447 0.649
Ti 9 1.138 0.185
F × MPs type 2 0.034 0.965
F × Ti 9 0.239 0.877
MPs type × Ti 18 0.478 0.962
Oxygen
concentrations
F1 11.358 0.006
MPs type 2 1.744 0.216
Ti 9 2.088 0.076
F × MPs type 2 0.314 0.735
F × Ti 9 1.296 0.247
MPs type × Ti 18 1.343 0.176
Chemical
parameters
Orthophosphates F 1 8.224 0.014
MPs type 2 0.183 0.834
F × MPs type 2 6.741 0.011
Ammonia F 1 3.982 < 0.001
MPs type 2 0.019 0.981
F × MPs type 2 2.943 0.091
Total nitrogen F 1 1.328 0.272
MPs type 2 1.728 0.219
F × MPs type 2 2.556 0.119
420 M. Pyznar et al.

Figure 2. Experimental conditions: temperature and oxygen concentration as the mean (± 1SD) for all of the experiments in each of the
experimental days, and orthophosphates, ammonium ions and total nitrogen concentrations at the end of the experiments (mean ± 1SD)
in all of the treatments: the control (c), and the presence of the sh (f), polyethylene (Pe), sh and polyethylene (F-Pe), polystyrene (Ps)
and sh and polystyrene (F-Ps). Statistically signicant differences between treatments are denoted with different letters.
Fish presence versus bacteria on microplastics 421

The Simpson 1-D index for the free-living bacteria
was relatively higher in the presence of sh. The effect
of MPs was only apparent in the case of Ps (Figure 5
(c)). The Simpson 1-D index for samples collected
from MPs’ surface was signicantly greater in the pre-
sence of sh compared to its absence for both types of
MPs (Figure 5(d)). In the absence of sh, the Simpson
1-D index for the taxa on MPs was lower compared to
its value for free-living bacteria. In the presence of sh,
this relationship was reversed.
The NMDS of the values of the Bray–Curtis
index calculated on the basis of the relative
Table III. The results of a two-way ANOVA to test the effect of the presence of the sh (F) and microplastics particles (MPs) on the
number of free-living bacteria in the experimental media and on the number of bacteria on MPs at the end of the experiment. Statistically
signicant differences (α = 0.05) are marked with bold (df – degrees of freedom, SS – Sum of Squares, F – F-statistics, P – p-value).
Parameter Factor; Interaction df SS F P
Number of free-
living bacteria
F 1 6.334 12.211 < 0.001
MPs type 2 1.310 1.262 0.004
F × MPs type 2 0.158 0.152 0.318
Number of
bacteria on MPs
F 1 0.371 31.884 < 0.001
MPs type 2 0.258 22.221 < 0.001
F × MPs type 2 0.007 0.655 0.002
Figure 3. The number of bacteria in water (A), and on the MPs’ surface (B) at the end of the experiments (mean ± 1SD) in all of the
treatments. Statistically signicant differences between treatments are denoted with different letters.
Figure 4. The relative abundances (top 13 most abundant ASVs) of the dominant bacteria taxa at the phylum level in water (a) and on the
microplastic particle surface (b) at the end of the experiments (mean ± 1SD) in all of the treatments. If the frequency was lower than 0.2%,
the taxa were assigned to the “Other” category.
422 M. Pyznar et al.

abundances of taxa on family level in water and on
the MP surface revealed ve distinct groups of sam-
ples: (1) control water samples, (2) water samples
from variants in the presence of sh and Ps or Pe
and in the absence of sh and presence of Pe, (3) PS
water samples in the absence of sh, (4) samples on
the surface of MPs of both types in the absence of
sh, (5) samples on the surface of MPs of both types
in the presence of sh (Figure 6). In general, at the
family level the taxonomic composition of the bio-
lm bacteria community is different from that of the
free-living bacteria community in the context of dif-
ferent proportions of the same bacterial families on
the particles and in water (Figure 6).
4. Discussion
The results of our study conrmed the rst hypothesis,
as the presence of a sh affects the quantitative (density)
and qualitative (taxonomy) characteristics of free-living
bacteria and bacterial communities colonising the MPs’
surface. The effects were accompanied by a large
increase in the concentrations of orthophosphates and
ammonium ions and the nitrogen concentration was
higher in the treatment with sh and Ps. The effect of
the sh on the increase in concentrations of dissolved
mineral phosphorus and nitrogen is consistent with the
numerous theoretical (e.g. Zimmer et al. 2006; Verant
et al. 2007) and experimental (Kitchell et al. 1975;
Threlkeld 1987; Vanni & Findlay 1990) studies on
the effect of sh egestion and excretion on water chem-
istry, which revealed that it can be an important source
of nutrients in aquatic ecosystems (e.g. Atkinson et al.
2017). The potential for signicant contribution of
a sh to phosphorus recycling in limnetic systems via
excretion was rst raised by Kitchell et al. (1975), who
concluded that phosphorus excretion by bluegill
(Lepomis macrochirus) in a small lake may support algal
populations during periods of low phosphorus availabil-
ity. In other studies, it has been revealed that nutrient
release by sh might play an important role in phos-
phorus cycling and algal production (Threlkeld 1987)
and that excretion of phosphorus by sh can be equal to
or even greater than that of zooplankton (e.g. Vanni &
Findlay 1990; Carpenter et al., 1992).
It has been revealed that sh activity may increase
the abundance of free-living bacteria directly
through released nutrients and indirectly through
Figure 5. The mean number of taxa at the family level (a, b) and Simpson 1-D index (± 1SD, c, d) in water (a, c) and on the microplastic
particles surface (b, d) at the end of the experiments in all of the treatments.
Fish presence versus bacteria on microplastics 423

trophic interactions (e.g. Jeppesen et al. 1998;
Villéger et al. 2019), although most of the previous
studies did not disentangle these effects. For
instance, in the long-term study of shallow and
hypertrophic Lake Sřbygĺrd (Denmark), it has been
revealed that temporal changes in sh stock affected
through cascading effects the abundance of bacterial
communities, although this effect was less apparent
than the cascading effect on zooplankton and phy-
toplankton (Jeppesen et al. 1998). In other study, it
has been revealed that the Mediterranean sh –
Sparus aurata had a positive effect of nutrient excre-
tion on the abundance of the microbial plankton,
including bacterioplankton in the absence of large
zooplankton when their growth was limited by nutri-
ent availability (Villéger et al. 2019). As the domi-
nant organisms in our experimental media were
bacteria, it could be assumed that the increase in
their abundance was only due to direct effect i.e.
due to nutrients released by the sh. We are aware
of only a single previous study in which a similar
observation was made (Maszczyk & Bartosiewicz
2012). In this study, chemicals originating from
sh activity fertilised the environment enough for
a signicant increase in bacterial abundance, which
positively affected the growth and other life history
parameters of their consumer – the planktonic cla-
doceran Daphnia. More importantly, in our study
the greater abundance of the free-living bacteria
was parallel to a greater abundance of bacteria on
the MPs surface. To our knowledge, this is the rst
evidence in the literature concerning this issue. As
only some bacterial strains possess the ability to
decompose plastics, an increased abundance of bac-
teria in the presence of sh exudates increases the
chance of more efcient MPs decomposition.
The results of our study also revealed that the
nutrient excretion by sh altered the taxonomic
composition of the free-living bacteria, which is con-
sistent with numerous previous studies (e.g. Schaus
& Vanni 2000; Villéger et al. 2019; Molina &
Fernández 2020). For instance, it has been revealed
that the nutrient excretion by the Mediterranean
sh – Sparus aurata altered the community structure
of the microbial plankton, including heterotrophic
bacteria in the absence of large zooplankton
(Villéger et al. 2019). A similar effect was observed
by Schaus and Vanni (2000), who found that the
gizzard shad (Dorosoma cepedianum) excretion
resulted in changes in the relative abundance of
Figure 6. NMDS of the values of the Bray–Curtis index calculated on the basis of relative abundances of taxa on family level in water and
on the microplastic particle surface at the end of the experiments (mean ± 1SD) in all of the treatments, separately in each of the two
experiments for which the analysis was performed (a and b in the subscript).
424 M. Pyznar et al.

different taxa within the phototrophic unicellular
organisms, including Cyanobacteria. Although the
aforementioned studies indicated that the presence
of sh exudates differentiates bacterial composition,
they did not disentangle the direct effect of nutrients
released by the sh or the indirect effect of the
metabolites excreted by other organisms present in
the experiments. Another example of the study con-
cerned the effect of dissolved organic matter and
inorganic nitrogen release through sh (Salmo
salar) epithelium mucus on the natural bacterial
assemblages from Northern Patagonia, Chile
(Molina & Fernández 2020). The study revealed
that sh mucus can cause rapid modications in
microbial assemblages, which was apparent in the
relative increase in the abundance of Proteobacteria
and Bacteroidota. The results of our study are not
consistent with this observation, as we did not nd
any difference in the relative contribution of
Proteobacteria and Bacteroidota between the sh
and no sh media. Instead, Verrucomicrobiota and
Actinobacteriota were more numerous in the sh
media. Moreover, Actinobacteriota was specic
only to the sh media. Overall, the number of taxa
at the family level of the free-living bacteria was
lower, but their diversity was greater in the sh
medium. It could be assumed that the change in
bacterial composition in the sh media was due to
the net effect of altered physicochemical water para-
meters, including nutrients and oxygen concentra-
tions, and enrichment of the media by introducing
sh-associated bacterial strains. As different bacter-
ial strains have different environmental require-
ments, it could be expected that altered
environmental parameters due to the presence of
sh stimulate changes in interactions within and
between different bacterial taxa, having a profound
impact on the outcome of their competition (e.g.
Hibbing et al. 2009). For the same reasons,
decreased oxygen concentrations in sh treatments,
which were caused by the sh’s respiration, might
have inuenced the composition of free-living bac-
teria as well. Overall, the changes in microbial com-
position obtained in our study in the sh medium
are consistent with the broad literature concerning
the composition of sh-associated bacteria, in which
Proteobacteria is the dominant phylum in all sh
species, and Actinobacteriota are also well repre-
sented (Cahill 1990; Li et al. 2014).
In our study, the change in the taxonomic compo-
sition of free-living bacteria was accompanied by
a change in the taxonomic composition of bacterial
assemblages on the surface of the MPs. Overall, in
the bacterial assemblages on MPs particles, there was
a lower contribution of Bacteroidota, Proteobacteria,
Actinobacteriota, and Cyanobacteria and a greater
contribution of Bdellovibrionota, Fusobacteriota,
Myxococcota, Acidobacteriota, and
Verrucomicrobiota than in free-living bacteria frac-
tion. The results are consistent with the numerous
studies revealing that biolm communities on MPs
differ from the communities in the surrounding envir-
onment (e.g. Rummel et al. 2017; Ogonowski et al.
2018; Miao et al. 2019; Rosato et al. 2020).
Therefore, MPs can be considered a distinct ecologi-
cal habitat for diverse microbial communities, the
“plastisphere” (Zettler et al. 2013), potentially char-
acterised by distinct microbial and ecological func-
tions (Miao et al. 2019). Moreover, our study seems
to be the rst one concerning the alteration of the
composition of bacterial assemblages on the MPs
surface in the presence of sh. It is clear from the
study that the number of taxa and their variability in
the bacterial assemblages on the MPs surface
increased in the presence of sh. This was evident
in the increase in the relative contributions of
Fusobacteriota, Myxococcota, Acidobacteriota,
Bacteroidota and Verrucomicrobiota, which was
accompanied by a decrease in the relative contribu-
tion of Proteobacteria on the MPs surface in the sh
media in relation to no sh media, which suggests
that the presence of the sh introduces new bacterial
strains with a greater potential to create biolms.
Dominant bacterial strains in the absence of sh are
potentially being displaced competitively by sh-
associated bacteria due to the competition for sur-
face. Due to the fact that only some strains possess
the ability to decompose MPs, it is not possible to
state whether the sh exudates modify the rate of MP
decomposition based on how it affects the relative
contribution of different taxa at the phylum level.
However, as the composition of the microbial com-
munities on MPs surface associated with sh pre-
sence is more diverse, it can be assumed that the
chance that such a community contains bacteria
with the ability to decompose MPs increases.
The results also conrmed predictions of
the second hypothesis as we observed apparent dif-
ferences in the sh effect on bacterial communities
associated with the surface of different types of MPs.
The positive effect of sh was greater on the abun-
dance of the bacterial community on the surface of
Pe than on Ps. Although our study seems to be the
rst one concerning the effect of sh exudates on the
bacterial communities associated with the surface of
different types of MPs, the results of our study are
Fish presence versus bacteria on microplastics 425

consistent with previous observations that the
microbial community on MPs’ surface depends on
the polymer type (e.g. Hori et al., 2010; Fotopoulou
et al., 2012; Hossain et al. 2019; Parrish &
Fahrenfeld 2019; Rosato et al. 2020; Tu et al.
2021). For instance, it has been revealed that within
2 weeks of incubation, the bacterial abundance was
greatest on PVC, lower on Pe, and lowest on PET,
Pp, and Ps pellets (Rosato et al. 2020). Also,
Hossain et al. (2019) showed that bacterial coloni-
sation of MPs is affected by the physicochemical
properties of MPs, including their surface roughness
and the type of plastic, although they did not nd
a difference in the bacterial density at the surface of
the types of plastics used in our study (HDPE and
Ps). However, it should be pointed out that in our
study, the effect of sh was similar in terms of the
number of taxa and diversity of taxonomic composi-
tion on the surface of both types of MPs. The only
differences were apparently greater effects of sh on
the contribution of Fusobacteriota on the surface of
Ps and a greater effect of sh on the contribution of
Myxococcota and Acidobacteriota on the surface of
Pe. This is visible also on the SEM photographs
(Figure 1(b)), which indicate that Ps has
a relatively smooth surface in comparison with Pe
with a more granular structure on the surface and
with that some Fusobacteriota have strong abilities
to attach to smoother surfaces (Moses et al. 2020).
Additionally, the presence of different MPs also
had a slight but signicant effect on water chemistry,
and this effect was different in the sh and no-sh
media. That is, the presence of Pe results in an
increase in the concentrations of orthophosphates
and the presence of Ps results in an increase in the
concentrations of ammonia only in sh treatments.
This is in accordance with studies indicating that the
presence of polystyrene increases ammonia excre-
tion of black rocksh (Sebastes schlegelii; Yin et al.
2019) or disturbs it in medaka (Oryzias melastigma)
gills (Zheng & Wang 2022). Another explanation
may consist of the differential effect of MPs on the
qualitative and quantitative compositions of free-
living and associated with MPs bacterial assem-
blages (e.g. Rosato et al. 2020), which may utilise
nutrients produced by sh to a different extent.
The last hypothesis, that the presence of the sh
indirectly (i.e., through alterations of the water
chemistry and bacterial composition on MPs parti-
cles) inuences the quality features of the MPs’ sur-
face, was not conrmed, as after 10 days, there was
no signicant change in the circumference and dia-
meter of the particles of any type. However, the
changes in the physicochemical parameters of the
water as well as the quantitative and qualitative
changes in bacteria in the presence of the sh exu-
dates observed in our study suggest that a longer
exposure to the sh exudates could accelerate the
deterioration or decomposition of the MPs.
5. Conclusions
Overall, the results revealed that the presence of
a sh affected the chemical parameters of the water
and the quantitative (density) and qualitative (tax-
onomy) characteristics of free-living bacteria and
bacterial communities colonising the MPs’ surface.
Although those changes did not affect the apparent
modication of the surface of MPs, they suggest that
longer than applied in our study, exposure to sh
may have an effect on the decomposition of MPs.
To the best of our knowledge, these results provide
the rst evidence of the direct effect of aquatic ani-
mals on the plastisphere and, in turn, their signi-
cance on the self-purication of water from plastics.
Acknowledgements
All applicable institutional and national guidelines
for the care and use of animals were followed.
Funding
The research described here was nanced by
grants [2019/35/B/NZ8/04523] and [2018/31/N/
NZ8/03269] from the Polish National Science
Centre.
Disclosure statement
No potential conict of interest was reported by the
author(s).
Supplementary material
Supplemental data for this article can be accessed
online at https://doi.org/10.1080/24750263.2023.
2217200.
ORCID
P. Maszczyk http://orcid.org/0000-0002-1738-419X
B. Kiersztyn http://orcid.org/0000-0002-8762-4873
M. L. Zebrowski http://orcid.org/0000-0002-1554-
6474
J.-S. Lee http://orcid.org/0000-0003-0944-5172
E. Babkiewicz http://orcid.org/0000-0003-2398-8448
426 M. Pyznar et al.

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