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ISJ 19: 136-149, 2022 ISSN 1824-307X
REVIEW
Current research on the effects of plastics pollution in marine and freshwater aquatic
invertebrates
V Pirillo, N Baranzini*
Department of Biotechnology and Life Sciences, University of Insubria, Via J. H. Dunant 3, 21100 Varese, Italy
This is an open access article published under the CC BY license Accepted October 18, 2022
Abstract
Plastics pollution in the aquatic environments represents one of the most critical worldwide issue.
Every year, million tons of waste products are reversed both in marine and freshwaters, persisting for
long timings and determining serious effects to living organisms. Here, these synthetic materials are
fragmented in small particles, known as micro- and nanoplastics, under the effects of both biotic and
abiotic factors. Due to their characteristics, smaller fragments are easier accumulated inside animal
tissues and organs, risking to enter in the trophic chain. To date, despite the current situation, only a
small amount of research has been conducted, especially on aquatic invertebrates, which can
represent a suitable model for better analyzing the possible plastics dangerous effects. For this
reason, in the present review we aim to collect the recent information about micro and nanoplastics
effects on both marine and freshwaters invertebrates. In particular, we do not only focus the attention
on the obtained results, but also, we report the main experimental methods and particle types used.
Regardless of the heterogeneity present in literature, the actual data result fundamental for setting up
the future research.
Key Words: plastic pollution; invertebrates; microplastics; nanoplastics; plastics uptake
Introduction
Plastics are lightweight, versatile, resistant, and
cheap materials. Thanks to their qualities, these
polymers can be easily manufactured for a variety of
civil and industrial applications. Since the middle of
the last century, their peculiar physical and chemical
characteristics made these synthetic materials
hardly replaceable and, for this reason, employed in
almost all industrial areas, such as food and textile
industries, healthcare, transports, electronics, and
telecommunications (Gourmelon, 2015). Moreover,
due to their multiple use and the low costs, the total
amount has significantly increased over the past 70
years, passing from 1,5 million tons in 1950 to 359
million tons in 2020 (Tournier et al., 2020).
However, the wide distribution and the
indiscriminate use, results into threatening
consequences, making the environmental plastics
pollution one of the most serious ecological
problems at the global level (Guzzetti et al. 2018;
Alimba and Faggio 2019; Prokić et al. 2019). In
particular, most of the monomers that constitute
_________________________________________
Corresponding author:
Nicolò Baranzini
Department of Biotechnology and Life Sciences
University of Insubria
Via J. H. Dunant 3, 21100 Varese, Italy
E-mail: nicolo.baranzini@uninsubria.it
these polymers derive from fossil hydrocarbons
resulting not biodegradable and less than the 20 %
are recycled, while most of them are burned, stored
in landfill sites or environmental dispersed
(Gourmelon, 2015). Furthermore, plastics not only
are extremely resistant, persisting for a long time
inside the ecosystems, but also result harmful for
the living organisms with which interact. In this
context, the aquatic environments are considered
the most affected, in which waste products deriving
from the human consumption or from the industrial
processing rapidly spread transported by currents
and winds (Barnes et al. 2009; Galgani, 2015;
Jambeck et al. 2015). Of the 360 million tons
produced, approximately between 4 to 12,7 million
are reversed into oceans every year, constituting a
significant part of the marine waste, even if this
value is considered lower (Andrady, 2011; Tramoy
et al., 2020). Unfortunately, with this rate it is
estimated that the total amount of dispersed plastics
may soon exceed that of numerous species, leading
to a great loss for many industrial sectors, not only
in terms of biodiversity but also in the economic
perspective (Letcher, 2020). Interestingly, plastics
have been already recognized as a possible
environmental problem for aquatic systems since
1960s, when the first studies have been conducted
focused on their ecological impact on marine species.
However, at the time any eventual considerations
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have been rejected, due to the inability to forecast
whatever future trends (Letcher, 2020).
Larger plastics, known as macroplastics, induce
different problems both in vertebrate and
invertebrate aquatic species when ingested,
blocking the digestive tract, damaging organs, and
leading to a decrease in food intake (Taylor et al.
2016; Burgos-Aceves et al. 2018; Faggio et al.
2018; Aliko et al. 2022; Lombardo et al. 2022).
Many organisms remain entangled in these debris
that cause injuries and limit movements, also
preventing the animal to feed on (Gregory, 2009).
However, macroplastics represent only a small part
of the plastics pollutants. In fact, when released in
marine or freshwater environments, these products
are subjected to biotic and abiotic processes, which
lead to the formation of small particles. Microplastics
(MPs) are in a range between 5 mm and 1 µm,
while nanoplastics (NPs) show a lower diameters
comprised between 1 µm and 1 nm and due to their
size these particles are easier ingested or
assimilated by animals, accumulating inside tissues
and entering in the trophic chain (Arthur et al., 2009;
Xu et al., 2020). Both types have been observed
from sediment to the surface, differently floated into
the water column (Van Cauwenberghe et al., 2013;
Lusher et al., 2015; Mistri et al., 2017; Honorato-
Zimmer et al., 2021). Furthermore, the different
chemical properties and the diverse density make
the comprehension of their fate highly complex
(Haegerbaeumer et al., 2019). To date, MPs and
NPs not only have been found in drinking water and
in 200 edible species, whose consume through the
diet can expose humans to an inevitable absorption.
Due to the ability in crossing the biological barriers
and penetrating inside tissues, many studies
showed as MPs and NPs are stored in biotic
samples, leading to the activation of many different
processes that tend to reduce vitality, promote
metabolic disorders and decrease the reproductive
fitness. Their assimilation is associated with many
toxic responses that occur in organisms such as
oxidative stress, activation of the immune system,
inflammations and growth inhibition (Alimba et al.
2021; Burgos-Aceves et al. 2021a, b). Other
research conducted in mammals reveal that the
uptake of some MPs induces dysbiosis, hepatic and
metabolic disorders in mice (Luo et al., 2019).
Moreover, another important aspect is determined
by the fact that, although these materials should not
be considered reactive for their biochemical
structures, plastics particles can act as carrier for
other pollutants, which are easier transported inside
organisms producing further harmful effects.
At any rate, the plastic degradation increases
the nature availability of these synthetic polymers
with severe impacts on living organisms, especially
in marine and freshwaters ecosystems (Tiwari et al.,
2020). Thus, it results essential to collect and
extend the current knowledge to successfully
understand their real impact and prevent the
potential risks. For this reason, in this review we
reported the most recent and relevant data available
in literature on the MPs and NPs effects both in
marine and freshwaters environments. In detail, we
aim to underly how plastics are produced and used
for the experimental applications and describe the
effects both at organism and cellular level in aquatic
invertebrate species. This work sets out to bring
new insights and provide useful information for the
future research.
Types of plastics
Despite, several emerging methods are
developing for a more sustainable recycling of
plastic (e.g., based on the enzymatic and microbial
biodegradation (Tournier et al., 2020; Lu et al.,
2022; Pirillo et al., 2022; Sonnendecker et al.,
2022), there are a few critical steps for the removing
process of high-density plastics from the
environment and many bench-scale experiments
are not enough for the application in large-scale
process (Patil et al., 2022). Indeed, due to their low
biodegradability, the plastic materials are high
recalcitrant in several environments and the deriving
debris widespread vary in terms of chemical e
physical properties (e.g., chemical composition and
density).
Starting from their initial dimension, the
engendering process that leads to formation of MPs
and NPs can be classified in primary and secondary
sources . With primary sources are identified all the
discarded particles that derive either from the
industrial production or that are generated by the
fragmentation of common objects used in everyday
life (e.g., fibers of synthetic textile products during
washing, personal care products, or cleaning
applications).
Instead, as secondary sources are considered
the total amount of micro- and nanofragments that
originate directly from a slow decomposition of the
former, which result in a fragmentation in smaller
size particles as effect of the environmental
exposition to several both biotic (i.e., living
organisms that influences its environment) and
abiotic factors (i.e., UV rays, temperature, salinity,
atmospheric events or ocean currents, and also
microbial degradation), enhancing the persistence in
natural ecosystems. Moreover, oxidation can be
also the cause of plastics physical abrasion (Arthur
et al., 2009) and some polymers result highly
inclined to this type of chemical process, which
impairs the strong molecular bonds between chains
(Tiwari et al., 2020).
From packaging to the manufacture of single-
used items, polyethylene terephthalate (PET), high-
density (HDPE) and low density (LDPE) polyethylene,
polyvinyl chloride (PVC), polypropylene (PP), and
polystyrene (PS) are the polymers which possess a
major industrial relevance and are the most
common identified in the investigated marine
environments (Table 1) (Auta et al., 2017; Danso et
al., 2019). The particles found in nature are
characterized by different both sizes and shapes
(e.g., spheres, fiber, film, irregular) (Chubarenko et
al., 2020; Patil et al., 2022), but also their density is
an important aspect to be considered for
determining their specific localization and their fate
in the in the water column. In fact, debris with a
density higher than seawater (1.020-1.029 g mL1)
(e.g., PET, PVC, and PS) would sink, on the
contrary polymers less dense (e.g., HDPE, LDPE,
and PP) would float on the surface (Table 1)
(Brignac et al., 2019). Considering all these aspects,
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Table 1 The most diffused synthetic plastics in the aquatic environments. For each type, resin code, abbreviation,
commonly use, decomposition rate for marine debris, density and toxicity levels are reported. aThe reported
chemical density referred to the pure plastic compounds in absence of additives (e.g., plasticizer, pigments,
stabilizers) as reported by Brignac et al., 2019
it results necessary shed light on the presence of
different types of MPs and NPs and their effects on
living organisms in freshwater and marine
ecosystems.
Overview on the MPs and NPs production and
potential applications
In literature are reported several types of MPs
and NPs for evaluating the in vivo effects that are
based on a chemical-mechanical production or are
commercially available (Fig. 1). Several techniques
of synthesis are reported by Lee et al., 2016 to
produce MPs and NPs, that can be divided into four
categories: (i) emulsion-based methods, (ii)
precipitation-based methods, (iii) direct compositing
methods, and (iv) new approaches including
microfluidic technique. However, emulsion and
precipitation are the most used to easily produce
MPs and NPs. For examples, (Grillo et al., 2021)
reported a simple emulsion/solvent extraction
synthesis method in chloroform (1 % m/V) and poly-
vinyl alcohol (PVA, 0.025 % m/V), producing a
range of PS microparticle sizes, where the 78 %
present an average diameter < 5 µm and a
spherical shape. Similarly, straightforward protocols
are available to produce PET NPs based on a
dissolution/precipitation method with water-soluble
solvent hexafluoro-2-propanol (Pirillo et al., 2021) or
two step of trifluoroacetic acid solution (TFA, 90 %
v/v and 20 % v/v) (Rodríguez-Hernández et al.,
2019; Pirillo et al., 2021), starting from commercial
PET (e.g., drink bottle, granulate, films, or fibers);
then, thanks to a filtration-step, it is possible to
obtain a particulate size that is usually in the 50- to
300-nm range. Moreover, the NPs so produced can
be colored with different dye, such as the Nile red,
which has been proposed by Maes et al., 2017 for
PET microparticles identification and exploited for
cell internalization experiments and ecotoxicological
studies (Rodríguez-Hernández et al., 2019).
Beyond the chemical synthesis that allow to
produce all spherical and in the same scale range
particles, easier protocol for MPs and NPs
production are also available based on a
mechanical processing. As proposed by (Romero-
Blanco et al., 2021), MPs can be obtained by
pulverization of PS tube-test, with a size ranged
between 10 nm and 514 µm. Likewise, other
reported protocols are based on the cutting and
fragmentation of commercially plastics fibers or row
pellets (Jemec et al., 2016; Kim et al., 2021). The
major advantage of this method is to produce
irregular fragments that are more similar to those
found in nature, in order to better simulate the
environmental conditions (Baranzini et al., 2022
accepted).
Regarding commercial MPs and NPs, several
modified particles exhibit fluorescence properties
Polymer name
POLYETHYLENE
TEREPHTHALATE
HIGH-DENSITY
POLYETHYLENE
POLYVINYL
CHLORIDE
LOW-DENSITY
POLYETHYLENE
POLYPROPYLENE POLYSTYRENE
Resin code
Abbreviation PET HDPE PVC LDPE PP PS
Use
Water bottles,
medicine jars,
clothing and
carpet fiber
Detergent
bottles, plastic
bags, toys
Credit
cards,
window and
door frames
Plastic wrap,
bread bags,
squeezable
bottles
Bottle caps,
potato chip bags,
packing tape,
drinking straws
Food boxes,
watering cans,
storage bins
Decomposition
rates for
marine debris
Up to 450 years Up to 450 years
Up to 450
years
500-1000 years 20-30 years 900 years
Density
(g mL-1)a
1.37−1.41 0.94−0.98 1.38−1.45 0.89−0.93 0.85−0.92 1.04−1.06
Toxicity level
High
Low
High
Low
High
Low
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and can be functionalized with carboxyl (-COOH) or amino (-NH2) groups on their surface. The most used
Fig. 1 Scheme representing environmental plastics dimensions, correlated to their production methodologies for
in vivo studies research and example of affected organisms. FT-IR, Fourier-Transform Infrared spectroscopy;
SEM: Scanning Electron Microscopy; TEM: Transmission Electron Microscopy
fluorescent MPs and NPs are those of PS, in
particular available as blue-dyed (345 nm excitation
and 435 nm emission, Phosphorex), red-dye (552
nm excitation and 580 nm emission, Micromer®-
redF), and yellow-green microspheres (441 nm
excitation and 486 nm emission, Fluoresbrite® Plain
YG) (Della Torre et al., 2014; Canesi et al., 2015;
Bergami et al., 2017; Gambardella et al., 2017;
Capolupo et al., 2018; Liu et al., 2019; Rist et al.,
2019; Cappello et al., 2021; Gonçalves et al., 2022).
Thanks to their easy detection, fluorescently labeled
and functionalized PS plastics are the most
common used in ecotoxicological studies, toxicity
bioassays and for the evaluation of the aquatic
organisms uptake.
However, independently from how have been
produced, MPs and NPs behavior is extremely
variable and an in-depth characterization is
required. A dynamic light scattering (DLS) analyses
combined with a Zetasizer Nano Series software
(ZS) should be necessary for determining several
key parameters, such as Z-average (nm),
polydispersity index (PDI, dimensionless) and zeta
(ζ-) potential (mV), which describe MPs and NPs
behavior in relation to the environmental media
(Bergami et al., 2017). In fact, salinity or pH can
alter the plastics bioavailability and distribution in
the water column, leading to polymers aggregation
and changing many physicochemical properties
(surface change or coating). Moreover, to evaluate
the potential effects, other aspects must be taken
into consideration. Concentration of MPs or NPs
used in the experimental conditions do not always
reflect the exposure scenario and the real
environmental conditions (e.g., using extremely high
concentrations). In this context, it is reported in
literature the needed to mimic as close as possible
the natural state, to obtain more accurate data on
the real toxicity of plastics dispersion. Moreover,
considering that the fate and the bioavailability of
MPs and NPs depends on size, shape, and charge,
it is important to have comparable methodologies
and approaches, to obtain a deeper knowledge that
considers all the physicochemical variables that can
interfere with interactions between plastics and
organisms (Oliveira and Almeida, 2019).
MPs and NPs effects on marine invertebrate
organisms
Phylum of Cnidaria
Cnidarians are benthic invertebrates that were
able to adapt to many different habitats. Living fixed
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on substrates in contact with sediments or floating in
the water column, these animals represent one of
the main targets for environmental pollutants. For
this reason, many studies have been conducted on
hydrozoan, scyphozoan and anthozoan marine
models to evaluate toxic effects deriving from
different compounds, such as chemicals or metals
(Muñoz-Vera et al., 2015; Lozano-Bilbao et al.,
2018). The presence of waste products can induce
morphological changes, affect vitality and alter the
expression levels of those gene related to
metabolism and oxidative stress (Kalsom and
Mehman, 2020). Due to their ability in the
incorporation of various anthropogenic materials,
both medusoid and polypoid forms are important
bioindicators of MPs and NPs impact in marine
ecosystems (Macali and Bergami, 2020).
Moreover, representing one of the first step of the
trophic chain, the comprehension about their ability
to accumulate synthetic polymers is extremely
relevant (Devereux et al., 2021). In nature, the
presence of plastic fragments has been detected in
different cnidarians species, such as Cyanea
capillata, C. lamarckii and Aurelia aurita
(Schyphozona), Cosmetira pilosella (Hydrozoan)
and Coelogorgia palmosa (Devereux et al., 2021;
Vencato et al., 2021), confirming as these
invertebrates are directly interested by MPs and
NPs pollution.
In the scleractinia coral species Porites porites,
PS MPs absorption was evaluated using chemically
produced spherical particles presenting a diameter
inferior to 5 μm, which were dispersed in seawater
in a range between 1 and 1000 mg/L. The analyses
conducted using both histological and enzymatic
techniques, in which the levels of catalase have
been evaluated, revealed that although the particles
uptake occurred in every condition, no significant
toxic effects on organisms behavior have been
registered in short time (96 h) of exposition. Based
on the initial concentration, PS microsphere have
been stored in gastrovascular tissue, mesenterial
filaments and coral tissue without affecting viability
or inducing bleaching and stress response (Grillo et
al., 2021). Contrariwise, long-term treatments (17
days) with PS cause potential harmful effects both
in polyps and ephyrae of the anthozoan jellyfish
Sanderia malayensis. Independently from the
dimensions, fluorescent FITC-conjugated
microbeads were able to instantly interact (already
after 24 h) with both epidermis tissue and digestive
cavity, in which they were clearly detectable.
Moreover, MPs can accumulate and persist until 52
days, altering feeding behavior and fitness. After 17
days, animals reproduction appeared reduced,
revealing as prolonged exposures to plastics,
although do not affect survival, can interfere with
polyps budding (Eom et al., 2022).
The effects of PE were evaluated in the coral
Stylophora pistillata, analyzing the as MPs inhibit
photosynthetic capacity of scleractinian
zooxanthellae symbionts. After 4 weeks, chlorophyl
reduced activity was analyzed by Pulse Amplitude
Modulated (PAM) fluorometry method, indicating a
condition of stress. Although this effect seemed to
be due to a direct contact between algae and MPs,
given the physical inability of this interaction,
authors suggested that MPs produced a signaling
interference between symbionts and hosts.
Moreover, by means of Nuclear Magnetic
Resonance (NMR) analyses, the metabolism of
different important metabolites has been detected
(Lanctôt et al., 2020). PE MPs effects were also
investigated in the Aurelia sp. ephyra stages using
fluorescent particles at different concentrations
(from 10 µg/L to 10 mg/L). By means of confocal
and tomographic analyses, it was observed as MPs
attached around mouth surface or were stored into
the digestive tract. Moreover, already after 24 h
from the initial exposition, in juveniles jellyfishes the
survival rate, behavior and radial symmetry resulted
significantly altered. Although after 72 h a total
recovery is restored in absence of MPs, these data
also confirmed a direct impact on larval immobility
and frequency of pulsations (AFp) that impair
animals viability (Costa et al., 2020).
Coral reef cnidarians were also used to test the
effects of PVC MPs. In the species Zoanthus
sociatus, commercial MPs, possessing an irregular
size, were dispersed in the aquarium under
continuously moving water condition, in which two
different PVC concentrations (1 and 10 mg/L) were
assessed. Plastics immediately adhered to
epidermis or gut cavity, stimulating stress response
and inducing photosynthetic events. As regards the
macroscopic effects, no variations in survival or
behavior have been detected, also after long term
exposure (Rocha et al., 2020).
Phylum of Mollusca
Given their commercial and ecologically
importance, many studies were conducted on
several molluscs species, in which the effects of
different plastic types have been tested with
different aims. In particular benthic and sedentary
organisms, such as bivalves, are the main models
employed in toxicological research (Pagano et al.
2020; Stara et al. 2020, 2021; Curpan et al. 2022).
Filtrating waters or directly feeding from the
substrate, they are excellent candidates for
investigating potential toxic effects, analyzing
plastics bioaccumulation and morphological or
behavioral changes (Rittschof and McClellan-Green,
2005; Jaeschke et al., 2015).
Spherical and colored PS MPs, with a diameter
of 3 µm, were administered to the marine mussel
Mytilus galloprovincialis and the several metabolic
parameters were analyzed by NMR-based
metabolomics spectra. Filtered MPs deposit into the
digestive gland and, especially after 48 h up to 72 h,
alter the production of various metabolites,
osmolytes and antioxidants. The levels of several
amino acids, lactate, glycogen, taurine, hypotaurine
and glutathione increased, suggesting a potential
toxicity for PS MPs (Cappello et al., 2021).
Significant change in the expression levels of
different genes was also observed after 3 µm-size
spherical MPs uptake in M. galloprovincialis larval
stages. Although plastics were assimilated and
retained, bioaccumulating inside the digestive tract
and risking persisting in the trophic chain, their
presence did not cause visible morphological
modifications during embryonic development up to
192 h. However, the upregulation of several genes
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related both to shell biogenesis and immune
response or the downregulation of those involved in
lysosomal activity were evident, highlighting as PS
plastics mostly act at the cellular level (Capolupo et
al., 2018). A similar result was also observed in the
Mytilus edulis larvae, in which although MPs were
faster stored into embryo tissues after ingestion, the
developmental stages were not altered, except
under extreme conditions (high concentration and
long exposition timings) (Rist et al., 2019).
Interestingly, similar potentially dangerous
effects were also detectable in mussels cells after
NPs chemical modifications. Indeed, cationic NPs
(PS-NH2) affected cellular processes and influenced
molecular intracellular pathways. Based on studies
conducted in sea urchin embryos and mammalian
cells, Canesi and colleagues demonstrated as the
addition of positive charges not only impacted on
the plastics uptake, but also on their final biological
targets. Indeed, M. galloprovincialis hemocytes
exposed to 50 nm PS-NH2 particles showed both a
reduced phagocytic activity and the increase of
lysozyme in a dose-dependent manner, together
with reactive oxygen species (ROS) and nitric oxide
(NO) production. In parallel, the apoptotic rate and
the loss of mitochondrial potential were evaluated
by means of flow cytometry (Canesi et al., 2015).
The PS NPs ability in impairing immune system was
also confirmed by electron microscopy (TEM) and
molecular analyses. The presence of numerous
laminar cytoplasmatic expansions and the diverse
modification of the p38 MAPK and PKC
phosphorylation state represented a symptom of
cellular stress in mussels hemocytes. In addition,
once entered inside tissues and cells, MPs and NPs
can be also conditioned by the chemical interactions
established with different proteins, known as
corona-protein, that interfere with plastics behavior.
The formation of a PS-NH2-corona proteins
complexes was investigated in the extracted
hemolymph, combining electrophoresis method and
HPLC-MS/MS techniques. Interestingly, the results
showed a specific binding with a C1q domain
protein, suggesting that although this interactions
makes more difficult the comprehension of plastics
fate, are essential to better assess their potential
impact (Canesi et al., 2016).
NPs toxic effects can be also calculated using
the Integrated Biomarker Response (IBR) index that
allows to compare and analyze the relations
between biomarkers and pollutants levels. This tool
has been used in M. galloprovincialis to better
interpretate the obtained results, in which a
concentration of 10 µg/L of PS particles caused a
chronic response characterized by a tissue-specific
genotoxicity. Among all the organs involved, gills
resulted the main affected. PS NPs aggregate and
interfere with the cell biological functions, damaging
DNA and impacting on cell viability. Moreover, the
antioxidant defenses, based on different enzymes,
result ineffective and after 14 days from the
beginning of exposure. The levels of ROS were so
high that lipid peroxidation (LPO) occurred, leading
to the disruption of cell membranes and causing a
chronic response (Gonçalves et al., 2022). Although
the PS registered effects are different in diverse
mussel species, the IBR index should represent a
valuable tool to simplify and clarify data
interpretation.
To date, although numerous investigations
have been performed on PS particles, only few were
conducted on the other plastic types. Among them,
the potential role of HDPE was analyzed in the
pacific oyster Crassostrea gigas larvae, in which
develop, morphological modifications and swim
ability were assessed. After 24 h from the initial
exposition, the smallest HDPE microparticles,
proposed at three different concentrations (100
µg/L, 1 and 10 mg/L), caused severe malformations
and arrest larval growth. Moreover, although the
speed during swim was not completely reduced,
trajectories appeared significantly altered. In the 70
% of oyster embryo, the interaction with MPs
produced more circular movements, decreasing
rectilinear proceeding. All these consequences on
their behavior not only interfere with the capacity of
larvae to feed or escape from predators, but also
with the possibility to naturally colonize new
substrates (Bringer et al., 2020).
Phylum of Arthropoda
Among all the invertebrate phyla, those of
arthropods is certainly the most numerous. Indeed,
it contains a great variety of species that, thanks to
their characteristics, conquered different types of
habitats and colonized quite all the ecological
niches (Kremen et al., 1993; Verma and Prakash,
2020). As for bivalve molluscs, the marine taxon of
crustaceans possesses a large importance both in
economic and biological terms and also recently
acquired a certain relevance in scientific and
ecotoxicological field (Anger, 2006). Therefore, it
results fundamental to investigate the plastics toxic
effects in these invertebrates, analyzing as MPs and
NPs could bioaccumulate inside tissues, not only
altering the development and life cycle, but also
risking to consequently enter in the tropic chain.
This the case of several classes such as Copepoda,
Cladocera or Branchiopoda, composed by small
crustaceans that form zooplankton and that
represent the main prey of fishes and other
invertebrates. Given that the chronic presence of
plastics in these organisms can be extremely
dangerous, numerous studies have been focused
proper on these animals.
In the brine shrimp Artemia salina, chronic
toxicity induced by PS MPs was determined using
both labeled and not-labeled plastics particles at
different concentrations (from 1 to 100 mg/L).
Moreover, the employ of greenly fluorescent MPs
better allowed to follow plastics fate and body
localization. Although MPs accumulate inside
tissues in all the developmental stages, organisms
growth was considerably reduced only with the
highest concentration. In this condition, a
considerable modification of the midgut cell
morphology was detected. In addition, to
characterize the way by which plastics influence
cells biology, transcriptome analyses have been
conducted in combination with Gene Ontology (GO)
analyses, confirming a change in the expression
levels of those genes involved both in metabolic and
catalytic activities, as already observed in other
arthropods species (Suman et al., 2020).
142
The PS plastics ingestion followed by harmful
effects was detected also in another crustacean of
the genus Artemia: Artemia franciscana. The
exposure to various MPs concentrations and sizes
revealed a variation in the activity of specific
proteins and enzymes, such as Heat Shock Protein
70 (HSP70), catalase, superoxide dismutase and
acetylcholinesterase, considered important markers
of the oxidative stress. However, the most relevant
data was represented by the increase in shrimps
mortality after 30 days of treatment. MPs impaired
survival rate in a dose-depending manner, by
inducing chronic and acute responses at different
biological levels (Eom et al., 2020).
A. franciscana brine shrimps have been also
treated in combination with the green microalgae
Dunaliella tertiolecta to assess as charged PS NPs
could induce diverse effects depending on the
associated chemical group. Interestingly, if on one
hand anionic carboxylate (PS-COOH) particles did
not impact on organisms health until highest
concentrations, although are easier internalized, on
the contrary the anionic PS-NH2 plastics induced
both the inhibition of D. tertiolecta algae growth and
the increase of A. franciscana shrimps mortality. All
these results are extremely important to better
understand the PS plastics role also in zooplankton
organisms that occupy a relevant place in the
trophic chain (Bergami et al., 2017).
Phylum of Annelida
Thanks to the possibility to amputate body
segments without compromise their survival, the
class of marine polychaeta constitutes an important
phylogenetic group to assess MPs and NPs effects
on tissues regeneration. Moreover, being prey of
many other animals and living directly on the
sediments, these invertebrates are continuously
exposed to environmental pollutants, representing
one the main channels for the entrance of
anthropogenic materials in the trophic chain (Pires
et al., 2022). However, despite their ecologic
relevance, only few studies have been performed to
determine how plastics could impact not only on
polychaetes life cycle and behavior, but also
interfere with the ability to regenerate tissues.
Two different species Hediste diversicolor and
Perinereis aibuhitensis were treated with different
concentrations of PS MPs and NPs, in which
behavioral change were determined after plastics
treatment. In detail, H. diversicolor worms exposed
to different concentration of PS NPs showed a
reduced burrowing ability. This parameter is
probably connected with a decrease in the
Cholinesterase activity, a fundamental enzyme that
regulates muscular functions. Furthermore, in the 50
% of the samples exposed to a NPs concentration
of 50 mg/L also the antioxidant defenses resulted
significantly reduced. However, LPO levels
remained lower than control groups, suggesting that
in H. diversicolor mechanisms of membrane repair
could be activated to cope with PS adverse effects
(Silva et al., 2020). Whereas Leung and Chan
analyzed the impact PS MPs during regeneration in
the polychaeta P. aibuhitensis. Authors
demonstrated as the smallest microparticles (8-12
µm) increased mortality and affected the ability to
regenerate removed segments (Leung and Chan,
2018). Moreover, these studies also revealed that,
in particular smaller beads, aggregated and
consequently compromised samples physiology and
behavior, acting at the cellular level by influencing
the expression of specific antioxidant enzymes.
H. diversicolor has been also used to
investigate the potential effects of other
environmental frequently found plastic types. PP
and PE MPs were added at different concentrations
into water (10 and 100 μg of MPs/L) and sediments
compartments (10 and 50 mg of MPs/kg),
demonstrating a different accumulation inside
worms tissues after 96 h. By means of flow
cytometry and enzymatic assays, the coelomocytes
viability, the phagocytic activity and the levels of
phenoloxidase (PO) and acid phosphatase (AcP)
were analyzed. Both a slight reduction of both cells
viability and PO and AcP were observed,
suggesting as different plastic types can interfere
with organisms integrity (Revel et al., 2020). A
similar result has been recently obtained mixing and
using together more type of MPs. Indeed, a mixture
of PE, PP, HDPE, LDPE, polyamide (PA) and
polyethylene/ethylene combined with vinyl acetate
copolymer (PEVA) was tested in H. diversicolor,
revealing that plastics accumulate, affecting animals
survival and growth rate at high concentration when
exposed for long time (Missawi et al., 2021).
Phylum of Echinodermata
Several studies have been also conducted in
echinoderms, in which the attention has been
mainly focused on the gametes formation or on the
embryonic and larvae phases. Thanks to the higher
sensitivity to pollutants then adults, juvenile stages
represent a perfect model to test possible
dangerous effects that can compromised the
development (Nobre et al., 2015).
In both eggs and spermatozoa of the sand
dollar Scaphechinus mirabilis, PS MPs induced a
significant DNA damage, in which more than the 20
% of the sequences resulted shattered. By means of
DNA comet assays, the genome loss was effectively
observed already after 1 h after 105 particles/L
exposure. However, despite this evident effect,
spermatozoa did not lose their capacity to fertilize
eggs (Mazur et al., 2021).
PS toxicity was also evaluated in the embryonic
stages of the sea urchin Paracentrotus lividus, in
which, as observed for A. franciscana brine shrimps,
positive and negative charged NPs showed different
properties. The structures and dimensions were
evaluated by means of TEM and Dynamic Light
Scattering (DLS) analyses. Although fluorescently
PS-COOH-labeled particles resulted more
aggregated and were easily accumulated inside
embryos gut, no particularly severe effects were
observed. On the contrary, positive PS-NH2
modified plastics, despite appearing more water
dispersed, caused serious developmental
deficiencies. This outcome is probably due to the
activation of apoptotic pathways, given that after 24
h from exposure, NPs lead to a significant
upregulation of the caspase 8 gene (Della Torre et
al., 2014). Interestingly, these data represent a
further confirm of how any plastics chemical
143
modification could not completely change the fate of
these synthetic polymers, but also lead to diverse
responses both at organism and cellular level.
MPs and NPs effects on freshwater invertebrate
organisms
It is estimated that a considerable part of the
plastics presents in seas and oceans arise from
freshwaters, in which a large amount of waste
products is reversed every year. Lakes and rivers
very often are the main environments affected by
plastics pollution, being in direct contact with urbans
and rural locations (Strungaru et al., 2019). Here,
MPs and NPs float in the water column or settle on
the sediments, entering in contact with living
organisms. Notwithstanding it results necessary to
better comprehend their impacts also in these
ecosystems, nowadays only few studies have been
performed compared to those conducted on marine
species (Imhof and Laforsch, 2016). Moreover,
freshwater benthic invertebrates are considered
useful biomarkers, thank to their extremely
vulnerability to pollutant, and can represent a
fundamental instrument to easily decode the
ecological conditions of a specific environment.
Phylum of Cnidaria
As marine cnidarians, also the freshwaters one
are an important source of food for many aquatic
species. Moreover, due to their anatomical and
physiological characteristics, these invertebrates are
extremely sensitive to chemical environmental
pollutants, representing a valid indicator of the water
quality (Beach and Pascoe, 1998). The presence of
toxic substances could have severe effects on the
body morphology and animals vitality. Among the
major critical outcomes, hydrozoans are subjected
to tentacles loss and impediment in food uptake. For
these reasons, also the toxic effects of PS NPs
have been tested on these invertebrate models,
mainly focused the attention on regenerating
processes (Auclair et al., 2020). In detail, the
freshwater Hydra attenuata was treated for 96 h
with different concentrations of PS fluorescent
particles (from 1.25 to 80 mg/L - 50 and 100 nm in
size), whose diameter has been evaluated by DLS
analyses. Despite NPs accumulation, depending on
the administered dose, following 24 h of depuration
severe loss of biological mass occurred. Moreover,
an important sign of oxidative stress condition was
determined by the fluorescent measuring of both
LPO and lipid-like liquid crystal (LCs) formation
inside cells cytoplasm. These data revealed as 100
nm particles were most effective then the smaller
one and highlighted as lipids metabolism could play
an important role in determining plastics toxicity in
freshwater cnidarians (Auclair et al., 2020).
Phylum of Mollusca
The effects of synthetic polymers have been
assessed in the mud snail Potamopyrgus
antipodarum, recreating the gastropods
environmental conditions. In detail, freshwater was
combined with river sands, in which MPs and NPs,
presenting sizes between 10 nm and 514 μm and
concentrations from 100 to 4000 mg/kg, have been
added. After 31 days, no critical impacts on both
mortality and reproduction rate were observed.
However, adverse behavioral responses were
detected analyzing animals immobilization, reaction
time and distribution at different conditions.
Interestingly, the highest concentrations of MPs did
not cause negative effects compared to the control,
while all the other treatments tent to increase the P.
antipodarum reaction time. Although different
hypotheses were developed, this factor is probably
closely related to the plastics intake, which risks to
block the digestive tract or to damage tissues.
Nevertheless, these organisms seem to well tolerate
plastics presence, independently from MPs and NPs
concentrations (Romero-Blanco et al., 2021).
Similar results were obtained by Imhof and
Laforsch, which examined the potential plastics
effects in P. antipodarum using a mixture of five
different common non-buoyant polymers (PA, PET,
PC, PS and PVC), directly added to food in two
different doses. The main focus was to analyze
possible morphological change in both adults and
juvenile stages, but no significant modifications
have been observed. Moreover, the shell formation
was not affected by the presence of polymers also
after 8 weeks at the higher concentration,
suggesting that the juvenile development was not
compromised. However, it must be considered that
in the present work large particles have been used
and the lack of effects should be due to plastics size
and shape (Imhof and Laforsch, 2016).
Phylum of Arthropoda
Among freshwaters arthropods, Daphnia spp.
are considered the main conventional model used in
freshwater ecotoxicological studies, recognized at
an international level (Taylor et al., 2018). Filtering
nutrients in a not-selective manner, these
crustaceans come into direct contact with numerous
contaminants, and, thanks to their simple body
anatomy, they allow to easily detect any possible
morphological or physiological modification. In
Daphnia pulex, fluorescent PS NPs immediately
entered inside digestive tract after 48 h from initial
exposure, showing lethal effects at the
concentration of 76.69 mg/L. Moreover, not only
survival rate was significantly reduced, but also both
the reproductive capacity and growth. The analyses
of the expression levels of several gene involved in
oxidative stress showed, as for other aquatic
invertebrates, a significant increase depending on
particles concentration. In particular, superoxide
dismutase (SOD), glutathione transferase (GST)
and HSP90 genes resulted more expressed than in
control samples. Also after long-term treatments, a
chronic response was visible, in which PS NPs
influence the reproduction, hatching time and future
offspring (Liu et al., 2019b). A comparable result
was also obtained in Daphnia galeata, in which 5
mg/L of PS NPs caused aberrant development and
a fitness reduction after 5 days (Cui et al., 2017).
Plastics particles aggregated with lipid droplets
located in the adult ovary, which play a fundamental
role in regulating crustacean embryos development
(Cai et al., 2019). Although the NPs effects were
also transient and did not impact on future offspring,
by means of Nile Red staining assay, Cui and
colleagues demonstrated that their presence led to
144
a decrease in lipid storage (Cui et al., 2017). These
variations revealed as nanoplastics could modify
metabolic pathways, becoming an effective barrier
to embryonic development.
As regards other synthetic materials, the effects
related to PET microfibers ingestion have been
examined in Daphnia magna after 48 h of treatment
and following 24 h of recovery. PET fibers were
characterized by FTIR spectrometer in ATR mode
comparing the results with spectral databases.
Although plastics found in the digestive tract
possessed a mean dimension of about 300 μm, also
particles with large dimensions (1400 μm) were
found in the gut. D. magna mortality appeared
significantly increased in no prefeeding animals,
while no effects were recorded in daphnids fed
before treatment. Subsequent experiments, in which
tissues were digested with H2O2 and the gut
contents were analyzed by means of scanning
electron microscopy (SEM), confirmed a
considerable amount of PET fibers in D. magna
tissues (Jemec et al., 2016). Also PP particles
accumulated in D. magna digestive tract, in a
similar concentration, though these polymers
caused a less response and minor lethal effects
after 96 h after the initial exposure (Kim et al.,
2021).
Phylum of Annelida
Differently from all the other invertebrates,
freshwater annelids that belong to the Oligochaeta
and Hirudinea classes are considered extremely
tolerant to waters contamination and very often their
population benefit of these compounds compared
with those of non-tolerant organisms (Sharma and
Chowdhary, 2011). Indeed, especially oligochaete
benthic species are generally the dominant in
muddy sediments of lakes or in the swampy areas,
able to exploit all the waste materials that
decompose on the bottom (Abubakr, 2018). Despite
the low number of studies, thank to their high
resistance, these organisms should be considered
extremely useful to understand how much plastics
could be dangerous. In fact, any effect caused by
these synthetic polymers represent a significant sign
of their potential toxicity.
In the oligochaete aquatic worm Allonais
inaequalis, the role of PE MPs was investigated.
Particles possessed a size between 40 and 48 μm
and were provided both in normal temperature (24
°C) and thermal stress (19 °C and 29 °C) conditions.
Independently from temperatures, after 96 h MPs
were ingested by worms without affecting animals
survival. Moreover, also chronic response did not
induce particular effects in terms of mortality and
reproductivity. However, as suggested by the
authors, although no specific effects were recorded,
exposition time, particle size, age and plastic type
could also produce different responses.
Nevertheless, thanks to its resistance properties, A.
inaequalis can represent a suitable model for
deepening microplastics effects in freshwaters
animals (Castro et al., 2020).
The resistance of oligochaete species to
contaminants was also confirmed in Tubifex spp.,
which inhabit areas in which the pollution levels are
extremely high (Sharma and Chowdhary, 2011). It
was demonstrated that in the species Tubifex
tubifex, MPs ingestion did not affect life cycle and
reproduction. As confirmed by FTIR analyses, a
wide range of synthetic polymers were found,
located in the digestive system and presenting
different sizes and concentrations. Although no
effects were registered, this study specifically
suggests as the bioaccumulation of MPs into tissues
and organs, make these oligochaetes a potential
risk for the other organisms in relation to the trophic
transfer (Hurley et al., 2017).
As concern Hirudinea, leeches are considered
a valid bioindicators, whose ability to accumulate
pollutants is comparable only with that of
oligochaetes. Is has been discovered that the
concentrations of contaminants in different species
of leeches were higher than in other analyzed
organism, vertebrates included. For example, in
Erpobdella punctata the levels of mirex, a
chlorinated hydrocarbon used in the past as
insecticide and nowadays banned due to its
significant environmental impact, were higher than
in other invertebrates and fishes. Whereas leeches
of the Glossiphoniidae family collected in the Tahoe
Lake (USA) presented a greater quantity of DDT
than filter-feeding clams of the genus Pisidium
(Metcalfe and Carey, 1984). In addition, leeches not
only are considered a suitable model for studying
innate immunity and regeneration (Grimaldi et al.,
2009, 2010; Baranzini et al., 2020, 2021), but also
they have been already used to investigate
nanoparticles effects (Girardello et al., 2015, 2017;
Bodó et al., 2020).
For all these reasons, the potential role of MPs
and NPs PP has been analyzed in the freshwater
leech Hirudo verbana, focused the attention on the
possible inflammatory response activation. To better
follow particles fate, mechanical-obtained
fluorescent plastics were administered to water at
the concentration of 400 mg/L and the effects were
analyzed both after short and long exposure timings
(1 h 6 h, 1 week, 1 month and 2 months). By means
of microscopy and DLS analyses, the size and the
fluorescent properties of manually created PP
fragments have been evaluated. Morphological,
histoenzymatic and molecular assays confirmed as
these synthetic polymers not only entered inside
tissues, passing the external cuticle, but also lead to
the activation of angiogenetic processes and
macrophage activation. Moreover, despite an initial
protection by mucous cells, their presence caused
an increase of the expression levels of important
pro-inflammatory markers, such as HmAIF-1 and
HvRNASET2 (Baranzini et al., 2019). Gene
upregulation was also observed for SOD and GST
antioxidant enzymes, suggesting as PP MPs and
NPs induced oxidative stress and confirming their
potential harmful effects, also at the cellular level
(Fig. 2) (Baranzini et al., 2022, accepted).
Concluding remarks
Plastic MPs and NPs, deriving from waste
materials or directly released during industrial
manufacturing, are widely present both in marine and
145
Fig. 2 Representation of PP plastics uptake and effects in the medicinal leech H. verbana. Leeches were exposed
to water resuspended plastic fragments that are able to pass external cuticle and accumulate inside tissues. Once
entered, the main effects immediately relevant induced by PP MPs and NPs involved angiogenesis, macrophage
recruitment and activation, and oxidative stress
freshwater aquatic ecosystems. The consequent
pollution represents a significant problem at the
global level, due to the potential risks not only for
organisms that inhabit a specific environment but
also for human health. In this context,
invertebrates are essential for better comprehend
these aspects. Living in all the ecological niches,
they are often the primary organisms that enter in
contact with plastics particles, also representing an
entrance for these synthetic materials in the trophic
chain. However, compared with the actual
situation, the number of studies present in
literature is still low and investigating plastics
effects must be considered crucial also in these
models. Moreover, although the highest
percentage of research is focused on PS MPs and
NPs, less is known about other plastics and the
different administering methods, particle types,
timings and the diverse concentrations used make
complicated to find uniformity in these results.
Notwithstanding these critical issues, the current
data are fundamental to lay the foundations to
understand the environmental consequences
caused by plastics presence and to direct the future
research to analyze these effects both at organism
and cellular level.
Acknowledgments
The authors would like to thanks the Biology
Invertebrates Lab and The Protein Factory 2.0 for
the support in the current publication.
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