Science of The Total Environment: Review

Science of the Total Environment 732 (2020) 138792
Contents lists available at ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
Review
Nano-plastics and their analytical characterisation and fate in the marine

environment: From source to sea
Sheeana Gangadoo a, Stephanie Owen a, Piumie Rajapaksha a, Katie Plaisted a, Samuel Cheeseman a,
Hajar Haddara a, Vi Khanh Truong a, Son Tung Ngo b, Van V. Vu c, Daniel Cozzolino a,d, Aaron Elbourne a,
Russell Crawford a, Kay Latham a, James Chapman a,⁎
a
School of Science, RMIT University, Melbourne, VIC 3000, Australia
b
Laboratory of Theoretical and Computational Biophysics, Ton Duc Thang University, Ho Chi Minh City 758307, Viet Nam
c
NTT Hi-Tech Institute, Nguyen Tat Thanh University, Ho Chi Minh City 70000, Viet Nam
d
Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Brisbane
H I G H L I G H T S G R A P H I C A L A B S T R A C T
• Polymer contamination has reached a

breaking point.
• Contamination has now been observed
in the Earth's most remote oceans.
• Macro and micro plastics have been
discussed comprehensively.
• Nanoplastics are an overlooked issue,
this review focuses on nanoplastics.
• Sources, breakdown, fates, uptake, and
characterisation will be discussed.
a r t i c l e i n f o a b s t r a c t
Article history: Polymer contamination is a major pollutant in all waterways and a significant concern of the 21st Century,
Received 31 October 2019 gaining extensive research, media, and public attention. The polymer pollution problem is so vast; plastics are
Received in revised form 16 April 2020 now observed in some of the Earth's most remote regions such as the Mariana trench. These polymers enter
Accepted 16 April 2020
the waterways, migrate, breakdown; albeit slowly, and then interact with the environment and the surrounding
Available online 5 May 2020
biodiversity. It is these biodiversity and ecosystem interactions that are causing the most nervousness, where
Editor: Damia Barcelo health researchers have demonstrated that plastics have entered the human food chain, also showing that plas-
tics are damaging organisms, animals, and plants. Many researchers have focused on reviewing the macro and
Keywords: micro-forms of these polymer contaminants, demonstrating a lack of scientific data and also a lack of investiga-
Plastic tion regarding nano-sized polymers. It is these nano-polymers that have the greatest potential to cause the most
Pollution harm to our oceans, waterways, and wildlife. This review has been especially ruthless in discussing nano-sized
Nanoplastic polymers, their ability to interact with organisms, and the potential for these nano-polymers to cause environ-
Microplastic mental damage in the marine environment. This review details the breakdown of macro-, micro-, and nano-
Water
polymer contamination, examining the sources, the interactions, and the fates of all of these polymer sizes in
Soil
the environment. The main focus of this review is to perform a comprehensive examination of the literature of
Plasticizers
the interaction of nanoplastics with organisms, soils, and waters; followed by the discussion of toxicological
⁎ Corresponding author.
E-mail address: [email protected] (J. Chapman).
https://doi.org/10.1016/j.scitotenv.2020.138792
0048-9697/© 2020 Published by Elsevier B.V.
2 S. Gangadoo et al. / Science of the Total Environment 732 (2020) 138792
issues. A significant focus of the review is also on current analytical characterisation techniques for nanoplastics,
which will enable researchers to develop protocols for nanopolymer analysis and enhance understanding of
nanoplastics in the marine environment.
© 2020 Published by Elsevier B.V.
Contents
1. Plastic in the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. Lifecycle of macro- and microplastic in the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1. Macroplastics (MaPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1.1. Sources of MaPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1.2. MaPs interaction with marine environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1.3. Fate of MaPs in marine environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2. Microplastics (MiPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2.1. Sources of MiPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2.2. MiPs interaction with marine environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2.3. Fate of MiPs in marine environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3. NaPs: sources and lifecycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3.1. Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.4. NaPs: bioaccumulation and fate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.4.1. Sediments, soil & streams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.4.2. Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3. Analysis of plastics in the marine environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.1. MaPs analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.2. MiPs analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.3. NaPs analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.3.1. Characterisation of plastic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.3.2. NaPs detection in sediments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.3.3. NaPs detection in water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.3.4. NaPs detection in marine organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.3.5. Analysis of NaPs degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.4. Challenges in NaP analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3.4.1. Challenges of NaPs characterisation in the natural environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4. Conclusions and future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Declaration of competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1. Plastic in the environment the classification of the material to fall under the “hazardous” (Rochman
et al., 2013). The extent and mechanisms of plastics' impacts on the envi-
The value of mouldable, resilient polymers has been recognised since ronment are still being established, potential areas under investigation in-
at least 1600 BCE. Initially, natural organic compounds such as latex- clude: the ability to travel through the food chain (Laskar and Kumar,
based rubbers and shellac were used to make balls, containers and water- 2019), the accumulation of toxic additives in living organisms, the pre-
proof coatings. The first synthetic polymers, based on nitrocellulose, were vention of gas-exchange of sea-beds;, and the creation of artificial
developed in the nineteenth Century. In 1907, came the first fully syn- grounds (Rochman et al., 2016; Wang et al., 2017). Beyond its ecological
thetic, petroleum-based polymer, Bakelite – its inventor, Leo Baekeland, impacts, environmental plastic pollution can also directly affect global
was the first to use the word ‘plastics’. Over the next few decades, plastics economies through tourism and the fishing industries, among others
were enthusiastically embraced by industry and the general public, as (Bhargava et al., 2018).
new types (such as PVC, acrylic, nylon etc.) were invented and new The excessive use of plastics, coupled with their inherent resistance
uses found for them, particularly during the resource demands of World to degradation, culminates in a significant environmental problem.
War II. Plastic production and plastic consumption is now at an all-time Once plastics are in the natural environment, they are susceptible to
high, and plastic pollution has become a major environmental concern degradation via abiotic or biotic processes (Canesi et al., 2015). Abiotic
(Andrady and Neal, 2009; Fenichell, 1996). Current estimates reveal factors primarily result in structural and mechanical degradation
that the use of plastic is set to increase rates of consumption in excess of which induces increased surface areas and/or active sites for microbial
260 million tonnes per annum (Thompson et al., 2009), making up 10% attack or physicochemical interactions (Gewert et al., 2015; Webb
of the waste discarded globally (Barnes et al., 2009). Plastic waste is et al., 2013). Some of the most important abiotic processes are
largely contributed to by land sources (~80%), with a small percentage thermal-, photo- and chemical-degradation. For example, mechanical-
(~20%) coming from marine environments. Plastic pollution has been degradation processes are facilitated by the action of waves, currents,
shown to affect both single-celled species, bacteria and fungi, and larger or attrition with rocks, sand, and other sediments (Gewert et al., 2015;
organisms, such as mammals and birds (Fu et al., 2018; Li et al., 2016a). Cooper, 2012). Floating plastic debris, which has greater exposure pe-
Many of the mass-produced modern plastics, such as polyethylene (PE) riods to sunlight, undergoes chemical changes driven by UV radiation
and polypropylene (PP) are based on saturated hydrocarbon polymers, exposure and the oxidative properties of the atmosphere. In the deep
which contributes to their chemical and biological stability (Glaser, ocean, where temperature and the rates of sunlight- and oxidation-
2019). The persistence of these plastics in the environment, along with mediated processes are reduced, abiotic degradation is much lower
the release of toxins and chemicals from their degradation, has led to than in comparison to near ocean surface exposures (Gewert et al.,
S. Gangadoo et al. / Science of the Total Environment 732 (2020) 138792 3
2015). This is also due to the reduced level of biodiversity, and smaller items such as plastic bags, plastic bottles, food packaging, and even plas-
microbial communities, and therefore the degradation processes are tic waste from the fishing industry. The polluting effects of MaPs is a
limited. For biotic processes, plastic degradation is initiated outside of prominent problem for the environment, particularly in marine settings
the cells. This is usually brought about by the action of enzymes, where MaPs have become rapidly dispersed and transported across
resulting in the cleavage of polymeric chains (Glaser, 2019; Gewert large distances (Barnes et al., 2009). Studies have indicated a multitude
et al., 2015). of detrimental consequences including physical harm to marine life
The degradation of plastics varies with type of plastic and composi- through direct and/or indirect contact with the polymers (Laist, 1987),
tion, and as such, can be categorised: plastics with a ‘carbon-carbon and its disintegration into finer particles such as micro- and NaPs
backbone’ (for example polyethylene (PE), polypropylene (PP), polysty- (Thompson et al., 2004). The latter have separate environmental conse-
rene (PS) and polyvinyl chloride (PVC), and plastics with ‘heteroatoms in quences and will be discussed in detail, later in this review. Ocean gyres,
the main chain’, for example poly(ethylene terephthalate) (PET) and formed from air currents between the tropics and the poles, have been
polyurethane (PU)) (Gewert et al., 2015). Plastics containing carbon- found to accumulate waste, particularly plastic waste; due to their
carbon backbones are the primary constituents of packaging materials, buoyancy and resistance to degradation (Wabnitz and Nichols, 2010).
which are typically discarded over short time frames, resulting in an in- Additionally, the largest of the ocean gyres, The “Great Pacific Garbage
creased probability of such plastics entering the environment in larger Patch”, is believed to be around ~80 t, 1.6 million km2 (approximately
quantities (O'Brine and Thompson, 2010). Abiotic degradation is the four times the size of Germany), and composed of N99% plastic, of
main process for carbon-carbon backbone plastic degradation (Gewert which ~75% is MaP, with a recent study by Lebreton et al. (2018) sug-
et al., 2015), and typically, initiation, propagation, and termination of gesting that it is rapidly accumulating more plastic.
the chemical bonds in the main polymer chain will occur and precede There are a range of sources of MaP pollution, with the largest pollut-
biotic degradation (Gewert et al., 2015; O'Brine and Thompson, 2010). ing contributors being single use items such as plastic bottles, plastic
Plastics with ‘carbon and heteroatomic chains’ have increased thermal bags, disposable plastic straws and cutlery (Hopewell et al., 2009), and
stability compared to their carbon-backbone polymeric counterparts, waste from the fishing industry such as netting (Li et al., 2016a). The dif-
and are susceptible to hydrolytic cleavage of the ester and amide ficulties associated with determining the original source of MaP pollut-
bonds (which may be prevalent in the polymer) (O'Brine and ants on a large scale has resulted in an inability to accurately identify
Thompson, 2010). and quantify the major sources of MaP pollution, and thus either track
This literature review will provide a comprehensive insight into the where these pollutants have originated from and/or prevent future re-
impact of nanoplastics (NaPs) in the environment, their sources, the ef- leases. However, it is thought that a significant portion is derived from
fects, interactions and fate of nanoplastics, along with current detection marine waste mismanagement and/or poor waste management sys-
methods and their challenges in reliable detection. Fig. 1 displays a tems on land, particularly in countries with large populations
schematic of plastic sources and their fate in the environment. (Jambeck et al., 2015).
It should also be noted many plastics are not composed of the same
2. Lifecycle of macro- and microplastic in the environment generic polymeric material and in fact have other compounds intro-
duced to bring about different physical characteristics for the polymer.
2.1. Macroplastics (MaPs) In fact, many plastics are chemically doped with non-covalent com-
pounds known as plasticisers (Brown et al., 2017; Chapman and
2.1.1. Sources of MaPs Regan, 2011; Navarro et al., 2010). These plasticizers are additives that
MaPs are commonly defined as “any plastic material with a size di- give plastics desirable properties. Plasticizers work by reducing the
mension greater than 5 mm”, with the most common sources including chemical affinity between the polymer molecules when embedded
Fig. 1. Schematic diagram to show the sources and fate of plastics and how plastics reach the marine environment (Geyer et al., 2017; Neufeld et al., 2016).
between chains of the raw materials which go into making plastic (or such as biological, anthropogenic, ocean currents and wind exposure
act as monomers in polycarbonate plastic). Many of these plasticizers (Ríos et al., 2018). Fig. 2 depicts the typical pathway of MaPs within
are not stable in these products, they can leach out and thus end up in the marine environment.
the environment. For example, PVC is particularly susceptible to ther-
mal and photodegradation, this ease of breakdown also causes these ad-
ditives to be released. Phthalates are a widely used group of plasticizing 2.1.3. Fate of MaPs in marine environment
chemicals that are primarily utilised in PVC polymers. Di-2-ethylexyl MaPs are readily degraded into smaller size polymer profiles (as
phthalate (DEHP) is the major plasticizer used in medical devices, tub- seen from the degradation of coffee cup lids into NaPs) (Lambert and
ing, blood bags, food protection for example. Further, polybrominated Wagner, 2016), through various processes including mechanical, chem-
diphenyl ethers, which are often added to plastics like PVC, can leach ical, and/or biological routes. The primary breakdown of long chain
from discarded food packaging's into the marine environment, also pre- polymers occurs through photo-oxidative degradation, which is initi-
senting leached as endocrine disruptors. In the review we will discuss ated from UV-radiation in combination with free oxygen available in
the importance and potential environmental impact of these plasticisers the water, and leads to the scission of the carbon-carbon backbone
and additives at the macro, micro, and nano level. (Gewert et al., 2015). The scission of these carbon chains significantly
reduces the mechanical properties of the plastics allowing them to
2.1.2. MaPs interaction with marine environment break down into smaller fragments, which is aided by the natural and
Given the magnitude of plastic availability in the environment, there mechanical processes such as wave and wind actions (Andrady,
are significant opportunities for interactions with organisms, plants, and 2015). These small polymer fragments are then further broken down,
bodies of water. MaPs can be ingested by marine vertebrates, such as through hydrolysis and biological degradation progressions (Gewert
whales (Lusher et al., 2015), turtles (Bugoni et al., 2001), pelagic fish et al., 2015). The degradation process of MaPs is typically generalised,
(Romeo et al., 2015), demersal fish (Murphy et al., 2017), and seabirds but due to the heterogeneous nature of the marine environment, the
(Žydelis et al., 2013) among many others (Laist, 1987). MaPs have primary factor responsible for degradation also relies on the surround-
been observed to harm marine life through a wide range of direct and ing specific conditions. A study on the degradation of high-density poly-
indirect effects including entanglement or capture, resulting in drown- ethylene, polypropylene and polystyrene in a salt marsh environment
ing, an inability to feed, a greater target for predation, or cause a suggested that the primary degradation factor of these plastic materials
wound infliction, promoting the chance of infection (Laist, 1997; involves the formation of a biofilm and subsequent microbial degrada-
Gregory, 2009) as well as physical rupture, and/or inhibition of an im- tion, in combination with other factors, initiating the breakdown
portant organ upon ingestion (Lusher et al., 2015; Gregory, 2009). through surface delamination (Weinstein et al., 2016). Additionally, a
MaPs can also function as a mode of transportation for invasive and/or recent study by Julienne et al. (2019) indicated that the presence of
pathogenic species (Masó et al., 2003; Barnes, 2002) as well as the water played a significant role in the degradation process, promoting
leaching of toxic compounds such as heavy metals (Nakashima et al., cracking in polyethylene films compared to those left in the air. A
2012; Li et al., 2016b) and trace metals (Munier and Bendell, 2018). large proportion of this research area acknowledges the multi-factorial
Lastly, the degradation and release of smaller micro- and nano- nature of the degradation process and inconveniently, the lack of a
particle sized polymers into marine environments showcases that the “one-size-fits-all” description of this process. It is thus important to con-
endpoint of MaPs is highly variable and difficult to predict as the poly- sider the combination of degradation components when studying or
mers are influenced by the interactions of a number of host factors implementing policy surrounding MaPs pollution, as different factors
Fig. 2. MaPs cycle from sediments to marine environment. A: entanglement of turtles with fishnet [National geographic]; B: satellite imaging of agricultural plastic waste (Lanorte et al.,
2017); C: ingestion of fishing equipment with sea birds [abc.net.au]; D: fur seal entangled (Barnes et al., 2010); E: ingestion of various plastic materials by whale [The Guardian]; F: remote
sensing of marine MaPs (Ritchie, n.d.).
are more or less important in relation to different environmental con- the fragmentation of MaPs debris, pre-production pellets (nurdles),
texts or plastic compositions. spillage during transportation and fabrication, waste-water debris, and
As previously mentioned, plasticized polymers are a cause for con- road-run-off fragments (Nelms et al., 2019), along with contribution
cern for any species which comes in to contact with them. Many from maritime activities (Smith et al., 2018). MiP sources can be
plasticisers and additives that leach from the polymeric matrix are of categorised as primary, occurring directly from domestic and industrial
an endocrine mimicking nature, thus resulting in biological functions applications and indirectly from surface run-off spillages, and secondary
being turned on, or cell functions being compromised through competi- consisting of the breakdown of MaPs to smaller MiPs due to various
tion, or toxicity towards the organism in high concentrations resulting degradation processes, as mentioned earlier (de Sá et al., 2018; Smith
in cellular death. In a study by Andrady (2011), “plasticizers tend to mi- et al., 2018). Fig. 3 shows the two contributing sources of MiPs and
grate slowly to the surface of the polymer and can therefore enter the their fate and interaction in the marine environment.
environment in low levels”. The effects of plasticizers at the macro A recent study indicated that the high amount of fibres from textiles,
level, would apply to higher trophic level organisms such as amphib- produced from increased washing use, can lead to increased production
ians, or fish. For example, plasticizers in amphibians have been shown of MiPs, with types ranging from polyester, polyamide, polypropylene
to be teratogenic and endocrine disrupting (Lee and Veeramachaneni, and acrylic (Smith et al., 2018; Browne et al., 2011). The different
2005). Given that polymers have travelled for many miles and over types of plastic determines its settling in the marine environment,
vast areas, the occurrence of plasticizers, and especially the most widely where materials such as polyamide, polyester, polymerizing vinyl chlo-
used, phthalates, aquatic species such as fish can also be exposed to ride (PVC) and acrylic sink to the bottom of the ocean, while polyethyl-
phthalates. For example, phthalates can bioconcentrate in fish, di-(2- ene, polypropylene and polystyrene tend to float on surface waters
ethyl hexyl-phthalate) (DEHP) was 120 kg−1 (wet wt.) in the carp (Smith et al., 2018). These MiPs debris accumulate in marine and terres-
and for BBP, 9.4 kg−1 (wet wt.), in bluegill sunfish, illustrating the prev- trial animals and contribute towards the presence of Persistent Organic
alence of the issue (Staples et al., 1998). Pollutants (POPs), such as polychlorinated biphenyls (PCBs), polycyclic
aromatic hydrocarbons (PAHs), and organochlorine pesticides (Eg: DDT
2.2. Microplastics (MiPs) or hexachlorobenzene (HCB)) (Browne et al., 2011). It has been re-
ported that sewage treatment plants are not equipped for the proper
2.2.1. Sources of MiPs disposal of MiPs, as contamination of fibres and polymeric MiPs were
MiPs are composed of plastic debris of sizes b5 mm (de Sá et al., found in sludge material (Browne et al., 2011; Zubris and Richards,
2018; Nelms et al., 2019; Smith et al., 2018) and have been recorded 2005; Kay et al., 2018).
in 18 shores across 6 continents, including the capital city of Portugal,
Faro (population census ~120,000), Sennon Cove, UK (population cen- 2.2.2. MiPs interaction with marine environment
sus ~200) with the highest count, and Port Douglas, Australia (popula- The UN reported that over 200 marine species, ranging from larger
tion census ~3500) with the lowest MiPs count (Browne et al., 2011). vertebrates, as mentioned above, to filter feeders, such as zooplanktons,
Global statistics show that the U.S. releases around 8 billion microbeads are affected by MiPs and their additives, through ingestion and accumu-
in aquatic environments daily, with the MiP debris mostly located in the lation onto the body surface (Nelms et al., 2019; Smith et al., 2018;
water column and sediments (Smith et al., 2018). The most common Browne et al., 2011). Similar to MaPs, MiPs can leach monomers and
MiP types include polyethylene and polypropylene (Smith et al., other toxins, allowing the direct and indirect transportation of these
2018), with 80% of MiPs originating from land-based activities, such as contaminants across organisms (de Sá et al., 2018; Smith et al., 2018).
Fig. 3. MiPs cycle from primary and secondary sources to marine environment. A: Raman analysis of plastic sample (Zhao et al., 2017); B: fluorescence microscopy of zooplankton (Cole
et al., 2014); C: fluorescence microscopy of mussel gut (Browne et al., 2008); D: polarized-light microscopy of mussel gut (Avio et al., 2015); E: fluorescence microscopy of crab gills (Farrell
and Nelson, 2013).
Studies have shown the presence of MiPs in the gut and the digestive tu- parameters of NaPs, resulting from macro- and micro-plastic degrada-
bules of mussels and are believed to cross onto the hemolymph and af- tion, alter regularly with a change in the environment (Gigault et al.,
fect the immune system (Canesi et al., 2010). The accumulation of these 2018). A literature search of the effect of NaPs on the marine environ-
particles additionally provides a route for hydrophobic organic contam- ment was conducted using Google Scholar with the following keywords
inants present in sea water (Browne et al., 2011), and can be further entered: sediments; marine; polystyrene; nanoparticles, and publications
transported across the food chain, eventually reaching humans. MiPs af- were chosen from 2009 to 2018.
fect marine organisms by reducing energy reserves, altering intestinal The presence of NaPs can be divided into two main sources: primary
and reproduction processes, and affecting brain tissue morphology sources, including plastics entering the environment at the nanoscale
(Nelms et al., 2019). MiPs of polyester and acrylic fibre materials also size from the inadequate disposal of specific products, and secondary
contain causative agents and monomers that can result in dermatitis sources, which result from the degradation of MaPs and MiPs (Ferreira
(Browne et al., 2011). et al., 2019). The degradation of plastics significantly reduces their mo-
Microplastics and plasticizers especially where phthalic acid esters lecular weight and increases their bioavailability to marine and land
(phthalates) have been used represent widespread contamination in biota (Santos et al., 2009). Research indicates NaPs are severely toxic
waters, sediments, and soils. The true knowledge of plasticizer concen- to humans as shown by disruption of the cell membrane of the intestinal
trations is still unknown and represent a secondary pollutant load in villi, as well as the release and wide distribution of chemical additives
this vast contamination issue. Wu et al. (2019) have performed a recent across the body (Mahler et al., 2012; Bouwmeester et al., 2015).
systematic study to ascertain the adsorption mechanisms of five com-
mon biphenyl analogues (BPA, BPS, BFP, BPB, and BPAF) on 2.3.1. Sources
microplastics. This is an important study, as the mechanism interpreta- NaPs are generated from a range of sources: primary, which includes
tions demonstrated that hydrophobic interactions, electrostatic forces, their release from various products and applications such as cosmetics
and noncovalent bonds are critical mechanisms affecting the adsorption (da Costa et al., 2016), paints (Oliveira et al., 2019), biomedical and
of five bisphenols on PVC microplastic, which can give an indication on drug products (Pohlmann et al., 2013), electronics (Blair et al., 2017);
the levels that may be leaching in future and more fundamental labora- a secondary source due to MiPs fragmentation – the physical friction
tory studies (Wu et al., 2019). Further, the importance of distinguishing and breakage of the lower energy bonds within polymer chains; and a
between microplastic and the containing leachable additives will be- third possible source – MiPs' degradation, which as mentioned earlier,
come another fundamental issue in the detection of these chemicals results from the deterioration of the MiPs' surface due to abiotic and bi-
and materials. otic weathering (Koelmans et al., 2015). The fragmentation of MiPs is a
major category, involving fragmentation at the surface of the MaPs or
2.2.3. Fate of MiPs in marine environment MiPs, followed by gradual size reductions due to natural or artificial
MiPs are highly versatile, able to resist multiple environmental deg- degradation processes (Koelmans et al., 2015). The major source of
radation pathways, and therefore have a slow degradation process and NaPs is associated with the production and application of plastic prod-
are persistent in the environment for a long time (Klein et al., 2018). The ucts and their life cycles, with physical abrasion of larger plastic particles
degradation process of synthetic MiPs can be categorised into physical, shown to be the main source process of NaPs, as shown by Fig. 4
chemical, biological and photodegradation processes, and vary due to a (Koelmans et al., 2015).
number of factors, such as polymer type, shape, density, weathering, pH, The behaviour of NaPs in the marine environment can be attributed
age, temperature, irradiation and its location in the ocean's water col- to properties such as agglomeration, aggregation, dissolution, and redox
umn (Smith et al., 2018). The physical process includes the exposure reactions, which are further believed to be regulated by their surface
of MiPs to friction forces, where the size of the plastic material is re- properties and ionic strength control (Quigg et al., 2013; Schirmer
duced and the surface area to volume ratio is increased, while the chem- et al., 2013). Studies utilising engineered NaPs found they typically re-
ical degradation is initiated from the aging process of the plastic main stable in demineralized and freshwater mediums (Brandts et al.,
material (Klein et al., 2018; Kooi et al., 2018; Duwez and Nysten, 2018a), whereas a continuous increase of particle size and dispersity
2001). The molecular level of degradation of MiPs is instigated by has been observed of NaPs exposed to different environments such as
photodegradation occurring from UV light exposure and chemical oxi- artificial sea water (Brandts et al., 2018a), natural sea water (Wegner
dation from hydrolysis (Klein et al., 2018; Andrady, 2011). This degra- et al., 2012; Marques-Santos et al., 2018), bacterial mediums (Nasser
dation process is fast and depends greatly on the chemical and Lynch, 2016) and biological fluids (Marques-Santos et al., 2018).
composition of the polymer (Klein et al., 2018). The biological degrada- Nasser et al. reported the adsorption of a thin protein layer onto the
tion of MiPs can occur through aerobic and anaerobic environmental NaP surface, resulting in agglomeration and an increase of size of over
conditions, and is caused by the biodegradation action of specific micro- 100 nm (Nasser and Lynch, 2016). This review paper has found that
organisms such as Bacillus cereus (Auta et al., 2018), Pseudomonas sp. while most research examined engineered NaPs to study their behav-
(Reddy et al., 2009), Micrococcus sp. and Corynebacterium sp. (Smith iour, fate and toxicity in the marine environment, very few studies
et al., 2018; Priyanka and Archana, 2011). The biodegradation of these have investigated naturally-occurring NaPs.
MiPs rely greatly on the environmental parameters present and can re-
sult in the complete or partial mineralization of the polymer, forming 2.4. NaPs: bioaccumulation and fate
various compounds, salts, minerals and biomass (Grima et al., 2000).
The degradation of MiPs leads to smaller plastic particles below the 2.4.1. Sediments, soil & streams
size of 1000 nm, termed NaPs, leading to this review's focus.
2.4.1.1. Transportation and interaction. The transportation of NaPs differs
2.3. NaPs: sources and lifecycle in various sediment material and stream types, with less mobility ob-
served in loamy sand than in quartz sand, and less mobility with
There is a debate in the environmental science literature in terms of biofilm-quartz sand than uncoated clean sand (Mitzel et al., 2016).
defining “nanoplastic” and its size-range, with 1–100 nm sizes generally The strong presence of ions in soil sediments resulting from high con-
assigned to intentionally-engineered NaPs, and 1–1000 nm range centrations of CaCl2 and KCl, alters the surface charge of NaPs rendering
assigned to plastic nanomaterial resulting from the degradation and them less negative, significantly increasing the attachment efficiency of
breakdown of aged-MiPs (Gigault et al., 2018). Their colloidal behaviour NaPs to other surrounding particles and influencing their movement
differs vastly from each other, with controlled surface and structure ob- across the soil component. Additionally, the hydrodynamic diameter
served from engineered nanomaterials, while the physical and chemical of NaP increases in monovalent solutions, with significant aggregation
Fig. 4. NaPs cycle from source to marine environment. A: Confocal images of PS-COOH NaPs (green) in sea urchin embryos (Della Torre et al., 2014); B: Optical microscopy images of PS
NaPs (green) in D. galeata (Cui et al., 2017); C: Fluorescence microscopy images of PS NaPs (green) in the gut of D. magna (Brun et al., 2017); D: Confocal images of PS NaPs (green)
(Rosenkranz et al., 2009).
observed in divalent solutions (Quevedo and Tufenkji, 2012). This ag- 2.4.2. Organisms
glomeration is believed to occur at the surface of the soil particle NaPs and their aggregates affect the following primary marine or-
(Awet et al., 2018). Most studies have modified NaPs with functional ganisms: phytoplanktons, filter feeders through surface waters, fish,
groups, carboxyl (COOH), amine (NH2), and sulphate (SO2− 4 ) to repli- mammals and deep-sea species, through different pathways. NaPs
cate the cationic (C+) and anionic (A−) surface charge of NaPs that have been shown to have the ability to permeate lipid membranes
can be observed in a marine environment, and have found that differing and to affect cellular function (Rossi and Monticelli, 2014). The physico-
charges interact with the side chains of lipids and amino acids distinctly, chemical properties of NaPs, such as size and surface charge are signifi-
altering the protein's secondary structures of extracellular polymeric cant parameters to their ingestion, accumulation, toxicity and excretion,
substances (EPS) in activated sludge of waste water treatment plants and affect organisms differently. Tables 1 & 2 below shows studies for
(Feng et al., 2018). the past 10 years conducted with various plastic compositions, size-
scales and surface modifications.
2.4.1.2. Toxicity and fate. NaPs have the ability to increase the transporta-
tion of contaminants of nonpolar and weakly polar composition by
physical adsorption and entrapment within the polymeric matrices, as 2.4.2.1. Ingestion & accumulation. NaPs and their additives enter and ac-
observed in soil sediment samples (Chen et al., 2018b). Pesticides, cumulate in marine organisms through various ways including deposi-
such as glyphosate, are adsorbed onto NaPs in soil and exert a strong tion, ingestion and endocytosis through cell membranes (Rossi and
toxicity effect in blue-green algae, Microcystis aeruginosa (Zhang et al., Monticelli, 2014; Salvati et al., 2011). NaPs can accumulate on biofilms
2018). A higher absorption of contaminant onto NaPs than MiPs has of a range of micro-organisms such as Alteromonas macleodii (Nevius
been observed, with NaPs enhancing the bioaccumulation of et al., 2012), Pseudomonas fluorescens (Nomura et al., 2016),
phenanthrene-derived residues onto the surface body of daphnids Ankistrodesmus angustus, Phaeodactylum tricornutum and Amphora sp.,
(Ma et al., 2016). NaPs have significant detrimental effect on the soil where they have been observed to accelerate the growth kinetics and
ecological community manifesting as a reduction of microbial biomass, assembly of exopolymeric substances (EPS) within the biofilm at con-
diversity and enzyme functional diversity (Awet et al., 2018; Zhu et al., centrations as low as 10 ppb (Chen et al., 2011). Ca-NaPs with NH+ 2
2018). NaPs have shown detrimental effect towards soil oligochaete functional groups, closely cover the surface of planktonic cells and are
Enchytraeus crypticus, key organisms significant for nitrogen cycling adsorbed at a higher degree onto cellulose films and the cell walls of
and organic matter decomposition, by reducing growth (Zhu et al., microalgae, than An-NPs with COOH functional groups, due to the
2018). Toxicity of NaPs towards bacterial communities is achieved latter's electrostatic repulsion to cellulose (Nomura et al., 2016;
through the adhesion of Ca-NaPs onto cell surfaces, resulting in cell dis- Bhattacharya et al., 2010; Nolte et al., 2017). The hardness of a medium
ruption in species such as Lactococcus lactis, whereas An-NaPs contrib- can significantly alter the surface charge of NaPs and therefore influence
ute very little to the toxic effect due to the charge repulsion exhibited their absorption kinetics with microorganisms (Nolte et al., 2017).
between the particles and bacterial cell surface (Nomura et al., 2015). Other studies show opposing results with An-NaPs, containing SO2− 4
The hydration properties of biofilms also contribute significantly to functional group, to accumulate at a higher rate onto Alteromonas
the retention and transportation of NaPs across a marine environment, macleodii biofilms than COOH and NH+ 2 functionalised NaPs (Nevius
as one study shows a clear difference of NaP deposition on two et al., 2012). NaPs are found to be typically ingested at a slower rate
P. aeruginosa strains that differed in cell surface hydrophobicity than MiPs, for example in suspension-feeding bivalves with their cap-
(Mitzel et al., 2016). turing efficiency of gills reduced with the smaller size of the particulate
Table 1
Studies of NaPs toxicity in soils, sediments & streams.
Type Surface Size Sample Major findings Reference

modifications
PS Carboxylated 24 nm Quartz & loamy sand • Difference in binding affinities of NaP to sediment constituents determine transpor- Quevedo I. R.
(COOH) tation and retention of NaPs 2012
PS Aminated (NH2) 100 nm Lactic acid bacteria • Toxicity to bacterial communities in sediments Nomura T.
2015
PS COOH; sulphated 25–40 nm P. aeruginosa biofilm • Biofilm increased retention and affect transportation of NaPs Mitzel M. R.
(SO2−
4 ) 2016
PS Non-functionalised ~30 nm Soil • Reduced microbial biomass and functional diversity of enzymes in soil Awet T.T.
(nf) • Increased in basal respiration and metabolic quotient 2018
PS nf 25–100 nm Wastewater sludge • Affect activity of activated sludge of wastewater treatment Feng L. 2018
• Protein secondary structures in EPS modified by NaP and caused bio-flocculation of
activated sludge
PS nf ~80 nm Soil • Enhance transport of non-polar and weakly polar contaminants Liu J. 2018
PS nf 50–100 nm Oligochaete Enchytraeus • Reduce growth of soil worms Zhu B. 2018
crypticus
(Ward and Kach, 2009). The ingestion and accumulation of the NaPs can critical for early development, which are then excreted or detached
be shown across the intestines of adult D. magna and its embryos from the brood pouch of the planktons onto their marine environ-
(Rosenkranz et al., 2009), mussels, Mytilus edulis and oysters, ments (Cui et al., 2017; Brun et al., 2017). NaPs can significantly af-
Crassostrea virginica, with aggregated NaPs more readily ingested than fect reproduction, affecting the size and numbers of lipid droplets
freely suspended NaPs (Ward and Kach, 2009). Rotifers, Brachionus resulting in their abnormal growth (Cui et al., 2017). One study sug-
manjavacas, have been observed to ingest NaPs from sizes gested the primary route of NaPs accumulation from adult crusta-
37–2980 nm, with larger sizes of N83 nm remaining in the stomach ceans to embryos does not occur through maternal NaPs uptake
and intestines, while smaller sizes were found crossing the intestinal (Brun et al., 2017; Pinsino et al., 2017). NaPs can travel up the food
wall, entering various tissues, affecting the immune system and passing chain and significantly affect fish behaviour (hunting activity & feed-
onto extruded eggs to the next generation of rotifer (Snell and Hicks, ing rate), liver and muscle metabolism, and crossing the blood-brain
2011). The ingestion of NH+ 2 NaPs occurs at a higher rate in Crassostrea barrier, altering brain tissue morphology (Cedervall et al., 2012;
gigas (Pacific oyster) than COOH NaPs (Cole and Galloway, 2015), due to Mattsson et al., 2014, 2017).
the stronger interaction of the positive charge with the negatively- The size of the NaPs contributes to their relative toxicity, with some
charged cell membrane (Nasser and Lynch, 2016). NaPs have the ability larger particles showing higher toxicity rates in certain organisms
to travel through the food chain across numerous trophic levels, from (Casado et al., 2013). However, the greatest contribution of NaPs toxic-
algae (Scenedesmus sp.) to zooplankton (D. magna) and finally to crucian ity is demonstrated by the surface charge parameter of NaPs, influenc-
carp (Carassius carassius) (Cedervall et al., 2012; Mattsson et al., 2014, ing its uptake, toxicity and fate prediction across trophic levels
2017). Skjolding et al. (2017) shows the presence of NaPs in the gastro- (Geitner et al., 2016). Ca-NaPs induce a higher toxicity rate than An-
intestinal tract of zebrafish (Danio rerio) after they have been fed brine NaPs (Nomura et al., 2016), as observed by severe developmental de-
shrimp exposed to NaPs, with accumulation occurring in the head and fects, growth inhibition and mortality in sea urchin embryos (Della
gills of the zebrafish. Larger particles, ~700 nm, have been shown to af- Torre et al., 2014; Pinsino et al., 2017), green microalgae (Bergami
fect biological pathways associated with immunological recognition et al., 2016, 2017) and methanogens (Chen et al., 2018a). Ca-NaPs in-
processes (Veneman et al., 2017). crease ROS and NO production, induced apoptotic processes and af-
fected immune parameters in Mytilus galloprovincialis (mussels)
2.4.2.2. Toxicological effects. The absorption of NaPs can lead to the pro- (Canesi et al., 2015, 2016) and brine shrimps (Bergami et al., 2016).
duction of reactive oxygen species in algae, and hinder important Ca-NaPs also result in high embryotoxicity and affect the shell formation
processes such as photosynthesis by blocking light intensity, reduc- in M. galloprovincialis, as shown by the dysregulation of genes involved
ing air flow (Bhattacharya et al., 2010) and decreasing chlorophyll in early shell formation (Canesi et al., 2016). Studies show that Ca-NaPs
concentrations (Besseling et al., 2014), leading to stunted growth and An-NaPs may not employ similar ingestion routes, accumulate in
(Besseling et al., 2014; Casado et al., 2013; Bergami et al., 2016). separate organs, and therefore activate different cellular pathways to in-
Studies demonstrated that even at concentrations as low as duce toxicity (Della Torre et al., 2014). The introduction of NaPs in
0.50 μg/ml, NaPs can reduce the population growth and reproduction media, such as sea water, result in the agglomeration and aggregation
rate, and induce mortality in various species including Brachionus of the plastic material, with Ca-NaPs forming nano-aggregates
manjavacas (rotifers) (Snell and Hicks, 2011), Thamnocephalus (b200 nm) and An-NaPs resulting in micro-aggregates (N1 μm)
platyurus (Casado et al., 2013), Paracentrotus lividus (sea urchin) (Manfra et al., 2017). Ca-NaPs and An-NaPs both showed clear accumu-
(Della Torre et al., 2014), Artemia franciscana (brine shrimps) lation in the gut of brine shrimps, however only the Ca-NPs caused tox-
(Bergami et al., 2016, 2017), Daphnia magna (planktonic crustacean) icity to brine shrimps (Bergami et al., 2016) and rotifers (Manfra et al.,
(Besseling et al., 2014; Casado et al., 2013; Booth et al., 2016) and 2017), and show the potential for trophic transfer with high accumula-
Daphnia galeata (planktonic crustacean) (Cui et al., 2017). Addition- tion of plastic from prey to predator (Bergami et al., 2017). This indi-
ally, NaPs can influence the behaviour of marine organisms, such as cates surface charge to be an important aspect in plastic NaPs' stability
reducing the filtering activity of Mytilus edulis (mussels) by produc- and interaction with the marine environment. Additionally, the pres-
ing pseudofeces (Wegner et al., 2012). Polystyrene and polycarbon- ence of a protein eco-corona formed onto the surface of NaPs have
ate nanoparticles can damage the antioxidant system, causing shown to increase their uptake and contribute to a longer retention in
oxidative stress and act as stressors to the innate immune system the gut of D. magna (Nasser and Lynch, 2016). NaPs have the ability in
of the fathead minnow Pimephales promelas (fathead minnow) adsorbing toxic chemicals such as phenanthrene, enhancing its bioaccu-
(Greven et al., 2016). The accumulation of NaPs was observed in mulation, toxicity and transfer through trophic levels and reduce its
the gastrointestinal tract of D. magna and lipid droplets of embryos, degradation rate in the marine environment (Ma et al., 2016).
Table 2
Studies of NaPs toxicity in marine organisms.
Type Surface Size Organism Major findings Reference

modifications
PS COOH 30 nm Daphnia magna • NP uptake passive Rosenkranz P. 2009

• Retention of 20 nm longer than
1000 nm
• Rapid ingestion and accumula-
tion in GIT
PS nf 100 nm Mytilus edulis (mussels); • Implications for toxicological Ward J.E. 2009
Crassostrea virginica effects and transfer of
(oysters) nanomaterial to higher trophic
levels
• Longer retention with NaP in GIT
than 10 μm
PS NH2; COOH 20 nm Chlorella; Scenedesmus • Higher adsorption NH2 than Bhattachary P. 2010
COOH
• Affected algal photosynthesis
• Promoted ROS production
PS nf 37 nm Brachionus manjavacas • Minimum concentration Snell T. W. 2011
(0.30 μg/mL) reduced 50% pop-
ulation
• Maximum concentration
(1.1 μg/mL) reduced 89%
populations
PS nf 23 nm EPS from Amphora sp.; • Affected EPS assembly Chen C. 2011
Ankistrodesmus angustus;
Phaeodactylum tricornutum
PS nf 30 nm Mytilus edulis (blue mussel) • Triggered production of Wegner A. 2012
pseudofeces
• Reduced organism's filtering
activity
PS COOH; SO2−
4 ; 20 nm; Biofilm from Alteromonas • Anionic particles associated Nevius B. A. 2012
NH2 20 nm; macleodii most strongly to biofilms
200 nm • Cationic particles associated
with biofilms at high ionic con-
ditions resembling those of sea-
water
• Lower Sorption of anionic parti-
cles NaPs
PS nf 24 nm Scenedesmus; D. magna; • Transfer of particles through Cedervall T. 2012
Carassius carassius aquatic food chain
• Affected lipid metabolism and
behaviour of the top consumer.
PS-PEI COOH 50 nm; P. subcapitata; D. magna; • Effects observed dependent in Casado M. P. 2013
110 nm T. platyurus; V. fischeri some cases on NaP size
• Higher effect being observed for
larger NaPs - greater effect
observed from 110 nm than
50 nm; algae and crustaceans
(Daphnids) most sensitive
organisms in aquatic exposure
to NaPs, same with D. magna
PS COOH 24 nm L. pneumophila • Did not affect biofilm Raftery T. D. 2013
morphology
PS SO2−
4 24 nm & From algae through D. • Affected feeding and shoaling Mattsson K. 2014
27 nm magna to fish; feeding and behaviour
shoaling behaviour • Affected metabolism of fish in
affected, as well as liver and muscle
metabolism • Changed morphology of brain
tissue
• Uptake of NaP through 3 food
chain levels
PS COOH; NH2 40 nm Sea urchin embryos • Cationic particles caused severe Della Torre C. 2014
(Paracentrotus lividus) developmental defects
• Anionic particles accumulated
inside embryo's digestive tract
while cationic particles were
more dispersed
• Differences in surface charges
affected their aggregation in
seawater and strongly affect
their embryotoxicity.
PS COOH 70 nm Green alga Scenedesmus • Reduced population growth & Besseling E. 2014
obliquus & zooplankter D. chlorophyll concentrations in
magna algae
• Exposed D. magna showed
(continued on next page)

Table 2 (continued)

modifications
Reduced body size and altered

reproduction of D. magna
• Reduced number and body size
of neonates were lower
• Induced malformations in neo-
nates up to 68% of individuals.
PS COOH; NH2; nf 70 nm–20 μm C. gigas (pacific oyster) • Cationic particles ingested and Cole M. 2015
retained more frequently
PS NH2 ND Bivalve Mytilus • Increased reactive oxygen spe- Canesi L. 2015
galloprovincialis cies (ROS) and nitric oxide (NO)
production
• Induced apoptotic processes
PS NH2 ND Mytilus galloprovincialis • In the presence of hemolymph Canesi L. 2016
serum, NaP increased cellular
damage and ROS production
PS COOH; NH2 ND Daphnia magna • Eco-corona around particle Nasser F. 2016
caused by proteins released by
D. magna
• Eco-corona caused heightened
uptake of NaP and increased
toxicity.
• Particles less efficiently removed
from the gut due to eco-corona
formation
• Affected rate of subsequent
feeding
PS COOH; NH2 40 nm & Dunaliella tertiolecta; • Anionic particles sequestered Bergami E. 2016
50 nm Artemia franciscana larvae inside gut lumen limiting food
intake
• Cationic particles adsorbed at
surface of sensorial antennulae
and appendages
• Affected feeding, behaviour and
physiology of brine shrimp
PS; polycarbonate (PC) nf 41 nm; Fathead minnow • Signification increase in degran- Greven A. 2016
148.7 nm (Pimephales promelas) ulations of primary granules of
neutrophils
PS nf 50 nm; D. magna • Increased absorption of toxic Ma Y. 2016
500 nm chemicals such as phenanthrene
and reduced its degradation rate
Poly(methylmethacrylate) (PMMA); poly nf ND D. magna; Corophium • Toxicity to D. magna Booth A. 2016
(methylmethacrylate-co-stearylmethacrylate) volutator; Vibrio fischeri
copolymer
PS latex NH2 100 nm Pseudomonas fluorescens • Cover negatively-charged bacte- Nomura T. 2016
rial surface of planktonic cells
• biofilm viable despite NaPs toxic
to bacterial cells
PS NH2 52 nm, Scenedesmus, D. magna & • Reduced survival of aquatic zoo- Mattsson K. 2017
53 nm, Carassius carassius plankton
57 nm, • Penetrated blood-brain barrier
58 nm, and caused behavioural
120 nm, disorders
180 nm,
330 nm
PS NH2 50 nm Mussell embryos • Affected development of mussel Balbi T. 2017
embryos
• High embryotoxicity/-
developmental arrest at high
concentration (_)
• Affected shell formation
• Induced dysregulation of gene
transcription involved in early
shell formation.
PS NH2; COOH 50 nm; Dunaliella tertiolecta; • Anionic formed micro-scale Bergami E. 2017
40 nm Artemia franciscana larvae aggregates and did not affect
growth of microalgae and brine
shrimps but were
• Anionic particles adsorbed on
microalgae and accumulated
(and excreted) in brine shrimps,
suggesting a potential trophic
transfer from prey to predator.
• Cationic particles NH2 formed
nanoscale aggregates, causing
inhibition of algal growth and
Table 2 (continued)

modifications
mortality in brine shrimps

• Cationic particles significantly
induced genes associated with
an apoptotic pathway in brine
shrimps
PS nf 25 nm Daphnia magna • Accumulation of PSNP particles Brun. 2017
in or on lipophilic cells in early
stages of development
• Uptake via brood pouch--
mediated accumulation
PS nf 52 nm Daphnia galeata • Transfer of PSNP from external Cui R. 2017
surface of the body to the inter-
nal organs, including thoracic
appendices, ovaries, caudal
appendices and brood chamber
and PSNP storage in lipid drop-
lets
• Decreased survival rates and
reproduction of adults
• Reduced hatching rates of
embryos and lipid droplets in
reproduction
• Increased in abnormal
development
PS NH2; COOH ND Brachionus plicatilis • Gut retention with anionic par- Manfra L. 2017
ticles but no lethal effects
• Lethal effects with cationic par-
ticles
• Higher toxicity of cationic parti-
cles in reconstituted sea water
than natural sea water
PS NH2; COOH ND P. subcapitata • Adsorption of cationic and Nolte T. 2017
unfunctionalised particles stron-
ger onto cell wall than anionic
particles
PS NH2 50 nm Sea urchin embryos • Affected development by modu- Pinsino A. 2017
(Paracentrotus lividus) lating protein and gene profile
PS COOH 20 nm Zebrafish (Danio rerio) • Particles observed in gut but not Skjolding L.M. 2017
within gut epithelia suggesting
no or limited uptake via intesti-
nal villi
• Particles observed in gills and
intestine and head region of fish
• Transfer of particles from brine
shrimp to zebrafish
PS nf 700 nm Zebrafish • Redistribution of particles Veneman W. 2017
throughout bloodstream
• Accumulation in heart region,
systemic region
• Activation of several biological
pathways related to an immune
response
PS 14C-radiolabelled 24 nm; Pecten maximus • Rapid and greater uptake for Al-Sid-Cheikh M.
250 nm 24 nm than 250 nm 2018
• Accumulation of 250 nm in
intestines while 24 nm dis-
persed throughout whole-body,
possibly indicating some trans-
location across epithelial mem-
branes
• Faster depuration faster for
24 nm
Poly (methyl methacrylate) (PMMA); nf ND Amphibalanus amphitrite • PMMA NaP ingested and Bhargava S. 2018
fluorescent perylene tetraester (PTE) translocated inside body of bar-
nacle nauplii within first 3 h
• Persist throughout stages of
growth and development
• Egestion observed through
molting and faecal excrement
PMMA nf 45 nm Dicentrarchus labrax • Sig. increased abundance of Brandts I. 2018
mRNA transcripts related to lipid
metabolism
• Decreased esterase activity
levels indicating compromised
(continued on next page)

Table 2 (continued)

modifications
immune system
• Sig. lower levels of alkaline
phosphatase in skin mucus
PS nf 50 nm Chlamydomonas reinhardtii • Adhesion to surface of primary Chae Y. 2018
(alga); D. magna (water producer and present in diges-
flea); Oryzias sinensis tive organs of higher trophic
(secondary-consumer level species
fish); Zacco temminckii • Affected fish activity
(end-consumer fish) • Induced histopathological
changes in fish livers
• Penetrated embryo walls and
present in yolk sac of hatched
juveniles
TiO2 + PS nf 10 nm; Caenorhabdiris elegans • Prolonged exposure to NaP Dong S. 2018
108 nm could enhance toxicity of TiO2 in
decreasing locomotion behav-
iour and induce ROS production
• Altered the molecular basis for
oxidative stress in nematodes
PS COOH; NH2 100 nm; Oyster Crassostrea gigas • Adhesion to oyster spermatozoa Gonzalez-Fernandez
actual gametes and oocytes C. 2018
~130 nm • Sig. increase of ROS production
in sperm cells with anionic
particles
PS nf 100 nm Caenorhabdiris elegans • Decreased survival rate and Lei L. 2018
body length
• Induced shortest average
lifespan in nematodes
• Induced size-dependent excit-
atory toxicity on locomotor
behaviour
• Downregulated expression
involved with cholinergic and
GABAergic neurons
• Elevated oxidative stress
PS nf 75 nm Daphnia pulex • Affected expression levels of Liu Z. 2018
genes encoding key stress
defence enzymes and proteins
PS NH2 45–65 nm Paracentrotus lividus • Affected pump's efflux activity Marques-Santos L.
• Role of TPP in triggering PS-NH2 2018
coelomocytes interaction and
toxicity
• Natural sea water increased
hydrodynamic diameter of par-
ticle
• Hydrodynamic diameter of par-
ticle increased in mammalian
and invertebrate systems
PS nf 51 nm Danio rerio • Accumulated in yolk as early as Pitt J. A. 2018
24 h
• Migrated to GIT, liver, pancreas,
heart and brain; slower rate of
depuration of NaPs in pancreas
and GIT
• Decreased heart rate
• Altered larval behaviour such as
swimming hypoactivity
PS COOH; NH2 50–55 nm Halomonas alkaliphila • Influenced growth inhibition, Sun X. 2018
chemical composition and
ammonia conversion efficiencies
• Promotion of ROS species
• Cationic PS induced higher oxi-
dative stress than anionic PS
PS COOH; NH2 50 nm; Crassostrea gigas • Induced sig. decrease in Tallec K. 2018
500 nm fertilisation success and
embryo-larval development
with numerous malformations
up to total development arrest
• Cationic particles had strongest
toxicity
PS; glyphosate; combination of PS & glyphosate NH2 200 nm Algae Microcystis • NPs sig. alleviated inhibitory Zhang Q. 2018
aeruginosa effect of glyphosate on growth
• Presence of glyphosate
enhanced stability of dispersion
system, allowing more NaPs to
Table 2 (continued)

modifications
adsorb on surface of organism

and result in greater enrichment
of NaP in food chain
PS; PS + carbamazepine nf NDa Mytillys galloprovincialis • Sig. alterations in expression of Brandts I. 2018
genes associated with
biotransformation, DNA repair,
cell stress-response and innate
immunity
• Combined PS + Cbz induced sig.
downregulation in gene expres-
sion
• Increased total antioxidant sta-
tus in digestive glands with
0.5 mg/L
• Total antioxidant capacity and
esterase activity observed for PS
50 mg/L in digestive glands and
gills
• Induced effects on
neutransmission, measured as
inhibition of cholinesterase
activity in haemolymph;
genotoxicity in haemocytes with
PS, Cbz & combo
• Lipid peroxidation with
0.05 mg/L showing NaP can
indce oxidative damage
(Brandts et al., 2018b)
PS COOH; NH2 100 nm Methanosarcina bareri • Cationic-NPs inhibited growth Chen R. 2018
• Methane production reduced
a
ND: Not described.
3. Analysis of plastics in the marine environment microscopic visualisation against reference materials and employing
the “hot needle” test by pushing a hot needle against the material deter-
3.1. MaPs analysis mining its melting capacity (Karlsson et al., 2017). Chemical character-
isation of MiPs can be done chemically using FTIR, Raman spectroscopy
The monitoring of plastic pollution in the marine environment is an or thermogravimetry (Miller et al., 2017; Claessens et al., 2011). A pres-
important, but highly difficult undertaking, and due to the different ac- ervation method with formalin or refrigeration is recommended for
cessibility, spatial and temporal heterogeneity, confounding factors and MiPs analysis of biological samples. The characterisation of MiPs within
a host of different monitoring techniques, data generated from which tissue material is similar to that used for seawater and sediment sam-
cannot necessarily be compared (Ryan et al., 2009). Due to the large ples' characterisation. It involves physical visualisation along with
size of MaPs which can be seen with the naked eye, the most popular chemical digestion, as plastic material is highly likely to be embedded
monitoring method is through visual observations. For example, some in tissue material. Alkaline and oxidative digestion is currently preferred
surveys collect data through describing observations from a vessel over acidic and enzymatic digestion, resulting in higher recovery rates
(Di-Méglio and Campana, 2017). This is useful for longitudinal studies of plastic as well as less damage to the structural integrity of the mate-
and the collection of large amounts of data, as these observations are in- rial. Pulsed ultrasonic extraction is an emerging method that uses high
expensive (i.e. they can be made by non-scientists) and the data gath- wave sonication to break apart tissues while maintaining the integrity
erer can quickly characterise key features of the individual MaPs, such of the plastic material. The solutions containing MiPs are further
as the approximate size, plastic source, location, as well as features of characterised using FTIR, Raman spectroscopy (Zhao et al., 2017; Lenz
the collective pollution such as the density of MaPs, total size of pollu- et al., 2015) or thermogravimetry coupled with differential scanning
tion (particularly relevant for ocean gyres) and tracking shifts in indi- calorimetry (Miller et al., 2017; Hidalgo-Ruz et al., 2012).
vidual MaPs characteristics across different areas. Interestingly, more
advanced techniques have been suggested recently, such as 3.3. NaPs analysis
hyperspectral remote sensing, which utilises characteristic reflectance
spectra generated from different surfaces dependent on composition, As mentioned in the previous section, most studies have made use of
size and shape of features in response to the sun's rays which can be de- engineered NaPs with controlled environmental factors, and therefore
tected by remote sensors (Goddijn-Murphy et al., 2018; Garaba et al., do not accurately reflect the impact of the actual marine environment.
2018; Goddijn-Murphy and Dufaur, 2018; Acuña-Ruz et al., 2018). NaPs are commonly characterised by their physico-chemical properties
– hydrodynamic diameter, dispersity & distribution, surface charge,
3.2. MiPs analysis morphology & shape, electrophoretic mobility, which can affect their
stability, aggregation, transportation, uptake, as well as adsorption and
The extraction of MiPs from seawater and sediment samples typi- desorption properties in the environment. Engineered NaPs typically
cally involves filtration, pressurised fluid extraction or flotation separa- have fixed sizes, shape and surface charge, unaltered from environmen-
tion methods, combined with elutriation, which is a simple technique tal conditions. These NaPs parameters are confirmed prior to being ex-
moving the fluid containing-MiPs in the opposing direction of the sedi- posed to various treatments, while any subsequent modifications to
ment flow. The particles are then characterised physically by NaP are disregarded in some studies (Chen et al., 2011; Rosenkranz
et al., 2009; Chen et al., 2018b; Pitt et al., 2018). The stability of NaPs are 2018; Mangalara and Varughese, 2016). The concentration of NaP can
reported as a measure of unchanged zeta potential or surface charge, be determined via various differing instruments including flow cytom-
and typically remain stable in demineralized and freshwater mediums etry (Cole and Galloway, 2015), fluorescence (Mitzel et al., 2016; Liu
(Liu et al., 2018a), whereas a continuous increase in particle size and de- et al., 2018b; Ward and Kach, 2009; Merzel et al., 2019) and UV–vis
crease in stability has been observed of NaPs exposed to different envi- spectrophotometer (Mitzel et al., 2016; Zhang et al., 2018;
ronments such as artificial sea water (Brandts et al., 2018a), natural sea Bhattacharya et al., 2010). A recent technique, Nanoparticle Tracking
water (Wegner et al., 2012; Marques-Santos et al., 2018), bacterial me- Analysis, has quickly paved its way as the new standard in
diums (Nasser and Lynch, 2016) and biological fluids (Marques-Santos characterising nanoparticles, due to its superfluous ability in combining
et al., 2018). Nasser et al. reported the adsorption of a thin protein layer the qualitative and quantitative characterisation of particles in a small
onto the NaP surface, resulting in agglomeration and an increase of size volume of sample, including the parameters mentioned above. The
of over 100 nm (Nasser and Lynch, 2016). Additionally, NaPs are used at analysis is done in a liquid suspension where rate of Brownian motion
high concentrations in toxicity studies and bare no overlap to the con- is related to particle size (Mattsson et al., 2014). Additionally, in some
centrations of NaPs as naturally found in the marine environment. studies determining NaP in soil samples make use of a dye absorption
Naturally-occurring NaPs collected, isolated and extracted from com- method to test the hydrophobicity of the particle (Mitzel et al., 2016).
plex media must also undergo a pre-concentration step prior to detec- Fig. 5 shows the most used techniques/instrumentation in
tion and quantitation. characterising NaPs and Table 3 provides an overview of prospects
and limitations regarding the techniques employed in characterising
3.3.1. Characterisation of plastic nanoparticles NaPs.
The most basic characterisation is performed by light scattering
technique, specifically Dynamic Light Scattering (DLS) and is used to
measure a range of NaPs characteristics including hydrodynamic diam- 3.3.2. NaPs detection in sediments
eter, particle dispersity, zeta potential and electrophoretic mobility The behaviour of NaPs in sediment is determined by their interac-
(Bhargava et al., 2018; Nevius et al., 2012; Bhattacharya et al., 2010; tions and transportation through mineral and rock particles, and essen-
Rosenkranz et al., 2009; Cedervall et al., 2012). The agglomeration and tially involves a separation and quantification process. Asymmetrical
following aggregation behaviour can be determined with DLS, UV–vis flow field-flow fractionation (AF4), combined with multiple-angle
spectrophotometry (Nolte et al., 2017) and electron microscopy (EM) light scattering (MALS) and DLS, can be used to isolate and determine
(Cole and Galloway, 2015). The electrophoretic mobility of NaPs in the occurrence and behaviour of NaPs in soil (Awet et al., 2018). More-
solid (soil, sand) and liquid (waste effluent, marine water) sample over, column transport experiments using glass chromatography col-
types is affected by the surface charge of the particle, and is necessary umns and spectrofluorometer, explore the retention of different
in investigating the speed at which the NaP travels based on the unpre- functionalised NaPs in samples affected by various factors including
dictable surroundings of different ecosystems and environments, which ionic strength and biofilm formation (Mitzel et al., 2016; Quevedo and
can be determined using DLS (Mitzel et al., 2016). DLS is, therefore, ef- Tufenkji, 2012). A study by Mitzel et al. investigated the electrophoretic
fective in monitoring changes in NaPs' physical properties within differ- mobility of engineered NaPs transported through clean and biofilm-
ent environmental conditions. Zetasizer Nano is currently the most coated sand, where a micro-well plate reader equipped with fluores-
employed DLS instrument (Bhargava et al., 2018; Brandts et al., 2018a; cence mode analysed the wavelengths of the effluents and the attach-
Nasser and Lynch, 2016; Awet et al., 2018; Feng et al., 2018; Cole and ment efficiency of NaPs, based on different environmental influences,
Galloway, 2015; Mattsson et al., 2014; Della Torre et al., 2014; Casado were determined (Mitzel et al., 2016). The stability of the NaPs through-
et al., 2013; Bergami et al., 2016; Bergami et al., 2017; Booth et al., out the column experiment can be measured using DLS (Liu et al.,
2016; Greven et al., 2016; Pinsino et al., 2017; Canesi et al., 2015; 2018b). The concentration of adsorbed contaminants onto NaPs can
Canesi et al., 2016; Manfra et al., 2017; Chen et al., 2018b; Raftery be measured using a ‘negligible depletion solid-phase microextraction
et al., 2013; Balbi et al., 2017; Chae et al., 2018; Dong et al., 2018; approach’, and liquid scintillation counter (LSC) during the column ex-
González-Fernández et al., 2018), followed by Nicomp (Al-Sid-Cheikh periment, detecting the radioactivity and degradation rate of the con-
et al., 2018), ELS-Z (Nomura et al., 2016; Mattsson et al., 2017; taminant. Further batch adsorption and desorption experiments can
Bergami et al., 2017) and Particle Size Analyzer (Fu et al., 2018). DLS be carried out investigating the absorption capacity and affinity of
measures the hydrodynamic diameter of the particle and does not NaPs (Liu et al., 2018b). The separation of organic matter and NaPs is
give the actual NaP size, whereas EM is able to provide the core size of importance for the proper qualification and quantification of NaPs
and shape of the particle but do not provide any information regarding in the environment. Wang et al. utilised biosolids and solid samples
the NaP behaviour in a solution. Therefore DLS and EM techniques are spiked with NaPs as a means to mimic environmental samples and the
often used in combination to confirm the physico-chemical properties removal of natural organic matter was performed with and without ox-
of NaP. EM instruments include Scanning Electron Microscopy (SEM) idation. While the application of oxidation successfully separated NaPs
(Bhargava et al., 2018; Feng et al., 2018; Liu et al., 2018b; Greven from biosolids and solid samples, removal of organic matter without ox-
et al., 2016; Chae et al., 2018; Lei et al., 2018), Transmission Electron Mi- idation was not able to remove NaPs from biosolids. Moreover, the use
croscopy (TEM) (Liu et al., 2018a; Brandts et al., 2018a; Marques-Santos of H2O2 caused surface modification and reduced extraction efficiency
et al., 2018; Awet et al., 2018; Feng et al., 2018; Ma et al., 2016; Cole and of NaPs. Samples were then separated by a flotation method and further
Galloway, 2015; Della Torre et al., 2014; Besseling et al., 2014; Bergami quantified using gravimetry and UV–vis spectrophotometry AT (Wang
et al., 2016; Bergami et al., 2017; Booth et al., 2016; Canesi et al., 2015; et al., 2018). Microbial communities form the ecological environment
Canesi et al., 2016; Raftery et al., 2013; González-Fernández et al., of soil components, and several other studies employed 16s rRNA ex-
2018; Al-Sid-Cheikh et al., 2018), Field Emission Scanning Electron Mi- traction and amplification techniques (Zhu et al., 2018) to investigate
croscopy (FESEM) (Canesi et al., 2016; Balbi et al., 2017) and Field Emis- the presence and/or abundance of particular bacterial species, as well
sion Transmission Electron Microscopy (FETEM) (Cui et al., 2017). SEM as CLSM and AFM in detecting shape and deformity resulting from
combined with energy dispersive X-ray spectroscopy and elemental NaPs toxicity (Nomura et al., 2015). Research is currently focused on
mapping can be used to detect the presence, composition and dispersity the separation and quantification of NaPs from the marine environment,
of NaP in sediment samples (Awet et al., 2018; Zhu et al., 2018). Fourier rather than its identification. This states the importance in focusing
transform infrared spectroscopy (FTIR) can further be used to analyse studies towards the qualitative assessment of NaPs by adopting previ-
the composition and chemical structure of NaPs by pelletizing with po- ously used instruments such as inductively coupled plasma mass spec-
tassium bromide (Liu et al., 2018b; Al-Sid-Cheikh et al., 2018; Lei et al., trometry (ICP-MS) or FT-IR.
3.3.3. NaPs detection in water 3.3.4.1. Investigation of NaP influence on Phytoplankton (marine algae) &
NaPs have been shown to alter the formation of dissolved organic EPS films. The adsorption of NaPs onto films formed by cellulose material
matter (DOM) and particulate organic matter (POM), crucial to the nor- and algal species can be determined by absorbance analysis using UV–
mal biogeochemical cycles of marine life. The monitoring of NaPs be- vis spectroscopy and microplate readers (Nevius et al., 2012;
haviour during DOM-POM assembly can be achieved first by filtering Bhattacharya et al., 2010; Nolte et al., 2017). Additionally, spectroscopic
seawater samples using a 0.22 μm fibre glass membrane, microbial bio- techniques and numerous toxicity assays, such as the ROS assay, can be
cide treatment and then analysed with a goniometer, which is used to used to determine the effect of NaPs on algal photosynthesis and gener-
measure scattered fluctuation signals corresponding to the size of the ation of ROS (Bhattacharya et al., 2010). The use of microscopy imaging,
suspended NaPs. The hydrodynamic diameters of microgels formation, such as fluorescence microscopy (Bergami et al., 2017), SEM (Bergami
from the presence of DOM-POM, can additionally be analysed using et al., 2016, 2017), Environmental Scanning Electron Microscopy
CONTIN method, where the aggregation state of NaPs can be measured, (ESEM) (Bhattacharya et al., 2010), Confocal Scanning Laser Microscopy
and the correlation of NaPs size and assembly size can be determined at (CSLM) (Bhargava et al., 2018; Nevius et al., 2012; Nomura et al., 2016;
specified time-points (Chen et al., 2018b). The biogas methane concen- Chen et al., 2011; Chen et al., 2018a) and Atomic Force Microscopy
tration of sewage sludge with NaP exposure can be determined using (AFM) (Nomura et al., 2016) are used to investigate NaPs adsorption
gas chromatograph, combined with a stainless-steel column and ther- onto algae and its alteration on structural networks of EPS microgels
mal conductivity detector (Fu et al., 2018). The impact of NaP on anaer- and biofilms. Fourier transform infrared spectroscopy (FT-IR) is a versa-
obic digestion present in sewage sludge can be determined by analyzing tile instrument that can determine the presence of specific functional
the hydrogen production from bacteria such as Acetobacteroides groups from functionalised NaPs, as well as altered protein secondary
hydrogenigenes, using gas chromatogram (Fu et al., 2018), or the reduc- structures of EPS with NaP exposure (Feng et al., 2018; Nevius et al.,
tion in dissolved oxygen (Feng et al., 2018). X-ray photoelectron spec- 2012). Additionally, alteration of functional groups and elemental com-
troscopy (XPS) can be used in determining the functional groups position of EPS can be determined using X-ray photoelectron spectros-
contributing to NaPs and EPS interaction, while FTIR can be used to copy (XPS) (Feng et al., 2018). The accumulation of NaPs within EPS
demonstrate the change in protein structures of EPS resulting from microgels can be measured using fluorescence microscopy (Bergami
the interaction (Feng et al., 2018). SEM analysis can be used to observe et al., 2016) and the growth kinetics of EPS assembly monitored using
the interaction of NaP between sewage sludge/bacterial communities. a laser spectrometer equipped with a goniometer feature (Chen et al.,
The effect of NaP on bacterial communities can be further extracted to 2011).
determine bacterial species present, with biogas slurries analysed and
representing microbial structure variations during anaerobic digestion
process (Fu et al., 2018). The detection of adsorbed compounds on 3.3.4.2. Investigation of NaP influence on filter-feeders & fish. The ingestion
NaP surface can be achieved using high performance liquid chromatog- and excretion of NaPs by organisms can be qualitatively assessed by use
raphy (HPLC) (Zhang et al., 2018). of fluorescent labelling of NaPs (Nasser and Lynch, 2016; Nomura et al.,
2016; Rosenkranz et al., 2009; Booth et al., 2016; Manfra et al., 2017),
coupled with microscopic instrumentations such as optical microscopy
3.3.4. NaPs detection in marine organisms (Ma et al., 2016; Cui et al., 2017; Greven et al., 2016; Pinsino et al., 2017),
The ingestion and accumulation of NaPs in marine organisms are in- fluorescence microscopy (Wegner et al., 2012; Chen et al., 2011; Snell
vestigated using a range of qualitative and quantitative techniques, and Hicks, 2011; Cole and Galloway, 2015; Cedervall et al., 2012; Della
where there are currently no set standards. Torre et al., 2014; Brun et al., 2017; Bergami et al., 2016; Bergami
Fig. 5. Common techniques/instrumentation involved in NaPs characterisation: (a) DLS; (b) SEM (Lei et al., 2018); (c) TEM (Awet et al., 2018); (d) NanoSight (Filipe et al., 2010); (e) UV–
vis spectroscopy (Carotenuto et al., 2003); and (f) FT-IR analysis (Feng).
Table 3 embryonic organisms using quantitative whole-body autoradiography

Prospects & limitations in NaP characterisation techniques (Bhattacharya et al., 2010; (Al-Sid-Cheikh et al., 2018) and assays such as Nile Red staining assay
Merzel et al., 2019; Maguire et al., 2018; Tomaszewska et al., 2013; Zucker et al., 2016).
(Cui et al., 2017; Brun et al., 2017).
Use/prospects Limitations The effects and interactions of the NaPs within organisms are inves-
Dynamic Light - Little sample preparation - Provides hydrodynamic tigated using a multitude number of techniques and instrumentation.
Scattering - Immediate results & diameter, and not actual size The most basic assessment of NaPs toxicity within an organism, include
reproducibility - Results are affected by rates of growth, reproduction, feeding and clearing rate of the NaPs from
- Monitor changes in NaP changing temperature and
the body (Nomura et al., 2016; Snell and Hicks, 2011), as well as visual-
behaviour and its surface solvent viscosity
modifications within - No information provided isation of morphological alterations induced by the NaPs (Cole and
different environments about the size-environment Galloway, 2015; Pinsino et al., 2017; Dong et al., 2018) using various mi-
- No calibration required relationship croscopy techniques include stereomicroscopy (Brun et al., 2017;
- Bias towards larger Manfra et al., 2017; Lei et al., 2018), optical microscopy (Ma et al.,
particles and aggregates
Microscopy - Provides physical - Long sample preparation
2016; Della Torre et al., 2014; Bergami et al., 2017; Balbi et al., 2017;
techniques information relating to - No information on the Chae et al., 2018), fluorescent microscopy (Bergami et al., 2016, 2017)
shape, size and dispersity properties of the NaP in a or SEM (Balbi et al., 2017). The monitoring of the organisms' behaviour,
of particles solution feeding rate, locomotion, motility and hunting confirm the proper func-
- No calibration required
tion of motor neurons, and can be conducted using video analysis
Nanoparticle - Measurements of single - Area of observation limited
Tracking Analysis particle to field of view of (Mattsson et al., 2017) or visual confirmation (Pitt et al., 2018; Dong
- Information about size, microscope: limits number et al., 2018). Additionally, the locomotion behaviour of organisms af-
size distribution and of particles tracked and fected by NaP can be determined by the translational expression of cho-
concentration duration of tracking linergic and GABAergic neurons, both involved with motor functions,
- Detection of secondary
using a fluorescence microscope (Lei et al., 2018). Various assays are
peaks
- No calibration required used in determining NaPs effect on cell functional parameters (Nasser
UV–vis - Concentration of NaP in - Wavelength shifts when and Lynch, 2016; Cole and Galloway, 2015; Casado et al., 2013;
spectrophotometer monodisperse solution size of NaP changes, but no Greven et al., 2016; Canesi et al., 2016; Raftery et al., 2013) and bio-
- No calibration required information of size is
chemical changes (Brandts et al., 2018a; Cedervall et al., 2012; Chae
- Easy sample preparation provided
- Can be used on et al., 2018). Proteomics and metabolomic changes in organisms can
environmental samples be investigated using various instrumentation. NaPs can bind to specific
Flow cytometry - Provides information - Calibration required to proteins in the biological body, which can be extracted from various bi-
about concentration and ensure separation of ological samples and separated using sodium dodecyl sulphate (SDS)
size of NaPs background noise and signal
polyacrylamide gel electrophoresis (PAGE) method (Nasser and
of smallest particle
- Dilution of sample and Lynch, 2016; Cedervall et al., 2012; Pinsino et al., 2017; Canesi et al.,
adjustment of concentration 2016) and analysed on mass spectrometry, such as matrix-assisted
is required to reduce noise laser desorption/ionization (MALDI) coupled with time-of-flight (TOF)
and “swarm” of particles
mass spectroscopy (Cedervall et al., 2012) and high-performance liquid
- More suitable for
submicron particles
chromatography (HPLC) with tandem mass spectrometry (MS/MS)
- Multiple variables need to (Canesi et al., 2016). Metabolites from various organs such as muscle,
be considered before analysis liver, brain and blood samples can be detected and quantitatively
including system electronics, analysed using 1H NMR analysis (Mattsson et al., 2014). CytoViva
reference particles, and
Hyperspectral Imaging System is a recent technique that has enable
particle number/mL
Fluorescence - Concentration of NaPs - Requires the use of the analysis and spectrally mapping of polystyrene NaPs in brain organs
- No calibration required fluorescent NaPs, which do of crucian carp (Mattsson et al., 2017). Real time (RT) Quantitative PCR
not translate to has been used in evaluating expressional changes in various cellular
environmental samples
functions including oxidative stress (Liu et al., 2018a; Dong et al.,
FTIR - Information on functional - Relatively long sample
groups attached onto the preparation
2018), cell cycle and signalling pathways (Della Torre et al., 2014;
surface of NaPs Pinsino et al., 2017), lipid metabolism (Brandts et al., 2018a), and target
genes involved with larval growth, molting (Bergami et al., 2016, 2017)
and shell formation (Balbi et al., 2017). Additionally, oxidative stress can
et al., 2017; Greven et al., 2016; Pitt et al., 2018) and confocal laser scan- be monitored by determining ROS production (Dong et al., 2018) and
ning microscopy (CLSM) (Bhargava et al., 2018; Nasser and Lynch, metabolic enzymes of the detoxification system (Lei et al., 2018) with
2016; Nomura et al., 2016; Rosenkranz et al., 2009; Della Torre et al., a fluorescent microscope. Histopathological analysis, using
2014; Cui et al., 2017; Brun et al., 2017; Chae et al., 2018; González- haematoxylin & eosin (H&E) dye, can be used to determine NaP's toxic-
Fernández et al., 2018). The quantitative assessment of ingested NaPs ity effect on cell morphology (Chae et al., 2018).
can be measured by absorbance analysis (Besseling et al., 2014) using
UV–vis spectroscopy (Wegner et al., 2012; Nasser and Lynch, 2016) 3.3.5. Analysis of NaPs degradation
and microplate readers or by fluorescence analysis (Nasser and Lynch, The biodegradation of NaPs occurs within organisms and the marine
2016; Ward and Kach, 2009; Rosenkranz et al., 2009). The visualisation environment and affects their physical and chemical characteristics. The
of ingested NaPs is also generally reported, and is performed by various change in surface charge can be determined by Zetasizer (Bhattacharya
microscopy techniques including Scanning Electron Microscopy et al., 2010; Casado et al., 2013) or resistive pulse sensors, which are able
(Bhattacharya et al., 2010) and Transmission Electron Microscopy to look at carboxylated nanopolystyrene (Vogel et al., 2012). The change
(Rosenkranz et al., 2009; Canesi et al., 2016; Raftery et al., 2013). Light in chemical structure and functional groups can be detected using
sheet microscopy (LSM) has recently proven to be a useful technique Fourier-transform IR (Nevius et al., 2012; Kundu et al., 2014), Raman
to track the uptake and accumulation of fluorescent NaPs through a Spectroscopy - identifying patterns of polystyrene biodegradation
live organism without compromising the integrity of the organs (Kundu et al., 2014) and Gas-Chromatography Mass Spectroscopy
(Skjolding et al., 2017). The accumulation and distribution of NaPs in (GC–MS) - looking at intermediates formed from degradation (Kundu
marine organisms can be detected in tissues and lipid droplets of et al., 2014) and gel permeation chromatography, recording changes
in molecular weight (Kundu et al., 2014). Other techniques used in- modified based on the requirements of the study (Roberts et al.,
clude: Field Emission Scanning Electron Microscopy (FESEM) to investi- 2012). DLS and AF4-MALS are reliable in discerning change in agglom-
gate biodegrading changes in biofilm surfaces; and Differential eration/aggregation state of NaPs from the presence of complex organic
Scanning Calorimetry (DSC) and thermogravimetry to analyse changes matter caused from the extraction and isolation protocols (Correia and
in the plastic material's thermal properties (Kundu et al., 2014). The bio- Loeschner, 2018). While an enzymatic digestion efficiently extracted
degradation of NaPs additionally leads to the leaching of substances spiked PS NaPs from fish, an acid digestion protocol resulted in larger
such as residual monomers, oligomers and other additives that can be aggregates of the particles (Correia and Loeschner, 2018). AF4-MALS,
analysed using Liquid Chromatography-Time Of Flight (LCTOF) coupled additionally demonstrated comparable detection of the stable NaPs
with Gas Chromatography (GC) (Björnsdotter, 2015). Ioakeimidis et al. from spiked fish extracts and ultrapure water. However, neither diges-
(2016) investigated the degradation of polyethylene terephthalate bot- tion methods were successful in isolating PE NaPs nor did AF4-MALS
tles (PETs) at the bottom of the Ionian Sea and demonstrated unique show an elution signal for these particular material particles. The lack
wavelengths and functional groups identification using ATR-FTIR. of surfactant from the polyethylene nanoparticles may have resulted
While this study utilised MaPs, the instrument's ability to characterise in its destabilisation during both the extraction and analysis processes
and differentiate the age of plastic materials in marine environment and the authors of this study concluded the necessity in adapting NaP
could also be applied to characterise and date NaPs (Ioakeimidis et al., isolation and extraction methods based on different polymeric material
2016). Schwaferts et al. (2019) describes the potential use and combi- studied (Correia and Loeschner, 2018). Moreover, the sample prepara-
nation of multiple spectroscopy and microscopy instruments, such as tion process is extremely crucial for allowing proper detection and
FTIR, Raman and Atomic Force Microscopy (AFM), in identifying the quantification of NaPs. Samples collected from seawater are typically fil-
chemical composition of NaPs in the environment (Schwaferts et al., tered, followed by the removal of biogenic matter and a
2019). While these instruments are typically used for micrometre and preconcentration step, prior to analysis (Ter Halle et al., 2017). The ex-
submicrometer particles, they can be adapted for NaPs analysis. Kiefer traction and quantification of NaPs from sediments and soil is particu-
et al. (2015) developed a fast and easy method in characterising the sur- larly challenging. Flotation is the most common method used to
face chemistry of nanoparticles, making use of various solvents and a separate plastic from mineral material. Larger beads (N100 μm) were
fixed bed of nanopowder, noting the molecular interactions between quantitatively extracted (~100%) while smaller beads had low extrac-
the particles and the hydrogen bonding network of the solvents tion efficiencies (avg. 20%). Extraction method using H2O2 oxidation
(Kiefer et al., 2015). The visualisation of surface topography and rough- (remove natural organic matter) negatively impacted extraction effi-
ness of the plastics, PETs and coffee cup lids, can be achieved using SEM ciency of NaP, but not larger beads. For soil, extraction with water
to classify the different stages of degradation (Lambert and Wagner, only, followed by flotation in ZnCl2 solution result in high extraction ef-
2016; Ioakeimidis et al., 2016). Liquid Scintillation Counter (LSC) and In- ficiencies (N75%) for larger beads than 1 μm, but low efficiencies (b30%)
strumental HPLC equipped with online radioactivity flow detector have for 0.05 and 1.0 μm beads (Wang et al., 2018).
been used to determine the presence of absorbed toxic chemicals onto Quality control and assurance involves the use of method blanks and
plastics and their degraded additives, by measuring the radioactivity controls within filtration, extraction and instrumental protocols (Ter
of the materials (Ma et al., 2016). Halle et al., 2017). Additionally, external and reference standards of
Van Der Pol et al. (2014) have shown the ability of NanoSight instru- polymeric material are used to provide chemical fingerprints and quan-
mentation to track NaPs in the marine environment through the use of titation measures for spectroscopic instruments, such as GC–MS and
their refractive index - to determine their presence and to distinguish FTIR, in detecting polymeric composition of NaPs collected (Ter Halle
different types of NaP material, such as silica and polystyrene beads, in et al., 2017).
different marine locations. This nanoparticle tracking analysis process The secondary characterisation of NaPs focuses on functionalised
can also be used in characterising the biodegradation of plastic material groups attached to their the surface and the change in surface charge
such as coffee cup lids into NaPs, and to determine the particle size and within different medium, enables a more comprehensive study of
size distribution within liquid samples (Andrady, 2015). their toxicity and hazard in the environment (Bergami et al., 2016).
The use of culture media and other in vitro cell culture conditions do
3.4. Challenges in NaP analysis not accurately embody real-life matrices, as the behaviour of NaPs do
not remain stable in natural mediums such as sea water. The accumula-
3.4.1. Challenges of NaPs characterisation in the natural environment tion of NaPs in sea organisms, such as rotifers, exhibit comparable toxic-
One of the most significant issues with NaP analyses is the inability ity in reconstituted sea water (RSW) and natural sea water (NSW),
to readily observe the material in the environment. There are issues concluding the importance in choosing an appropriate medium when
with the most recent analytical techniques and also the current investigating the influence of NaPs on organisms (Manfra et al., 2017).
methods used to isolate these polymers from the complex matrices or The presence of high ionic strength, proteins, salt and natural organic
tissues they are resident in. Therefore, nanopolymer characterisation matter (NOM) in sea water are absorbed onto the surface of NaPs,
has many challenges; (i) being able to “see” the NaPs; (ii) their colloidal forming complexes, and significantly altering their parameters, such
behaviour; (iii) their binding ability to soils and organic material; (iv) as the surface charge and size. In-turn this change in the surface charge
their interaction with ions; (v) their dispersion and gravimetrically set- strongly influences the surface interactions, and dispersion properties
tling; (vi) and their residence within tissues of organisms. The ability to (Gigault et al., 2018; Casado et al., 2013) and can lead to the aggregation
qualify and quantify polymer within a complex, interactive environ- of the NaPs within the surrounding environment (Wegner et al., 2012).
ment is no easy task, even with the most advanced analytical tech- Studies reporting the rapid increase in hydrodynamic diameter of An-
niques. This section of the review aims to detail these challenges, but NPs with COOH functional groups compared to Ca-NPS with NH2 func-
also to showcase some of the emerging methods to assist in this tional groups (Della Torre et al., 2014; Manfra et al., 2017). This differ-
characterisation. ence in aggregation and dispersion of different functionalised NaPs
The primary characterisation of NaPs is particularly important when impact their ingestion and fate within an organism and further ecotox-
investigating its fate in the environment and involves analyzing basic icological studies involving the interaction of these functional groups
parameters such as hydrodynamic diameter, agglomeration, polydis- with cellular structures should be investigated. The presence of multiple
persity and electrophoretic mobility. While various analytical tech- constituents in NSW, such as proteins, polysaccharides and other NOM,
niques exist in studying these properties, the latter constantly change are adsorbed onto the NaPs surface and result in the formation of an
with the surrounding environment and complex matrices, affecting its “eco-corona”, a translucent coating that contributes towards the aggre-
behaviour and detection. The techniques are therefore routinely gation state, bio-interaction, and bioavailability of NaPs. This eco-corona
is prevalently observed in NSW than RSW and other laboratory me- and environmental pollution detection of plastics. The power of sensors
diums (Canesi et al., 2016; Canesi and Corsi, 2016) and increases the which are tailored specifically for plastic composition, and size profiles
size and affect the stability of NaPs resulting in an increase of uptake, re- which could then be coupled with rapid diagnostics such as NIR or
tention and toxicity in filter feeders such as D. magna (Nasser and Lynch, MIR techniques will also greatly assist in determining the true impacts
2016). Fluorescent labelling has been the standard technique for NaPs of plastic toxicity and their interactions in the environment (Chapman
visualisation in ecotoxicological studies, with no difference observed et al. 2020) and potentally in organisms. However, the ability to resolve
in the uptake and excretion between labelled and non-labelled NaPs in the nano-domain will remain a challenge, and will require some ex-
(Bergami et al., 2016; Booth et al., 2016). However, the detection of tremely dynamic innovations in sensor technology, chemometrics will
smaller particles, b20 nm, in organisms appear to be a prevalent limita- still remain imperative to the whole process. Resolving and
tion with impracticability in differentiating NaPs from biological fea- characterising NaPs is vital if we are to understand the true environ-
tures such as lipid droplets (Cole and Galloway, 2015). The adsorption mental impact these particles are having. Furthermore, laboratory-
of various proteins onto the surface of NaPs can result in different eco- based assays should first be fully characterised rather than going for
or protein-coronas, creating either a negative or positive surface charge the complex matrix. If more parameters can be controlled experimen-
and affecting their uptake into cells differently, with a higher response tally, the depth of understanding of the negative impacts of nanoplastics
observed with positively-charged rather than negatively-charged NaPs in a marine environment and their interactions with biological systems,
(Baier et al., 2011). The process of centrifugation, gel electrophoresis will be significantly enhanced.
and nano-HPLC-ESI-MS/MS was combined in detecting the protein co- The need for reliable, robust, and reproducible data seems to still be
rona formed around NH2 NaPs, and confirm the origin derived from in the infancy for the topic of nanoplastic contamination and the envi-
the marine organism it was incubated with (Marques-Santos et al., ronmental importance. Many researchers are still reaching for the envi-
2018; Canesi et al., 2016). Polyacrylamide gel electrophoresis (PAGE) ronment as a hypothesis-testing site, when really, fundamental
is increasingly used in qualifying the proteins of NaPs eco-corona experiments in controlled environments will be the telling results of
(Marques-Santos et al., 2018; Canesi et al., 2016; Docter et al., 2014). the future to deal with impact of plastic contamination. Furthermore,
Finally, most studies investigating the effects of NaPs in the marine the tendency to overuse analytical instrumentation just to drive limits
environment have focused on PS material, overlooking other represen- of detection is also not a good approach to understanding the environ-
tative plastic material in natural scenarios such as PE or PP. Polymeric ment and what plastics are truly doing. It is not only the understanding
materials display different transportation mechanisms as well as stabi- but comprehensively grasping the impact of plastics, their chemical
lization, as shown by the clear difference of polymer interaction with breakdown, biological importance, plasticizers and additives
cellular features with one study showing the stabilization of lipid mem- (Chapman et al., 2010), and their potential to harm the environment
branes with PS, while PP showed a lipid phase separation and PE mod- that is holistically thought about rather than one variable at a time,
ified the phase boundaries topology and contributed to cholesterol that will generate true environmental sense. It will be the use and devel-
depletion (Bochicchio et al., 2017). Currently, about ~40% of plastic opment of innovative tools using chemometrics, instrumentation, and
waste is currently comprised of PE material, with few commercially PE fundamental laboratory studies which will better inform for the future
nanoparticles available to investigate its fate. This provides another of our environmental health and beyond.
drawback towards investigating the fate of naturally-occurring poly-
meric nanoparticles in the environment (Correia and Loeschner,
2018). El Hadri et al. (2020) investigated a top-down approach for the Declaration of competing interest
synthesis of representative polyethylene nanoparticles by mechanical
degradation of primary and secondary microplastics, and the resultant The authors declare that they have no known competing financial
particles showed heterogenous shapes and sizes, mimicking particles interests or personal relationships that could have appeared to influ-
from the marine environment (El Hadri et al., 2020). ence the work reported in this paper.
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Science of the Total Environment 732 (2020) 138792

Contents lists available at ScienceDirect

Science of the Total Environment

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

Nano-plastics and their analytical characterisation and fate in the marine

• Polymer contamination has reached a

1. Plastic in the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Type Surface Size Sample Major ﬁndings Reference

Type Surface Size Organism Major ﬁndings Reference

PS COOH 30 nm Daphnia magna • NP uptake passive Rosenkranz P. 2009

(continued on next page)

Type Surface Size Organism Major ﬁndings Reference

Reduced body size and altered

Type Surface Size Organism Major ﬁndings Reference

mortality in brine shrimps

(continued on next page)

Type Surface Size Organism Major ﬁndings Reference

Type Surface Size Organism Major ﬁndings Reference

adsorb on surface of organism

Table 3 embryonic organisms using quantitative whole-body autoradiography

4. Conclusions and future References

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