Trends in Analytical Chemistry 114 (2019) 196e201

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Trends in Analytical Chemistry

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The why and how of micro(nano)plastic research

M. Oliveira*, M. Almeida

rio de Santiago, 3810-193 Aveiro,
Department of Biology & CESAM e Centre for Environmental and Marine Studies, University of Aveiro, Campus Universita
Portugal

a r t i c l e i n f o a b s t r a c t

Article history: The presence of small plastic particles in the environment, reported for the first time in the 1970's, has
Available online 15 March 2019 only recently been recognized as a global issue. Although environmental awareness continues to grow, so
does its consumption and associated risks. The number of studies reporting the presence of micro-
Keywords: plastics, has grown exponentially as did the concern over plastic degradation into smaller particles like
Effects nanoplastics, a potentially more pernicious form of plastic pollution. The reported effects of micro(nano)
Characterization
plastics on biota range from depletion of energy reserves and altered metabolism to immunological,
Methodologies
neurotoxic effects and behavioral effects. This paper presents a critical review of current scientific
Marine environment
Research needs
knowledge in terms of reasons to study the effects of small plastics present in the environment, what has
been assessed so far; most common methodologies. Research and technical developments requirements
are also presented. Overall, it is clear the need for standardization of procedures and communication of
results.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction quantify the environmental contamination by plastic particles,

Over the last 50 years the use of plastics allowed a considerable
improvement of human everyday lives [1]. Routinely used in simple
daily requirements (e.g. fresh food requirements, transport of
shopping goods, aseptic medical products, preparation of meals),
plastics may be considered a pillar material in a global “throwaway
culture” economy. Plastics allowed the use of more cost effective,
light weight products and production of materials with character-
istics required for a wide range of human needs. Among the life
quality improvements that may be attributed to the use of plastics,
food packaging allows the reduction of wastage and food borne
bacterial infections. As world plastics production continues to
experience an increase (approximately 335 million tons in 2016
[2]), plastics are, deliberately and/or accidental, being released into
the environment, having been found even in remote areas like
Antarctica [3], mountain-tops [4] and deep ocean [5], becoming
available for ingestion to a wide range of organisms (e.g.
zooplankton, bivalves, shrimps, lugworms, fishes and whales).
Thus, the entire planet may be under pressure by the presence of
plastic particles. Nevertheless, the overall level of environmental
contamination remains largely unknown. There is the need to
* Corresponding author.
E-mail address: migueloliveira@ua.pt (M. Oliveira).
their ecological effects and overall risk to the environment and
ultimately, human health. This is, however, a difficult task,
complicated by several factors associated with plastics intrinsic
properties (e.g. nature: different polymers with distinct additives
and potential for sorption of environmental contaminants; size:
original and of entry in the environment), sources (ranging from
wastewater treatment plants, to open air dumpsites), dispersion
potential and fragmentation into increasingly smaller particles. A
realistic risk assessment requires data of abundance, size distri-
bution and chemical composition of microplastics [6]. This paper
aims to present a critical discussion of the facts that make plastics a
potential environmental risk and the methodologies currently
available to quantify, characterize and test effects.
2. Plastics in the environment
Understanding the global mass inventory is one of the main
challenges in present research on plastic marine debris [7]. The
plastic particles found in the environment differ in terms of source
(via different pathways associated with land and marine-based
activities), size (macro, meso, micro and nano), density
(16e2200 kg m 3), chemical composition, and shape (e.g. spheres,
fibre, film, irregular) [8]. Land-based sources are considered the
most important, including industrial and domestic drainage sys-
tems, wastewater treatment effluents, storm sewers, wind, water

https://doi.org/10.1016/j.trac.2019.02.023
0165-9936/© 2019 Elsevier B.V. All rights reserved.
 M. Oliveira, M. Almeida / Trends in Analytical Chemistry 114 (2019) 196e201
systems currents and run off soils from agricultural practices like
plastic mulching or treated with sewage sludge, often used as fer-
tilizer. The use of polymers (e.g. polystyrene and polyurethane),
added to compost to improve soil properties and increase crop
yields may also be considered as a recognized source of micro(-
nano)plastics in the environment [9]. The marine-based sources are
associated with transportation of products, recreational cruises,
extraction of oil, illegal dumping, and fishing activities. Regardless
of the sources, contamination by plastics is putting the environ-
ment under a considerable pressure, particularly oceans, the ulti-
mate recipient of most environmental contaminants, adding to
other pressures like ocean acidification and temperature rise.
Despite the recognition of the potential of soils to retain and
accumulate plastic particles, there is limited knowledge in terms of
micro(nano)plastics levels in this environmental compartment. It
has been estimated that between 63 000 and 430 000 tons of
microplastics are annually added, to European farmlands, via
sewage sludge [10]. However, plastics have also been found in
agricultural soils, not subjected to microplastic-containing fertil-
izers (e.g. sewage sludge and organic fertilizers) nor agricultural
plastic applications (e.g. 0.34 microplastic particles per kilogram
dry weight of soil) [11].
Although the problem of the presence of small plastic particles
in the environment has only recently attracted public awareness,
the first reports in coastal areas and oceans, dates from the 1970's
[12]. Plastic characteristics like resistance, durability, that support
its use in a wide range of industrial applications, are also reasons for
environmental concerns and a challenge for remediation, as the
particles already present in the environment may persist for cen-
turies [13]. Additionally, it is now recognized that plastics may
suffer multiple degradation and disintegration procedures, via bi-
otic and/or abiotic processes, that lead to the formation of smaller-
sized plastics, such as microplastics (<5 mm) and nanoplastics
(defined as < 1000 nm according to some authors or < 100 nm
according to others [14,15]), making their removal increasingly
harder. The physical process involved in plastic degradation is more
favoured in beaches, subjected to high UV radiation, physical
abrasion induced by waves and high oxygen availability. At some
locations, microplastics can constitute over 80% of intertidal plastic
debris [16]. Micro(nano)plastics are also used in several products
like personal care products (e.g. facial cleansers, tooth paste,
shower/bath gels, scrubs, peelings, makeup, insect repellents and
sunscreen), abrasives in cleaning products, and biomedical appli-
cations that have, as end destination, their release into sewage
systems and/or environment. Although it is known that nano-
plastics are being released and transported through air, soil, and
water compartments [17], the available data concerning its pres-
ence, variation of characteristics and impacts on natural systems is
extremely scarce, largely due to the difficulty of its detection and
characterization. Nonetheless, its levels are expected to increase
consistently over time in the environment.
In the aquatic environment, in addition to characteristics
like resistance and durability, the distribution of plastics is
modulated by characteristics like its buoyancy. In seawater,
particles with higher density tend to sink and accumulate in
sediments, becoming more available to benthic organisms. The
less dense particles may remain at sea surface until biofouling
processes by biota or entrainment in biological aggregates
(including faecal pellets after ingestion) occur, leading to
increased densities and their sinking [18], or be degraded into
smaller particles. At the nanometer size, many physicochemical
(e.g. surface charge, coating) may be altered by variations in
the surrounding media (e.g. salinity, pH) and lead to changes in
size (agglomeration/aggregation processes) and/or hydropho-
bicity, thus influencing its distribution in the water column.
197
Ultimately, marine sediments may be considered long-term
sinks for micro(nano)plastics.
The available information in terms of the levels of plastics
found in the aquatic environment vary, with most studies quan-
tifying particles in the marine systems. Examples of levels re-
ported in freshwater and marine systems are presented in
Table SI1. The available data may, however, be underestimating
contamination as environmental sampling frequently resorts to
300 mm nets mesh. A clear example is presented in Table SI1,
where abundance of microplastics sampled using a 330 mm net
mesh is considerably lower than using an 80 mm mesh (0.45
versus 106 particles/m3).
The assessment of the potential consequences of the presence of
micro(nano)plastics on the environment should also account for its
role on the dispersion of environmental contaminants from places
with high concentrations to areas of low concentrations, acting as a
dispersion agent. Their large surface area to volume ratio endow
them a high association potential for environmental contaminants
[19] although different polymers have shown different sorption
capacities. Microplastics are able to concentrate environmental
contaminants [20], increasing the concern on their associated
environmental hazard.
3. Interaction of micro(nano)plastics with biota
The widespread use of plastic materials and their dispersion in
the environment create a decentralized environmental risk [21]. An
increasing number of studies are trying to understand the effects of
plastics to biota, mainly in estuarine/marine environments, ex-
pected to be the ultimate sink of microplastics. However, the
establishment of cause-effect linkages of risks associated with
environmental microplastics is difficult, leading to great uncer-
tainty in their scientific assessment [21].
Recently published reviews summarized the available studies
on microplastic presence, behavior and effects on aquatic [2] and
soil [22] organisms. Significant mismatches between the types of
microplastics most commonly found in the environment and
those used in laboratory experiments has been highlighted [2].
Overall, available studies demonstrated that microplastics may be
ingested by different marine organisms from zooplankton to fish.
Fish and crustacea are the most studied groups, followed by
molluscs and annelid worms but there are very few studies with
other groups of organisms. In organisms like fish, ingestion has
been associated with confusing plastic particles with preys. Re-
ported levels in field sampled organisms show that the number of
plastic vary considerably (e.g. 0.03 particles/fish were found in
animals caught in the North Baltic sea whereas 21.8 particles/fish
were reported in fish from the Persian Gulf, Table SI2). Data from
feeding experiments with zooplankton show that plastic particles
ingestion depends on factors like size, abundance and deposition
in the environment, predator's feeding mode and anatomy of
feeding/digestive organs [23]. The levels found in zooplankton
from Northeast Pacific Ocean are in the same range of fish in the
North Baltic Sea (0.02 particles/copepod and 0.05 particles/eu-
phausiids, Table SI2). There is, however, a need for standardization
of the units expressing microplastics content in biota to allow
comparison between studies (e.g. levels have been expressed as
particles per gram of dry weight or particles per animal). For
laboratory studies, the concentrations of micro(nano)plastics and
how they are expressed is also very important. The same unity of
mass may represent huge different in terms of number of particles
available to the organisms.
A decrease in particle size leads to an increased number of or-
ganisms able ingest them (Fig. 1), as well as in the processes of
leaching of chemicals, desorption of additives and adsorption of
198 M. Oliveira, M. Almeida / Trends in Analytical Chemistry 114 (2019) 196e201
Fig. 1. Diagram representing methodologies to sample and identify micro(nanoplastics) and examples of affected organisms. AFM - Atomic force microscopy; Cars - Coherent anti-
Stokes Raman scattering; FT-IR - Fourier-transform infrared spectroscopy; Phyto-Pe Phytoplankton; SEM - Scanning electron microscopy; SRS - Stimulated Raman scattering
microscopy; TEM - Transmission electron microscopy; Zoo-P e Zooplankton.
environmental contaminants [24]. At a nano size, additional path-
ways of incorporation in biota (e.g. through gills and skin) are
possible, increasing the number potential target tissues. Micro-
plastics, at high concentrations, have shown the ability affect the
base of a trophic web Microplastics have the ability to negatively
influence microalgae growth [25] and photosynthesis [26], and
zooplankton feeding and reproduction [27]. At a higher trophic
level, in fish, they can affect neurotransmission [28] and behavior
[29]. In benthic organisms, reduced feeding activity, decreased
energy reserves, inflammation [30] and body weight decrease have
also reported [31]. Available data from laboratory studies suggest
that particles incorporated by organisms in a lower trophic level
(e.g. zooplankton, mussels) may be passed up the food web [32,33],
and be a vector for the transfer of environmental contaminants that
may eventually reach the humans.
Humans may be exposed to micro(nano)plastics through
different sources (e.g. contaminated food, sea salt, airborne parti-
cles, personal care products). The ability of organisms like mussels,
oysters, clams and prawns to incorporate microplastics (Table SI2)
may have serious consequences to man as these organisms, unlike
most fish species, are consumed as a whole. However, the potential
for micro(nano)plastics of environmental origin to cause harm to
human health remains understudied [1]. Data concerning trans-
location events of micro(nano)plastics across biological compart-
ments together with known effects in neurotransmission in aquatic
organisms emphasize the need of studies on the effects of
micro(nano)plastics to humans.
The behavior of nanoparticles, their fate and effects once
internalized by cells is a key factor influencing cytotoxicity
which may not only dependent on the nature of the core ma-
terial but also on surface modifications (e.g. hydrophobicity,
charge, functional groups and size [34e36]). As for other
nanoparticles, surface charge, size, agglomeration and aggrega-
tion of polystyrene nanoplastics are altered in environmental
media [37], which can thus alter its biological effects. Although
the effects of nanoplastics on marine organisms are poorly
studied, recent studies have shown that polystyrene nano-
particles can induce neurotoxic and genotoxic effects and
interact with pharmaceuticals effects [38]. High levels of poly-
styrene micro and nanoplastics have been reported to induced
mortality in zooplanktonic organisms like the marine copepod
Tigriopus japonicus [27]. In addition to the potential pernicious
effects of the particles by themselves, the fact that they are
present in environments contaminated with substances re-
ported to adsorb onto surface of plastic particles (e.g. poly-
chlorinated biphenyls, polycyclic aromatic hydrocarbons (PAHs),
metals, some of which known carcinogens, and/or endocrine
disrupters) and the ability of micro(nano)particles to modify
distribution, biotransformation and effects of other environ-
mental contaminants (e.g. PAHs and pharmaceuticals) upon
simultaneous exposure [28,38] motivate research on the ability
of micro(nano)plastics to act as transport vectors for environ-
mental contaminants [17,28,38]. These effects should take into
consideration processes like plastic aging/weathering, that may
increase surface area and polarity [39,40].
An important aspect in the study of the consequences of the
presence of micro(nano)plastics in the environment is the link
cause/effect response. If laboratorial assays allow such studies, in
the environment several factors may contribute to effects to biota,
from the particle itself, release of additives and chemicals used in
their synthesis, background contamination of plastics and presence
of pathogenic organisms. It is difficult to assess the risk of envi-
ronmental “contaminated plastics” due to the difficulty of knowing
all associated chemicals.
 M. Oliveira, M. Almeida / Trends in Analytical Chemistry 114 (2019) 196e201
4. Methodologies to quantify and assess the effects of
micro(nano)plastics
Research on micro(nano)plastics may be considered in early
stages. Fate and effects are frequently assessed using different ap-
proaches and exposure scenarios, often with limited environmental
relevance [2,41,42] (e.g., using extremely high concentrations and/
or other non-environmentally relevant conditions). Considering
that there are a high number of variables that may modulate effects
of micro(nano)plastics, there is the need to standardize method-
ologies/approaches to assess effects using, whenever possible, also
environmentally relevant conditions. This aspect is even more
important for nanoparticles as mobility, fate, and bioavailability
depend on their characteristics such as size, shape, charge, and
other properties [38,43], modulated by the surrounding media.
Studies of metallic nanoparticles behavior in artificial seawater
revealed that the time nanoparticles took to aggregate, decreased
with increased particles concentration, an expected output because
the probability of nanoparticles collisions, at a given temperature,
will increase with increasing number of particles per volume [43].
In this perspective, a complete characterization of micro(nano)
plastics is determinant to truly understand their distribution in the
water column as well as their potential pernicious effects. However,
little is known about the fate and behavior of synthetic nano-
materials in the environment, and appropriate methods to detect
them in complex environmental media are not yet available [44].
4.1. Quantification and identification
An accurate assessment of the consequences of the presence of
micro(nano)plastics in the environment requires the knowledge of
its abundance, size distribution and chemical composition. How-
ever, despite the efforts to establish effective analytical procedures,
detection of nanoplastic in environmental substrates is not yet
possible [9,45].
One of the first challenges in the quantification of micro(nano)
plastics in the environment is the sampling procedure. In the ma-
rine environment, the approaches vary according to the position in
the water column. Manta or neuston nets are frequently used in
surface waters, bongo nets on mid-water levels and benthic trawls
usually used for seabed (Table SI1). The most commonly used nets
have a mesh size approximately of 300 mm [7], which compromises
the possibility to quantify and identify the smaller fractions and
leads to an underestimation of the content in aquatic
environments.
For soils, adopted approaches include grounding and sieving
(which, to allow comparison with results obtained in sediments
and water, should use sieves with mesh <5 mm). The mineral phase
of soils may be removed by density fractionation methods, also
used for sediment analysis. However, for soils, procedures to
remove organic moieties must be optimized to remove inference of
soil organic matter in the detection methods [46].
In biological matrices, quantification of microplastics required
extraction and degradation of biogenic matter, detection of the
particles, characterization and quantification. The removal of
organic material in the samples can be achieved through degra-
dation or digestion (e.g. degradation with H2O2 (30%); KOH (10%);
HNO3 (22.5 M); mixture of HNO3 and HClO4), methods that present
different efficiencies as well potential to damage polymers. Thus,
there is the need to standardize procedures that do not compro-
mise the integrity of plastics, in order to accurately estimate of
microplastics content (number of particles, size and type) in bio-
logical materials [23]. In this perspective, enzymatic digestion of
biogenic material has been proposed as an effective approach that
avoids damage to plastic polymers [47], although this process
199
requires specialized equipment, sample desiccation and grinding
[23].
The visual examination of the isolated microplastics is
frequently performed to sort microplastics from other materials.
The use of optical microscope, that allows visualization in the low
micrometer range (Fig. 1) and the use of Nile red, a fluorescent dye
that selectively stains most synthetic polymers (except rubber),
using a UV wavelength that induces green fluorescence [48], allows
optimization of the isolation procedure for posterior identification
of the polymer types. The identification of plastics may be achieved
through different processes but Fourier Transform Infrared Spec-
troscopy (FT-IR) and Raman techniques are among the most used
methodologies, as they do not destruct the material, require low
amount of sample and allow the possibility for high throughput
screening. Furthermore, they may be considered more environ-
mentally friendly than other techniques like differential scanning
calorimetry and pyrolysisegas chromatographyemass spectrom-
etry. Compared to FTIR, Raman techniques show better spatial
resolution (down to 1 mm versus 1e20 of FTIR), wider spectral
coverage, higher sensitivity to non-polar functional groups and
lower water interference and narrower spectral bands [6] but the
proneness to fluorescence interference and requirement of long
measurement time are technical handicaps. Raman is effective in
the identification of polymers but may not be as sensitive as FTIR
when studying polymer degradation. The processes of degradation
and weathering of plastics may cause variation in the spectroscopic
spectra of polymers making identification more difficult.
For nanoplastics, size-based discrimination may be achieved by
ultrafiltration or other separation methods like flow field frac-
tionation and hydrodynamic chromatography. Nanoparticle
tracking analysis [14], dynamic light scattering (which may also
give information of surface charge) [38] and electron/super reso-
lution microscopy may be used as techniques to assess size. Tech-
niques like scanning electron microscopy with energy dispersive X-
ray spectrometry and flow field fractionation coupled to multiangle
light scattering with pyrolysis have also been reported [49]. Com-
bination of atomic force microscopy, super-resolution and infrared
spectroscopy (SRS/CARS) has also been proposed to characterize
material at the nanoscale (Fig. 1) [50].
Although identification and quantification of micro(nano)
plastics on biota are extremely important, the analysis of data of
plastic particles on organisms caught in the environment has to be
carefully examined, as laboratory studies suggest that the absolute
number of ingested microplastics may not accurately reflect the
level of microplastics in seawater, as a consequence of possible
biodilution [23], in which microplastics become less available due
to competition with a growing number of individuals.
4.2. Methodologies to assess effects of Micro(nano)plastics?
The selection of the most adequate approach to test the effects
of environmental contaminants like micro(nano)plastics is depen-
dent on several aspects of the research (e.g. research question and
degree of information required, and model species selected for the
study). Biological effects of micro(nano)plastics may be assessed
using in vivo studies, which involve exposure an organism as a
whole, and allow assessment of lethality, effects on behavior,
feeding, bioaccumulation, depuration, trophic transfer, and
biochemical, physiological and histopathological responses. The
assessment of effects on fish embryos is gaining increasing
importance in the field of ecotoxicology, having been proposed as
an alternative to adult in vivo tests. Among the different approaches
that may be used to assess the effects of micro(nano)plastics is
behavior, which may be considered the outcome or endpoint of
many complex developmental and physiological processes.
200 M. Oliveira, M. Almeida / Trends in Analytical Chemistry 114 (2019) 196e201
However, few studies are addressing effects on these endpoints or
mainly focusing on feeding impairment despite effects on behavior
parameters like movement and avoidance may have ecological
consequences (e.g., altering prey-predator relationship and
decreasing competitive advantages). In vitro methodologies like
primary cell culture and cell lines, allow the study of particular
mechanisms of action, although data from these methodologies do
not provide information on the feedback systems involved in
regulation of organismal responses to environmental stress. Cyto-
toxicity, genotoxicity, distribution within a cell and molecular re-
sponses like expression of genes involved in a metabolic pathway
may be assessed using in vitro systems. These methods are ethically
recommended, whenever they can be used to replace animal
experimentation, with the advantage of allowing a high throughput
approach to investigate specific modes of action and species-
specific responses (e.g. fish and humans).
Molecular endpoints are considered sensitive and efficient tools
to detect effects of xenobiotics and can be assessed in in vivo and
in vitro studies. They are used based on the principle that changes at
a molecular level occur prior to manifestations at higher levels of
biological organization (e.g. reproduction, behavior) and on the
information that they may provide in terms of mechanisms of ac-
tion (through the analysis of genomic modifications; transcription
of genetic information to mRNA; translation to proteins synthesis;
and metabolic products). Molecular endpoints may be used in a
first approach to rank the toxic effects of different micro(nano)
plastics. However, despite the sensitivity of these endpoints, com-
plex regulatory mechanisms in the organism as a whole, may result
in the lack of effects at higher levels of organization. Thus, effects at
higher levels of organization (e.g. behavior) should be also assessed.
5. Research needs
There is an increased need of development of analytical
methods for micro (nano)plastics and their standardization to
allow detection, identification of polymers and quantification in
environmental matrices. This applies not only to water and sedi-
ments but other matrices like biological media as well as food
items. The knowledge on how plastic particles of different poly-
mers are distributed in the water column and the role of abiotic
conditions on their environmental distribution, behavior and
bioavailability is poorly known and should be studied. Limited in-
formation is also available in terms of nanoplastics, particularly in
terms of potential for formation of different polymers, under
environmentally relevant conditions, their stability/aggregation
and deposition.
Soils are being neglected in terms of microplastics research (e.g.
their effects to soil organisms and degradation), despite the
recognition of soils potential to retain and accumulate plastic par-
ticles, and the use of plastics in agricultural. Although some labo-
ratorial studies are available, there is no field data [22]. It is, thus,
imperative to develop this aspect of microplastic research.
In terms of biological effects, plastic age-related effects should
be studied considering that weathering processes alter the struc-
ture of the particles, their structural consistency, ability to release
chemicals used in their synthesis and to adsorb environmental
contaminants. Despite the technical challenges, studies with
nanoplastics should be performed to establish the role of size on
biological effects, bioaccumulation, trophic transfer and trojan
horse effect. This is particularly important considering the size
dependent effects reported for other types of nanoparticles in
laboratory studies. Pathways of incorporation and distribution of
particles once incorporated by organisms should be studied (e.g.
using fluorescent labelled particles and histology), to determine
potential main target tissues and modes of toxic action. Most of the
available studies are focusing on short-term exposures. Considering
the persistent nature of plastics in the environment, long-term
studies involving multigenerational exposures should also be
considered. Furthermore, identification of more sensitive phyla and
life stages is also important to detect areas of increased risk, rank
polymers in terms of noxious effects and ultimately suggest
controlled production/use of polymers. The study of the role of
non-chemical factors in the effects of micro(nano)plastics (e.g.
ocean acidification, biotic stress) should also be studied.
Effects on humans should also be assessed. In this perspective,
in vitro systems may be valuable tools as they allow the study of
modes of actions in organisms like fish and humans.
6. Final considerations
The presence of small plastic particles in the environment has
motivated research in the field of chemistry (e.g. methodologies to
isolate, identify and characterize polymers) and biology (e.g.
studies of incorporation, translocation, biochemical, behavioral,
histological effects and trophic transfer). Nanoplastics appear as a
new challenge requiring improved methodologies to isolate,
quantify and characterize and relate biological effects with particles
characteristics. There is also the need to standardize methodology
to allow integration of field and laboratory data. The implementa-
tion of new approaches to study the effects in the biota and humans
(e.g. in vitro) are needed.
Acknowledgments
CESAM (UID/AMB/50017/2019) received financial support by
FCT/MEC through national funds. Project MO had financial support
of the program Investigador FCT (IF/00335/2015), co-funded by the
Human Potential Operational Programme and European Social
Fund.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.trac.2019.02.023.
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