Science of the Total Environment 631–632 (2018) 1440–1456

Contents lists available at ScienceDirect

Science of the Total Environment

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

Review

An overview of graphene materials: Properties, applications and toxicity

on aquatic environments
Lucia De Marchi a,b,⁎, Carlo Pretti c, Bárbara Gabriel b, Paula A.A.P. Marques b, Rosa Freitas a, Victor Neto b
a
Department of Biology & Center for Environmental and Marine Studies (CESAM), 3810-193 Aveiro, Portugal
b
Centre for Mechanical Technology and Automation, Mechanical Engineering Department, University of Aveiro, 3810-193 Aveiro, Portugal
c
Department of Veterinary Sciences, University of Pisa, San Piero a Grado, Pisa 56122, Italy

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

• Up-to-date information on properties,

synthesis, and applications of GFMs
• Different characterization methods of
GFMs are reviewed.
• Update of the existing knowledge about
the ecotoxicity effects of GFMs
• Overview about research studies on
GFMs effects toward aquatic organisms

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

Article history: Due to unique chemical and physical properties, nanomaterials from the Graphene family are being increasingly
Received 5 January 2018 introduced in all fields of science. The specific roles they can occupy within different applications are attracting
Received in revised form 10 March 2018 increased attention by several industrial sectors. These carbon nanoparticles are released into the environment
Accepted 11 March 2018
especially accumulating in aquatic systems. Since the discovery of graphene, a number of research actives are
Available online xxxx
being conducted to find out the toxic potential of the Graphene family materials to different organism's models.
Editor: D. Barcelo Although their toxicity effects are well described for biomedical applications, few data were produced with the
specific aim of assessing the toxic effects of these carbon nanomaterials in the aquatic environment. The purpose
Keywords: of this review is to compile up-to-date information on properties, applications and characterization methods of
Graphene family materials graphene family materials in aquatic environments and identified biological toxic impacts of these NMs, with
Graphene oxide applications special focus on graphene oxide based on the most recent literature.
Characterization techniques © 2018 Elsevier B.V. All rights reserved.
Aquatic environment
Toxicity

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1441
1.1. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1442
1.2. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1443

⁎ Corresponding author at: Department of Biology & Center for Environmental and Marine Studies (CESAM), 3810-193 Aveiro, Portugal.
E-mail address: lucia.marchi@ua.pt (L. De Marchi).

https://doi.org/10.1016/j.scitotenv.2018.03.132
0048-9697/© 2018 Elsevier B.V. All rights reserved.
 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456 1441

2. Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1443
2.1. Toxicity of GFMs in aquatic environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1444
2.2. Toxicity of GFMs toward aquatic organisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1445
2.2.1. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1445
2.2.2. Photosynthetic organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1445
2.2.3. Invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1445
2.2.4. Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450
3. Methods of GFMs characterization and detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1450
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1452
4.1. Environmental safety guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1452
4.2. Levels of exposure concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1452
4.3. Interaction with biological systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1452
4.4. Studies in the field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1452
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1452
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1453
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1453

1. Introduction Woodrow Wilson database: http://www.nanotechproject.org/

Carbon nanostructures such as diamond, fullerenes, carbon nano-
tubes (CNTs), carbon black and graphene, are among the most impor-
tant carbon NMs (Neto et al., 2008, 2011; Dinadayalane and
Leszczynski, 2010; Dowling et al., 2004). The industrial production
and the number of scientific, environmental, industrial, and medical ap-
plications of carbon NMs have been increasing rapidly due to their im-
portant properties such as electromagnetic, optical, catalytic,
mechanical, thermal, and pharmacokinetic. Many carbon NMs have al-
ready been incorporated into numerous consumer products (see the
inventories/consumer/), and the number of products containing carbon
NMs are expected to increase in future years (Petersen and Henry,
2012).
Graphene is a carbon based NM assuming a 2D flat sheet conforma-
tion rather than a rolled up carbon (Peres et al., 2006). Few-layer
graphene, multi-layer graphene, graphene nanosheets, graphene
oxide (GO), and reduced-graphene oxide (r-GO), as illustrated in
Fig. 1 (Jastrzębska et al., 2012) and defined in Table 1, are considered
graphene related materials and all of them can be included in the
graphene family materials (GFMs) (Castro Neto et al., 2009; Zhang

Fig. 1. Graphene families including few-layered graphene (a), graphene nanosheet (b), graphene oxide (c), and reduced graphene oxide (d). (Jastrzębska et al., 2012) Copyright 2012,
Springer-Verlag Dordrecht.
1442 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456

Table 1 concentration measurement, the use of non-standardized experimental

Definition of GFMs. conditions and the absence of specific quantification analysis which
GFMs Definition References may influence the determination of the GFMs in the environment.
Graphene Graphene is a Bianco et al., 2013
This review paper aims to highlight and discuss the issues
single-atom-thick sheet with concerning properties, applications, characterization methods of GFMs
sp2-bonded carbon atoms that as well as the toxicological effects of these NMs, with special focus on
are suspended or adhered to a the effects of GO to aquatic organisms based on the current literature.
foreign substrate. The lateral
dimensions of graphene can
vary from nanometers to the 1.1. Properties
macroscale.
Graphene layer Graphene layer is a Bianco et al., 2013 Elastic modulus (Lee et al., 2008), very high thermal conductivity
single-atom-thick sheet with (5.1 × 103 W m−1 K−1) (Balandin et al., 2008), highest intrinsic electri-
sp2-bonded carbon atoms
occurring within a carbon
cal conductivity compared with other carbon NMs (6 × 105 S m−1) (Du
material structure. The et al., 2008), high aspect ratio of flakes (ratio of lateral dimensions to the
“graphene layer” is a structural thickness of 104 and higher) and high intrinsic flexibility (Fasolino et al.,
unit that has been used for 2007; Zhang et al., 2011) are some of the most important characteristics
many years to describe the
of GFMs. These characteristics attribute functional properties to these
structure and texture of 3D
carbon materials with primary NMs such as electrical (semi-) conductivity, unique photonic/optical
sp2-hybridized bonding. transportation, anisotropic transport, low permeability, and fluores-
Multi-layer A 2D (sheet-like) material Bianco et al., 2013 cence quenching. For all these reasons applications of GFMs are increas-
graphene consisting of a number ing rapidly (Britnell et al., 2013; El-Kady and Kaner, 2013; Tetsuka et al.,
between 2 and about 10 of
2012; Mao et al., 2013; Shahil and Balandin, 2012). Table 2 presents a
stacked graphene layers of
extended lateral dimension. set of GFMs' applications related to their properties. GO is one of the
Carbon films containing most popular graphene derivatives (Bianco et al., 2013). It exhibits var-
fragments of small lateral ious properties that make them attractive for different applications. An
graphene layers called “carbon
important feature of GO is its chemical reactivity and its capacity for
thin films” or “multi-layer
graphene”. chemical functionalization. Their functionalization involves the surface
Graphene A single-atom-thick sheet with Bianco et al., 2013 oxygen functional groups that can participate in various chemical reac-
nanosheet sp2-bonded carbon atoms that tions allowing for example the growth of metallic nanoparticles
is not an integral part of a
carbon material, but is
Table 2
suspended or adhered to a
Properties and applications of GFMs.
foreign substrate and has a
lateral dimension less than 100 Property Application References
nm
Graphene oxide GO is characterized by a Hummers and Offeman, Optical Anti-reflection coatings Y. Lin et al., 2013; P.C. Lin et al.,
(GO) distinctive structure of sp2 1958; Schniepp et al., Tailored refractive index of 2013
carbon, oxygen, and hydrogen 2006; Marcano et al., surfaces
in variable ratios. In GO, the 2010; Mas-Ballesté et al., Magnetic Increased density storage Singh, 2016; Reina et al., 2017
carbon atoms covalently 2011; Bianco et al., 2013 media
bonded to oxygen functional Nanomagnetic particles to
groups such as hydroxyl, improve detail in Magnetic
epoxy, and carboxy containing Resonance images
sp3 hybrids groups which are Thermal Improve efficiency of Cabaleiro et al., 2017
displaced above or below the coolants
graphene plane Mechanical Improve wear resistance Nine et al., 2015; Tapasztó et al.,
Reduced-graphene The reduced GO (rGO) sheets Pei and Cheng, 2012; New anti-corrosion 2017
oxide (rGO) are usually considered a kind of Bianco et al., 2013 properties
chemically derived graphene. New structural materials,
The GO can be reduced to composites
graphene-like sheets by Electronic High performance and Secor et al., 2015; Asadi et al.,
removing the smaller components (e.g. 2015
oxygen-containing groups with mobile phones)
the recovery of a conjugated High conductivity materials
structure by thermal, chemical Energy High energy density and Long et al., 2013; Sun et al., 2017;
or electrical treatments durable batteries Acik and Darling, 2016
Electrocatalysts for high
efficiency full cells
Ultra-high performance solar
et al., 2011; Sanchez et al., 2012). In recent years, several studies have cells
been conducted to understand the development of GFMs. Specifically, Catalysts for combustion
the value of the graphene market was estimated to be around US$12 engines to improve efficiency
Biomedical Sensor for diseases detection Dubey et al., 2015; Elkhenany
million in 2013 (Zurutuza and Marinelli, 2014), reaching almost US (quantum dots) et al., 2015; Singh, 2016; Reina
$195 million by 2018, US$1.3 billion by 2023, with a 5-year compound Programmed release drug et al., 2017
annual growth rate (CAGR) of 47.1% from 2018 to 2023 (BBS delivery systems
Research, 2013; Ahmed and Rodrigues, 2013; Nazarpour and Waite, Environmental Clean up soil contamination Cohen-Tanugi and Grossman,
and pollution (e.g. oil, 2014a, 2014b; Nicolaï et al.,
2016). The market projection indicates that a significant expansion
biodegradable polymers) 2014; Konatham et al., 2013
and production is to be expected, driving down the costs and driving Treatment of industrial
up the production scale (Zurutuza and Marinelli, 2014). emissions
Despite the increasing use and production of GFMs, only limited ex- Water filtration
perimental data are available in the literature concerning their environ- Personal care Inorganic sunscreens Qiu et al., 2014; Benítez-Martínez
Dye protection et al., 2016
mental impact, possibly because of the difficulties of NMs preparation,
 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456
(Gonçalves et al., 2009) or the growth of organic moieties (Gonçalves
et al., 2010). Moreover, GO surface can be also modified using covalent
bonding between nanoparticle precursors and metal oxides, as de-
scribed for titanium dioxide (TiO2) by Peter et al. (2015) that tried to de-
termine the influence of synthesis conditions and thermal treatment of
two GO–TiO2 composites on their morphostructural and photocatalytic
characterization. TiO2 was also used by Jastrzębska et al. (2016) describ-
ing the influence of the modification of the electrostatic properties of
TiO2 nanoparticles by their deposition on the surface of rGO. The same
authors investigated the effects of six key synthesis on morphology of
rGO/alumina (rGO/Al2O3) nanocomposites in terms of reducing the un-
desired agglomeration of rGO/Al2O3 nanocomposite flakes to a signifi-
cant minority and obtaining the uniform coverage of the rGO surface
with Al2O3 nanoparticles (Jastrzębska et al., 2017a). Apart from the
mentioned studies, large numbers of metal oxides and complex oxides
such as zinc oxide (ZnO) (Min et al., 2012; Zhou et al., 2012b; Khan
et al., 2012), copper (II) oxide (CuO) (Yusoff et al., 2013), copper
(I) oxide Cu2O (Gao et al., 2012), manganese (II, III) oxide (Mn3O4)
(Yao et al., 2013), manganese (III) oxide (Mn2O3) (Chandra et al.,
2012), tin dioxide (SnO2) (Seema et al., 2012), zinc tungstate
(ZnWO4) (Bai et al., 2012), bismuth molybdate (Bi2MoO6) (Wang
et al., 2012), bismuth vanadate (BiVO4) (Yan et al., 2013), barium chro-
mate (BaCrO4) (Gawande and Thakare, 2013) have also been tried in
combination with graphene and their derivates to serve as
photocatalysts and adsorbents for removal of pollutants.
The presence of covalent oxygenated groups in GO serve as sites for
modification of GO and in turn allows the tunability of its physicochem-
ical properties (Eda et al., 2008; Loh et al., 2010; Mattevi et al., 2009). For
example, by tuning the oxidation or reduction parameters to better con-
trol the structural disorder, GO can be made into an insulating, semicon-
ducting, or semimetallic material (Eda and Chhowalla, 2010). For
example the electronic properties such as conductivity of GO sheets de-
pend strongly on their chemical and atomic structures that can be mod-
ified via chemical and thermal treatments (Gilje et al., 2007; Eda et al.,
2008). In addition to electronic characteristics, GO is also expected to
exhibit optical properties as evidenced by the recent demonstration of
photoluminescence (PL) from GO (Loh et al., 2010) confirming the use-
fulness of this material for biosensing, fluorescence tags and optoelec-
tronics applications (Sun and Zhou, 2008; Eda et al., 2008).
1.2. Applications
Recent years witnessed the high growth of GFMs synthesis and the
exploitation of their wide applications, mainly due to their striking
physicochemical properties, particularly associated to high electronic
and thermal conductivity (Zhu et al., 2017). Incorporating GFMs into
solar cells is a cost-effective possibility to boost stability, optical trans-
mittance and also general performance as studied by Ubani et al.
(2016). Sensing applications is also a field in which graphene is applied.
In Alberto et al. (2015) GO–optical fibre coatings are characterized for
sensing applications. The coatings were deposited using the electro-
static self-assembly method, considering different parameters (average
sheet size of the GO colloidal solutions, the number of the deposited
layers and the coating methodology) and characterized through SEM
and Raman analyses. Aiming to explore this scheme for sensing applica-
tions, GO-coated tilted fibre Bragg gratings were characterized based on
refractive index variations and an improvement of the sensitivity was
obtained. This result suggests that this could be a promising research
field for the development of a new line of sensors. Zhu et al. (2017)
highlighted recent efforts in the design of graphene-like 2D
nanomaterials and their derived biointerfaces, including possible appli-
cations of their research on fluorescent sensors and a series of electro-
chemical sensors, including amperometric, electrochemiluminescence,
photoelectrochemical and field-effect transistor sensors.
Focusing on graphite and its allotropes, graphene, GO, and reduced
rGO, these NMs have considerable applications in quantum physics,
1443
nanoelectronics, catalysis, nanocomposites, sensor technology and
water filtration (Zhang et al., 2011; Upadhyay et al., 2014). For example,
GO foam is proposed for water remediation/purification as described in
Henriques et al. (2016) with improved performance and high selectivity
for the removal of mercury in water. Lujanienė et al. (2017) studied the
use of magnetic GO based nano-composite (MPBGO nano-composite)
for the removal of radionuclides and metals from contaminated fluids
indicating that the NPs can effectively adsorb some metals even at
high concentration of K+, Na+ and other ions naturally occurring in sea-
water. Furthermore, Santhosh et al. (2016) and Das et al. (2015)
reviewed the potential of graphene as an adsorbent for the removal of
twenty-two organic pollutants and fifteen heavy metal ions with possi-
ble implications for environmental remediation, specifically in pollution
monitoring and sustainable agriculture respectively. Recent works have
shown that graphene composites can perform multiple roles as antimi-
crobial agents making them an effective agent against all major water
pollutants including organic molecules, heavy metal ions and water
borne pathogens (Upadhyay et al., 2014). The composites of GO and
rGO hybrids with metal oxides such as Iron (II, III) oxide (Fe3O4) alumi-
num oxide (Al2O3) and TiO2 have been already explored as an antimi-
crobial agent for the disinfection of water. Gollavelli et al. (2013) have
synthesized Fe3O4 graphene composite and demonstrated the good dis-
infection properties against E. coli; Akhavan and Ghaderi (2009) re-
ported that GO/TiO2 can photo-inactivate E. coli 25% more efficiently,
as compared to bare TiO2; Jastrzębska et al. (2017b) showed that GO
can be used as adsorbent for bacteria removal showing that the bacterial
cells were so strongly adsorbed on the surface of GO that the morphol-
ogy of single cells was difficult to observe (fuzzy cell borders), and the
cells were characterized by a ‘glued’ morphology. The authors, synthe-
sizing nanocomposite flakes (rGO/Al2O3-M (where M = Silver (Ag),
Gold (Au), or Palladium (Pd)) in relation to various bacterial species,
i.e. Escherichia coli, Staphylococcus aureus, Bacillus sp., and Sarcina sp.,
demonstrated that all the synthesized nanocomposite flakes had good
adsorption properties toward Bacillus sp. and E. coli bacteria in the
near neutral pH range. In particular, the rGO/Al2O3-Ag nanocomposite
was considered the most effective material, due to the fact that it pos-
sessed advantageous biosorption and biocidal properties, which were
related to its nano silver content confirming that their properties were
applicable for water filtration.
GO use is also reviewed and highlighted by Yang et al. (2015) and
Maharubin et al. (2016) for hydrogen storage; lithium (ion and air) bat-
teries; anode, cathode, lithium sulfur batteries; supercapacitors man-
agement; and capture and conversion of harmful gases.
Graphene has also a great potential application as energy storage, re-
newable energy sources and energy transmission. Li et al. (2017) sum-
marized the evolution of graphene nano-batteries, with special focus
on electric vehicle battery technology as well envisage the graphene
battery applications. Kant et al. (2017) presented a study on heat trans-
fer of phase change materials, using graphene nanoparticles for thermal
energy storage. The applications of graphene-encapsulated materials
were highlighted by Morales-Narváez et al. (2017). Rasheed et al.
(2016) reviewed the graphene based nanofluids and nano-lubricants,
particularly their synthesis, thermal conductivity, critical heat flux, rhe-
ological studies and tribology.
2. Toxicity
Considering the increasing use and production of GFMs, and the con-
sequent discharge in the environment, their toxic impacts are becoming
an urgent issue as reviewed by Jastrzębska and Olszyna (2015). GFMs
will inevitably be released into the environments during manufacturing,
transportation, use and disposal. Upon being released into waters, sed-
iments, and soils, they will interact with a variety of physicochemical
and biological factors, thus possibly causing significant adverse effects
to the environment with implications at the ecosystem level (He et al.,
2017). Organisms are able to intake carbon NMs dispersed in the
1444 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456
environment by different ways: direct ingestion or from contaminated
prey, water filtration, inhalation, and surface contact (Cattaneo et al.,
2009) and, when in the organisms, recent studies have shown that
GFMs exhibit several unique modes of interaction with biomolecules
such as nucleic acids, lipids and fatty acids, proteins and peptides as
well as sugars (Sanchez et al., 2012) causing a generation of ROS (reac-
tive oxygen species) leading to oxidative stress in target cells
(Oberdörster et al., 2005; Marques et al., 2013; De Marchi et al.,
2017a). Giving the high relevance of this subject, the present article
aims to update the existing knowledge about the ecotoxicity effects of
GFMs.
2.1. Toxicity of GFMs in aquatic environment
To our knowledge, there is no current information regarding the
concentration of GFMs present in the aquatic environment. The ERCs
(environmentally relevant concentrations) of ENMs in water as deter-
mined based on a stochastic/probabilistic material-flow computer
model, are in the μg/L or ng/L range (Sun et al., 2016), however the
ERCs of GFMs are not reported yet. Nonetheless, the PECs (predicted en-
vironmental concentrations) of GO (one of the most used GFMs' in eco-
toxicological testing) in aqueous systems reported from the most recent
literature (see for example P. Zhang et al., 2017; X. Zhang et al., 2017)
was considered similar to other CNMs such as multi-walled carbon
nanotubes (MWCNTs) (projected to approximately 0.001–1000 μg/L)
because these two CNMs have many similarities (e.g. nanometer size,
carbon based structure and applications in consumer electronic de-
vices) (Nouara et al., 2013; Lanphere et al., 2014; P. Zhang et al., 2017;
X. Zhang et al., 2017). As a result, transport of GFMs from landfills may
cause a potential threat to nearby groundwater or surface water envi-
ronments. Moreover, there are growing concerns that ENMs such as
GFMs will enter natural water bodies at one point in their life cycle
(Hammes et al., 2013).
The toxicity of graphene has been demonstrated to be strongly re-
lated to the concentration, variety within the graphene family (two-di-
mensional size, 3D structure and thickness), manufacturing methods
and the types of functional groups (reviewed in Guo and Mei, 2014).
Studies focused on the toxicity of GFMs in the aquatic environment
showed the GFMs toxicity depends of their nature, exposure concentra-
tion, shape, size, and surface charge (Sun et al., 2016; Jastrzębska and
Olszyna, 2015; Jiang et al., 2009; Hoshino et al., 2004) as well as by
the time of exposure, medium composition, route of particle adminis-
tration and target species (Khosravi-Katuli et al., 2017).
In detail, size and shape of the nanoparticles appear to influence the
interactions with biological systems, including absorption, distribution,
metabolism, and excretion (Jiang et al., 2009). For example, the rod-
shaped nanoparticles can have larger contact area (in terms of interfa-
cial energies) with the cell membrane receptors than the spherical
nanoparticles when the longitudinal axis of the rods interacts with the
receptors. This could reduce the number of available receptor sites for
binding with over-recruitment via high ligand density, and therefore
could lower overall cell binding nanoparticles' capacity (Chithrani
et al., 2006). Indeed the uptake of nanoparticles by organisms is also af-
fected by their hydrodynamic size and surface charge (Hoshino et al.,
2004). Lockman et al., 2004). Regarding the GFMs, different distribution
of the benzene rings in the two-dimensional structure of graphene de-
termines the shape, size, edges and number of layers and additional co-
valent or noncovalent bonds with other atoms which results in
modifications of the electrical or chemical properties of graphene
which further modulate the biological effects on living cells (Skoda
et al., 2014). Also, the changes in the reconstruction type or sustained
removal of carbon atoms from the edge provides the defects which
can directly break the membranes of cells and living organisms regard-
less of graphene properties (Skoda et al., 2014).
Another main issue to consider in the environmental risk assess-
ment of carbon-based NMs in aquatic environment, is represented by
their adsorption capacities. Several studies (Apul et al., 2013; Ersan
et al., 2017; Jiang et al., 2017) have assessed the capacity to adsorb or-
ganic contaminants by different forms of graphene. Natural organic
matter (NOM) can have a significant effect on the fate and transport
of ENMs in groundwater and surface water environments (Akaighe
et al., 2013; Furman et al., 2013). Commonly present in natural waters,
NOM is a byproduct of decaying plant material and is typically present
in its dissolved form. The NOM levels found in most natural waters
range from 0.1 to 20 mg/L (Hemond and Fechner-Levy, 2009;
Rodrigues et al., 2009) and the presence of NOM has been reported to
influence on the fate and transport of ENMs (Aschberger et al., 2011;
Pelley and Tufenkji, 2008). In the work of Apul et al. (2013), graphene
nanosheet and GO were used, and compared with other carbon NMs.
The presence of NOM in the natural environment showed to negatively
influence the uptake of two organic contaminants (phenanthrene and
biphenyl) by all NMs. However, the degree at which NOM influenced
the uptake of organic contaminants was lower for graphene compared
to other NMs. The absorption of organic contaminants by graphene
nanosheets has been recently reviewed by Ersan et al. (2017). Based
on the example shown, graphenes may be considered as alternative ad-
sorbents for removing organic contaminants from water in engineering
applications. Due to their high surface to volume ratio, graphenes can
enhance the reactivity of adsorbents with environmental contaminants.
Therefore, the materials can be used to treat water/wastewater which is
generally not possible by conventional technologies (Amin et al., 2014).
However, if these are released in the environment, graphenes will also
adsorb organic contaminants influencing their fate and ecotoxicological
impact and the molecular size of organic contaminants is an important
factor that affects their capacity for adsorption. The available adsorption
sites on CNMs are determined mainly by the molecular size and shape of
the organic contaminant (Peng et al., 2014). Smaller-sized molecules
have a greater number of available sites on graphenes and therefore,
higher adsorption in comparison with larger-sized molecules. More-
over, the mobility and transport of dispersed graphenes and
graphenes-adsorbed organic contaminants could be promoted in natu-
ral aqueous environments, potentially increasing the spread of various
organic contaminants and their associated environmental risks and
this should be considered while assessing the potential environmental
impacts.
Similarly, Jiang et al. (2017) studied the adsorption of two estrogen
contaminants (17β-estradiol and 17α-ethinyl estradiol) by graphene
NMs comparing them to other carbon-based NMs like CNTs, biochars
and activated carbon in ultrapure water and in the competition of
NOM. Graphene nanomaterials showed adsorption ability, however
the effect of NOM was smaller on graphenes compared to other NMs.
The competition of NOM lowered estrogen adsorption by all adsorbents.
Summarizing, in environmental conditions graphene and other
carbon-based NMs could act as an organic contaminants
accumulation-matrix, even if NOM partially inhibits the adsorption.
This issue must be taken into account in the environmental risk assess-
ment of NMs, particularly those graphene-based.
The stability of NPs has also been reported to increase in the pres-
ence of humic acid and decrease in the presence of polysaccharides,
both of which are forms of NOM (Lanphere et al., 2014). In a study con-
ducted by Lanphere et al. (2014), the effects of groundwater and surface
water constituents (i.e., NOM and the presence of a complex assortment
of ions) on GO were investigated to provide additional insight into the
factors contributing to fate and the mechanisms involved in their trans-
port in soil, groundwater, and surface water environments. Stability re-
sults showed that the hydrodynamic diameter of the GO at a similar
ionic strength (2.1 ± 1.1 mM) was 10 times greater in groundwater en-
vironments compared with surface water and NaCl and MgCl2 suspen-
sions. Transport results confirmed that in groundwater, GO are less
stable and are more likely to be removed during transport in porous
media. In surface water and MgCl2 and NaCl suspensions, the relative re-
covery was 94% ± 3% indicating that GO will be highly mobile in surface
 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456
waters. Moreover, additional experiments were carried out in monova-
lent (KCl) and divalent (CaCl2) salts across an environmentally relevant
concentration range (0.1–10 mg/L) of NOM using Suwannee River
humic acid (SRHA), showing that the transport and stability of GONPs
was increased in the presence of NOM. This study confirmed that planar
“carbonaceous-oxide” materials follow traditional theory for stability
and transport, both due to their response to ionic strength, valence,
and NOM presence and is the first to look at GO transport across a
wide range of representative conditions found in surface and ground-
water environments.
In the natural environment, most GFM will occur in a reduced form
(Chowdhury et al., 2015). Spontaneous reduction (Kim et al., 2012;
Dimiev et al., 2012), UV irradiation (Williams et al., 2008) natural reduc-
tants (e.g. bacteria (Salas et al., 2010)) can accelerate graphenes' reduc-
tion in the environment. A study conducted by Hou et al. (2015) showed
that GO readily photo-reacts under simulated sunlight and photo-
disproportionates to CO2-reduced materials similar to rGO which are
more fragmented low molecular-weight species. Moreover, in environ-
ment the naturally available metal-based reductants (e.g. Al and Fe (Fan
et al., 2010, 2011)) can reduce GO to rGO.
Despite the different methods to evaluate the toxicity of GFMs, their
concentration in nature, physical and chemical properties, as well as
time of exposure and medium composition, the information of real
toxic effect of these carbon nanomaterials to organisms is still incom-
plete (Liu et al., 2011; Akhavan and Ghaderi, 2010; Zhang et al., 2011;
Misra et al., 2012; Sasidharan et al., 2011). Due to the expected large-
scale production of GFMs (Montagner et al., 2017), it is reasonable
focusing on their fate in the environment, which is in certain cases
unknown, but are likely to end up in water treating systems, affecting
and/or modifying aquatic community.
2.2. Toxicity of GFMs toward aquatic organisms
Toxicity to aquatic organisms may be an important concern after en-
vironmental GFM release. Studies performed to assess the effects of
GFMs toward aquatic organisms are presented in Table 3. Toxicity
data have been ranked and summarized according to organisms' taxo-
nomic group, type of graphene-based nanomaterials, exposure condi-
tions, mechanisms of interaction and effects (Table 3).
2.2.1. Bacteria
Bacteria are the main component of activated sludge, involved in the
biological wastewater treatment process. The presence of contaminants
in the wastewater influent may adversely affect the functions of these
microorganisms. Therefore, GO and other functionalized forms of this
nanomaterials on a wastewater microbial community have been inves-
tigated, showing that bacteria from sludge could be significantly com-
promised (Akhavan and Ghaderi, 2010; Hu et al., 2010; Liu et al.,
2011; Wu et al., 2013) as well as the community's metabolic activity,
bacterial viability, and nutrient biological removal capacity (Ahmed
and Rodrigues, 2013). Moreover, it has also been demonstrated that
the presence of these NPs can cause effects on bacterial survival
(Wang et al., 2014) and cell membrane integrity (Ristic et al., 2014).
Specifically, it was observed that rGO was more toxic compared to GO
in terms of antibacterial activities in Escherichia coli and Staphylococcus
aureus (Akhavan and Ghaderi, 2010), however a study conducted by
Liu et al. (2011) demonstrated opposite results showing the highest an-
tibacterial activity in E. coli by GO (69.3 ± 6.1% cell inactivation)
followed by rGO (45.9 ± 4.8%), Gt (26.1 ± 4.8%), and GtO (15.0 ±
3.7%). In agreement with the present finding Ahmed and Rodrigues
(2013) confirmed the high toxic effects of GO, showing impact on bac-
terial metabolic activity, bacterial viability, and biological removal of nu-
trients, such as organics, nitrogen and phosphorus by these carbon NPs.
Moreover, in a study conducted by Wang et al. (2014) GO helped the
dispersion of ZnO NPs, slowed the dissolution of ZnO, acted as the stor-
age site for dissolved zinc ions, and enabled the intimate contact of
1445
E. coli with ZnO NPs and zinc ions as well. The results of the studies pre-
sented in Table 3, have also shown that functionalized form of
graphenes can be considered more toxic compared to the non-
functionalized ones as demonstrated by Wu et al. (2013). The authors
functionalized rGO with glutaraldehyde (MRGOGA) showing to be
more rapid and effective photothermal agent toward S. aureus and
E. coli compared to non-functionalized rGO. The irradiation of graphines
was also demonstrated to increase the toxicity of the GFMs as demon-
strated by Ristic et al. (2014) where the irradiated graphene quantum
dots (GQD) with blue light photodynamically disrupted S. aureus and
E. coli cell membrane integrity, entered the damaged bacteria, and in-
creased the numbers of green fluorescent ROS-containing cells.
2.2.2. Photosynthetic organisms
In cyanobacteria and different species of green algae growth inhibi-
tion (Pretti et al., 2014; Wang et al., 2016; Du et al., 2016), ROS genera-
tion (Tang et al., 2015; Nogueira et al., 2015) and oxidative stress
(Nogueira et al., 2015; Ouyang et al., 2015; Wang et al., 2016) were in-
vestigated as potential effects of exposure to GFMs as well as cell divi-
sion (Hu et al., 2015; Nogueira et al., 2015; Ouyang et al., 2015) and
cell permeability. In detail, Guo et al. (2016) investigated the toxicity
of graphene to the algal cells Chlorella pyrenoidosa measured as cell sur-
vival rates in the presence of dissolved organic matter showing that the
toxicity of graphene was concentration-dependent, with a decrease of
the survival rate of cells with the increase of graphene concentration
leading to the hypothesis of a possible shading effect of the Carbon
NPs CNPs. A concentration-dependent growth inhibition of graphene
was also demonstrated by Wang et al. (2016) in the unicellular green
alga Scenedesmus obliquus. Moreover, these CNPs generated oxidative
damage in the algae by causing ROS to be produced. The effects of func-
tionalized form of graphene such as graphine nano-powder grade C1
(GNC1) and graphene monolayer flakes (PGMF) were also investigated
by Pretti et al. (2014) showing that GNC1 was more toxic compared to
PGMF in terms of growth inhibition of the green alga Dunaliella
tertiolecta.
GO has been observed to generate toxic effects in terms ROS gener-
ation and membrane damage (cell viability) in green algae Raphidocelis
subcapitata (Nogueira et al., 2015) and inhibition of cell rates division in
another green algae Chlorella vulgari (Hu et al., 2015). Different effects
by GO were observed in cyanobacteria Microcystis aeruginosa for
which none of the tested GO concentrations exhibited significant toxic-
ity, however the presence of GO significantly enhanced Cd2+ toxicity
(demonstrated with the generation of ROS as toxic mechanism)
confirming that GO can enhance the toxicity of Cd2+ by introducing
more Cd2+ into the cell or attaching itself onto the surface (Tang et al.,
2015).
The possible toxic effects in photosynthetic organisms by a function-
alized form of GO were also investigated. Ouyang et al. (2015), using the
green alga Dunaliella tertiolecta demonstrated that internalization of GO
quantum dots GOQD was 10–80 times higher than nano-GO (GONS)
causing higher cell division, cell permeability and oxidative stress. Du
et al. (2016) evaluated the phytotoxicity of rGO to microalgae
Scenedesmus obliquus and showed first of all that the treatment with
rGO suppressed microalgae growth. Furthermore, rGO has inhibited
Chlorophyll a and b levels in the algal cells.
2.2.3. Invertebrates
Invertebrates are considered the ultimate recipients of most con-
taminants released into the environment (Torre et al., 2013; Faggio
et al., 2016; Pagano et al., 2017; Savorelli et al., 2017). Although an in-
creasing number of studies on invertebrates has been published on
NPs toxicity, comprehensive knowledge on the toxic effects posed by
GFMs to these aquatic organisms is still poor. What has been already
demonstrated from the literature is that composites of graphene hy-
brids with metal oxides (FePO4/dodecylamine hybrid nanosheets), pris-
tine graphene nanoparticles, few-layer graphene (FLG), GO, rGO and
1446 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456

Table 3
Summary of in vitro and in vivo studies of most frequently used/found GFMs in aquatic-related species. Toxicity data have been ranked according to organisms' taxonomic group, species,
type of graphene based nanomaterials, exposure conditions, mechanisms of interaction, effects, characterization techniques.

Group of Species Graphene based Exposure conditions Mechanisms of Effects Characterization References
organisms nanomaterials interaction techniques

Bacteria Escherichia coli Graphene oxide Spread of powder (1 Direct contact Antibacterial activities: SEM Akhavan
nanowalls (GONWs) mg/mL) in 100 μL of the after 1 h, 41 ± 8% of the and Ghaderi
diluted bacterial bacteria survived (2010)
Staphylococcus Graphene oxide suspension for 24 h Antibacterial activities:
aureus nanowalls (GONWs) after 1 h, 26 ± 5% of the
bacteria survived
Escherichia coli Reduced graphene Antibacterial activities:
nanowalls (RGNWs) after 1 h, 16 ± 3% of the
bacteria survived
Staphylococcus Reduced graphene Antibacterial activities:
aureus nanowalls (RGNWs) after 1 h, 5 ± 1% of the
bacteria survived
Escherichia coli Nano-graphene oxide Incubation with 20 and Internalization Decreased of cell FT-IR spectra; Hu et al.
(GO) 85 μg/mL for 2 h within cells metabolic activity to 70% TEM (2010)
and 13% at 20 and 85
μg/mL respectively
Escherichia coli Graphite (Gt) Incubation with 40 Direct contact Cell inactivation SEM; DLS Liu et al.
μg/mL dispersions in percentage of 26.1 ± 4.8% (2011)
Graphite oxide (GtO) isotonic saline solution Cell inactivation
for 2 h percentage of 15.0 ± 3.7%
Graphene oxide (GO) Cell inactivation
percentage of 69.3 ± 6.1%
Pseudomonas Graphene oxide (GO) Incubation with 0, 25, 50, Direct contact Decreased of viability UV–Vis; X-ray Gurunathan
aeruginosa 75, 100, 125, and 150 progressively with diffraction; SEM; et al. (2012)
μg/mL in isotonic saline increased of GO Raman
solutions for 2 h concentration (observed spectroscopy
at concentrations of 75
μg/mL)
Wastewater Graphene oxide (GO) Exposure of 10, 20, 50, Adsorption Dose-dependent toxic AFM; XPS Ahmed and
microbial 100, 200 and 300 mg/L effects of GO on microbial Rodrigues,
community for 5 h communities, especially 2013
at concentrations
between 50 and 300 mg/L
showing significantly
impact on bacterial
metabolic activity,
bacterial viability, and
biological removal of
nutrients (organics,
nitrogen and phosphorus)
Escherichia coli Graphene oxide (GO) Agar disk diffusion Direct contact GO helped the dispersion TEM; XRD Wang et al.
method: approximately of ZnO NPs, slowed the (2014)
106 CFU/mL cells dissolution of ZnO, acted
inoculated on as storage site for the
Sabouraud's agar plates dissolved zinc ions and
and 10, 20, or 40 μL of enabled the intimate
ZnO/GO− contact of E. coli with ZnO
nanocomposite NPs and zinc ions as well
suspension (100 μg/mL)
on the surface
Pseudomonas Reduced graphene oxide Incubation with 0, 25, 50, Direct contact Decreased viability UV–Vis; XRD; Gurunathan
aeruginosa (rGO) 75, 100, 125, and 150 progressively with SEM et al. (2012)
μg/mL for 2 h increase of GO
concentration (observed
at concentrations of 75
μg/mL)
Escherichia coli Incubation with 40 Direct contact Cell inactivation AFM; SEM Liu et al.
μg/mL for 2 h percentage at 45.9 ± 4.8% (2011)
Staphylococcus Magnetic reduced Turbidity measurements Absorption 99% killing efficiency FT-IR spectra; Wu et al.
aureus graphene oxide (after every 10-min photothermal agent TEM (2013)
Escherichia coli functionalized with vortex) with addition of toward both bacteria
glutaraldehyde 50 mg MRGOGA to 5 mL
(MRGOGA) of bacterial suspensions
Escherichia coli Graphene quantum dots Incubation with 200 Incubation and GQD generated ROS when AFM; TEM Ristic et al.
(GQD) mg/mL for 10 min and photoexcitation the bacteria were (2014)
Staphylococcus Graphene quantum dots irradiated with blue light photoexcited (470 nm, 1
aureus (GQD) (465–475 nm, 1 W) W)
Photosynthetic Dunaliella Pristine graphene Suspension with 10 mg/L Mechanical The LOEC (lowest DLS Pretti et al.
organisms tertiolecta monolayer flakes (PGMF) to 0.675 mg/L for 72 h interaction observed effect (2014)
concentration) of PGMF
was 0.675 mg/L
Graphene nanopowder LOEC of GNC1 was 1.25
gradeC1 (GNC1) mg/L
 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456 1447

Table 3 (continued)

Group of Species Graphene based Exposure conditions Mechanisms of Effects Characterization References
organisms nanomaterials interaction techniques

Microcystis Graphene oxide (GO) Exposure to combined Internalization with No significant toxicity due DLS; Zeta Tang et al.
aeruginosa effects of GO and Cd2+ cells to the combined effects of potential; SEM; (2015)
solution: 0.1, 0.3, 0.5, 1.0, GO and cadmium ion Cd2 TEM
+
2.0, and 5.0 mg/L for 24 solution on the algae
h. was observed. The
presence of GO even if at
low concentrations,
significantly enhanced
Cd2+ toxicity revealed
that GO, with Cd2+, both
attached to and entered
the algae with consequent
ROS generation
Raphidocelis Exposure to 0; 0.5; 2.0; Attached to the algal ROS generation and DLS; Zeta Nogueira
subcapitata 5.0; 10.0; 20.0; 50.0; surface membrane damage (cell potential; AFM; et al. (2015)
70.0; and 100 μg/mL for viability) started from Raman
96 h 10.0–20.0 μg/mL spectroscopy;
UV–Vis
Chlorella Exposure to 0.1; 1.00 and Direct interaction GO inhibited the rates of DLS; zeta Hu et al.
vulgaris 10.0 mg/L for 96 h cell division by 0.08–15% potential; XPS; (2015)
at 10 mg/L and covered TEM; EPR
the cell surface.
Metabolomic analysis
revealed significant
differences in the
metabolic profiles
Chlorella Graphene oxide quantum Spike of 0.02, 0.2, 2.0, Direct interaction Internalization of GOQD EPR; XPS; TEM; Ouyang
vulgaris dots (GOQD) and 20.0 μg for 96 h (smaller than GONS), AFM et al. (2015)
Nano-graphene oxide resulted 10–80 times
(GONS) higher than GONS, and
ecotoxicity resulted was
higher with different
effects (e.g. cell division,
cell permeability and
oxidative stress)
Scenedesmus Graphene nanoplatelets Inoculation at 0.5, 1, and – The GNP suspension TEM; DLS; Zeta Wang et al.
obliquus (GNP) 5 mg/L for 96 h caused potentials (2016)
concentration-dependent
growth inhibition: 5 mg/L
(34 ± 1%); 1 mg/L (22 ±
2%); 0.5 mg/L (17 ± 7%).
GNPs may have caused
oxidative damage in the
algae by causing ROS
production
Chlorella Graphene Inoculation at 0, 5, 10, 20, – Survival rate of cells TEM; SEM Guo et al.
pyrenoidosa 30, 50, 70, 100, 150, 200 decrease with the (2016)
mg/L of Graphene and increase of graphene
addition of citric acid concentration. Median
(CA) solutions (5, 10, 20 effective concentration
mg/L) into algal cells (EC50) was 41.35 mg/L.
with the treatment of 40 Cell survival rate by
mg/L graphene for 24 h. graphene (78.44%)
increased in the presence
of CA (37.07%)
Scenedesmus Reduced graphene oxide Exposure to 0, 10, 20, 50, Direct contact All the treatment with SEM; AFM; FT-IR Du et al.
obliquus (rGO) 100, 150, 200, 250, and rGO suppressed spectra (2016)
300 mg/L for 72 h microalgae growth. The
72 h IC50 values of rGO
evaluated were 148 and
151 mg/L with significant
inhibition of Chlorophyll a
and b levels in the algal
cells
14
Invertebrate Daphnia magna C-labeled graphene Exposure to 250 μg/L for Accumulation into Nanomaterial's HPLC; GC–MS; Guo et al.
24 h the gut and transfer concentration was nearly SEM; TEM; (2013)
to the neonates 1% of the organism dry Raman
mass and the spectroscopy
accumulated graphene in
adult Daphnia was likely
transferred to the
neonates
Artemia salina Pristine graphene Exposure to 0.10 and Mechanical Presence of PGMF and DLS Pretti et al.

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Table 3 (continued)

Group of Species Graphene based Exposure conditions Mechanisms of Effects Characterization References
organisms nanomaterials interaction techniques

Graphene nanopowder 1.00 mg/L 48 h interaction GNC1 aggregates into the (2014)
grade C1 (GNC1) gut; a 48 h exposure
monolayer flakes (PGMF) experiment revealed an
altered pattern of
oxidative stress
biomarkers (increase of
catalase activities in both
PGMF and GNC1 at 1.00
mg/L); increase of
glutathione peroxidase
activities in PGMF (0.1
and 1 mg/L) and
increased levels of
membrane lipid
peroxidation was also
observed at PGMF 1 mg/L
Litopenaeus Few-layer graphene Exposure to 500 Incorporation through the FLG exposure led Raman
vannamei (FLG) mg/kg of ration food to an increase in spectros-
mixed to the feed for ROS copy; TEM;
8 days concentrations, DLS
modulation of
the activity of
antioxidant
enzymes
glutamate
cysteine ligase
and
glutathione-S--
transferase and
reduced
glutathione
levels and total
antioxidant
capacity
Fernandes
et al. (2017)
Limnodrilus Exposure to 1 mg/L for 36 Uptake from the The FLG body burden UV–Vis; DLS; XPS; FT-IR Mao et al.
hoffmeisteri h sediment and in the organism was spectra (2016)
accumulation in the gut nearly 60 ng/mg (on
a dry mass basis).
Protein-coated FLG
demonstrated
different
bioaccumulation
behaviors in the
organisms
Amphibalanus Graphene oxide (GO) Exposure to 0.001; 0.01; Mechanical Increased concentrations TEM; SEM; DLS Mesarič
amphitrite 0.1; 0.5; 0.75 and 1 interaction with and increased exposure et al. (2013)
mg/mL for 24, 48 and 72 larvae times resulted in the
h decreased of nauplii
swimming speed and
increased in their
mortality
Artemia salina Graphene oxide (GO) Exposure to 0–700 mg/L Ingestion Acute mortality at the TEM; SEM; DLS Mesarič
for 72 h highest concentration in et al. (2015)
A. salina larval stages at
the end of 72 h
Diopatra Graphene oxide (GO) Exposure to 0.01; 0.10 – Organisms exposed to SEM; IR De Marchi
neapolitana and 1.00 mg/L for 28 higher concentrations spectroscopy; et al.
days took longer periods to Raman (2017a)
completely regenerate spectroscopy
the entire body. GO also
seemed to alter
energy-related responses
and induce cellular
damage, despite higher
activities of antioxidant
and biotransformation
enzymes in individuals
exposed to GO
Tubifex tubifex Graphene oxide (GO) Exposure to 20 and 180 Uptake of No mortality of T. tubifex TEM; AFM P. Zhang
μg GO/g in dry weight sediment-associated was observed at both GO et al. (2017)
sediment for 5 days NPs concentrations, whereas and X.
burrowing activity was Zhang et al.
reduced at 20 and 180 μg (2017)
 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456 1449

Table 3 (continued)

Group of Species Graphene based Exposure conditions Mechanisms of Effects Characterization References
organisms nanomaterials interaction techniques

GO/g
Mytilus Graphene oxide (GO) Exposure to 0.001, 0.01, Endocytosis and/or GO, GO-PVP and rGO-PVP TEM; EDX Katsumiti
galloprovincialis 0.1, 1, 10, 25, 50 and 100 phagocytosis uptake showed et al. (2017)
mg/L for 24 h of NPs by mussel concentration-dependent
hemocytes cytotoxicity. rGO-PVP
Reduced graphene oxide (LC50 = 29.902 and 33.94
(rGO) with mg/L depending on the
polyvinylpyrrolidone-PVP origin) was more toxic
(rGO-PVP) than GO (LC50 = 49.84
Graphene and 54.51 mg/L
oxide with depending on the origin)
polyvinylpy- and GO-PVP (LC50 =
rrolidone-- 43.72 mg/L). GO, GO-PVP
PVP and rGO-PVP caused
(GO-PVP) membrane damage and
their toxicity is
ROS-mediated.
Fish Danio rerio Graphene oxide (GO) Exposure to 0, 3.4, 7.6, Coverage of the GO induced toxicity to Extinction Chen et al.
12.5, 25 and 50 mg/L for whole embryo body organisms since it spectra; SEM (2012)
24 h induced minor (about
20%) cell growth
inhibition and slight
hatching delay of
zebrafish embryos at a
dosage of 50 mg/L
Graphene oxide (GO) Suspension of 100, 10, Absorption to the GO (10 and 100 mg/L) Zeta potential; Chen et al.,
1.0, 0.1 or 0.01 mg/L in surface of chorions adhered to and enveloped AFM; TEM; SEM; 2015
embryo culture medium and embryos the chorion of zebrafish FT-IR spectra
for 24 h embryos and caused
marked hypoxia and
hatching delay. GO
damaged the
mitochondria and
circulatory system of
zebrafish inducing
generation of ROS
(oxidative stress, DNA
damage and apoptosis)
Graphene oxide (GO) Exposure to 2, 10 and 20 Interaction with cell GO exposure after 24 and AFM Souza et al.
mg/L of GO for 24 h, 72 h membranes 72 h led to an increase in (2017)
and 14 days the number of gill cells
that were in early
apoptotic and necrotic
stages. Generation of ROS
in gill cells. Genotoxicity
was not observed in blood
cells after 14 days
Graphene oxide (GO) Exposure to trace Direct contact DNA modification, protein AFM, Zeta P. Zhang
concentrations (1–100 carbonylation, ROS potential et al. (2017)
μg/L) for 7 days generation, especially the and X.
superoxide radical. Also, Zhang et al.
the transcriptomic (2017)
analysis revealed that
disturbing collagen and
matrix metalloproteinase
(MMP)-related genes
affected the cardiac
development of zebrafish
Ultra-small graphene Exposure to N/P ratios Transfection into the Plasmid DNA was AFM; FT-IR Zhou et al.
oxide (PEI-USGO) (the molar ratio of embryos transfected into zebrafish spectra; zeta (2012a)
nitrogen in PEI-g-USGO embryos with 90% potential
to phosphate in DNA) efficiency and high
from 2.3 to 46 for 24, 48 embryo viability after 72
and 72 h h, increasing with the
increase of N/P ratios
Graphene oxide quantum Exposure from 12.5 to Transfer of the NPs The results showed a TEM; PL; FT-IR Wang et al.
dots (GOQD) 200 μg/mL for 4-96 h from the solutions decrease of movement spectra (2015)
into the heart and exposure to GQDs at
intestinal region of concentrations of 50, 100,
the embryos and 200 μg/mL. The larval
behavior testing showed
that the total swimming
distance and speed

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Table 3 (continued)

Group of Species Graphene based Exposure conditions Mechanisms of Effects Characterization References
organisms nanomaterials interaction techniques

decreased
dose-dependently.
Embryos exposed to 12.5
μg/mL showed
hyperactivity while
exposure to higher
concentrations caused
hypoactivity in the
light-dark test
Poeciliopsis GO nanoplatelets Exposure to 0.125–16 Internalization and GO nanoplatelets AFM; TEM; DLS Lammel and
lucida μg/mL for 24, 48 and 72 h intracellular fate in penetrated through the Navas
the fish cell line plasma membrane and (2014)
Carboxyl graphene Exposure to 0.25–32 accumulated in the
(CXYG) μg/mL for 24, 48 and 72 h cytosol. Cells
demonstrated
significantly reduced
mitochondrial membrane
potential (MMP) and
increased ROS levels at 16
μg/mL GO nanoplatelets
and CXYG (72 h)

their hybrids form (GO-polyvinylpyrrolidone-PVP) (GO-PVP and Rgo-
PVP) have been already explored as causes of toxicity in invertebrates'
due to accumulation and transfer from adults to neonates (Guo et al.,
2013), accumulation in the organs (Pretti et al., 2014) oxidative stress
(Pretti et al., 2014; Fernandes et al., 2017; De Marchi et al., 2017a), alter-
ation on physiology, behavior and death (Mesarič et al., 2013, 2015; P.
Zhang et al., 2017; X. Zhang et al., 2017). In detail, FLG was able to gen-
erate an increase in ROS concentrations, influenced antioxidative en-
zyme system by regulating the activities of glutamate cysteine ligase
and glutathione-S-transferases, and lowered glutathione levels and
total antioxidant capacity in shrimp Litopenaeus vannamei exposed to
these contaminates (Fernandes et al., 2017). Moreover, FLG demon-
strated different bioaccumulation behaviors in the aquatic oligochaete
Limnodrilus hoffmeisteri (Mao et al., 2016). Hybrids form of graphene
were also used to evaluate possible toxic responses in different inverte-
brate species: Guo et al. (2013) using synthesized graphene by means of
graphitization and exfoliation of sandwich-like FePO4/dodecylamine
hybrid nanosheets and 14C, showed that these CNPs, accumulated in
adults, were subsequently transferred to the neonates in the crustacean
Daphnia magna. Also, other forms of synthesized graphene (such as
PGMF and GNC1) showed the same behavior by accumulating in the
gut of crustacean Artemia salina generating an oxidative status in the or-
ganisms (Pretti et al., 2014). Toxic effects of GO were also investigated in
different invertebrate organisms. The results showed that these CNPs
were able to generate alteration on the survival rate and inhibition of
swimming behavior of crustacean Amphibalanus amphitrite's nauplii
(Mesarič et al., 2013). GO also showed the capacity to attach on larvae
surface including on gills and appendages, causing an alteration of
swimming behavior in A. salina (Mesarič et al., 2015). Moreover P.
Zhang et al. (2017) and X. Zhang et al. (2017) showed that burrowing
activity of oligochaete, Tubifex tubifex was significantly reduced after
GO exposure. A recent study also demonstrated that GO caused alter-
ation in terms of biochemical performances. As an example, in the
polychaeta Diopatra neapolitana GO seemed to alter energy-related re-
sponses and induce cellular damage, despite higher activities of antiox-
idant and biotransformation enzymes in individuals exposed to these
contaminates (De Marchi et al., 2017a). Comparing the toxic effects of
different forms of GO (with and without polyvinylpyrrolidone-PVP as
stabilizing agent: GO and GO-PVP) and rGO (with and without
polyvinylpyrrolidone-PVP as stabilizing agent: GO and GO-PVP)
Katsumiti et al. (2017) demonstrated rGO was more toxic than GO
and GO-PVP in mussels Mytilus galloprovincialis. However, all these
CNPs were able to cause membrane damage and their toxicity were
ROS-mediated in the exposed organisms.
2.2.4. Fish
Invertebrates, in turn, are consumed by fish, making possible the
process of bio magnification of NPs. The majority of studies concerning
GFMs impacts to aquatic organisms are devoted to freshwater species,
including fish species in which zebrafish (Danio rerio) is among the
most commonly used (Chen et al., 2012; Zhou et al., 2012a; Wang
et al., 2015; Souza et al., 2017; P. Zhang et al., 2017; X. Zhang et al.,
2017). These studies are based on the evaluation of bioaccumulation
and concentration levels in different tissues and organs as well as excre-
tion rates, oxidative stress and mortality as a consequence of GO and
GQDs exposures. In all the above cited studies GO caused growth inhibi-
tion and slight hatching delay of D. rerio embryos (Chen et al., 2012).
Furthermore, GO were able to adhere to the chorion of zebrafish em-
bryos causing envelopment, hypoxia and hatching delay (Chen et al.,
2015). Biochemical responses were also observed in organisms exposed
to these CNPs inducing excessive ROS generation and increased oxida-
tive stress, DNA damage and apoptosis in the mitochondria and the cir-
culatory system of zebrafish embryos (Chen et al., 2015; P. Zhang et al.,
2017; X. Zhang et al., 2017). Bioaccumulation and ROS generation in an-
other fish species (Poeciliopsis lucida) were also observed after GO expo-
sure (Lammel and Navas, 2014). Transcriptomic analysis revealed that
disturbing collagen and matrix metalloproteinase (MMP)-related
genes affected the cardiac development of zebrafish (P. Zhang et al.,
2017; X. Zhang et al., 2017). Histopathology damage were also caused
in D. rerio exposed to GO, generating an increase in the number of gill
cells that were in early apoptotic and necrotic stages, but genotoxicity
was not observed in blood cells. The authors also observed damage at
the level of the hepatocytes (non-uniform shape, picnotic nuclei, vacu-
ole formation, cell rupture, and necrosis) demonstrating that GO could
be harmful to fish species representing risks for the aquatic food chain
(Souza et al., 2017).
Toxic effects of GQDs were also investigated zebrafish embryos
showing that these CNPs induced embryonic malformations and larval
hyperactivity (Wang et al., 2015).
3. Methods of GFMs characterization and detection
There are several techniques that can be applied for GFMs character-
ization and detection. These include scanning and transmission electron
 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456
microscopy (SEM and TEM), atomic force microscopy (AFM), nuclear
magnetic resonance spectroscopy (NMR), Fourier transformed infrared
spectroscopy (FT-IR), inductively coupled plasma mass spectroscopy
(ICP-MS), atomic absorption spectroscopy, X-ray photoelectron spec-
troscopy (XPS), X-ray diffraction (XRD), X-ray absorption near edge
structure (XANES), Brunauer-Emmett-Teller analysis of surface area
(BET), thermal gravimetric analysis (TGA), zeta potential analysis, parti-
cle size measurement using dynamic light scattering (DLS) or
electrospray scanning mobility and flow field flow fractionation,
among others (Huang et al., 2011). Table 4 (Y. Lin et al., 2013; P.C. Lin
et al., 2013) summarizes the available data found in the literature re-
garding different modalities of GFMs characterizations depending on
the physicochemical characteristics of nanomaterials. Considering the
several specific characteristics of GFMs such as size, surface composi-
tion, surface energy, surface charge and shape few specialized tech-
niques for characterization of GFMs have been used in ecotoxicity
studies including SEM, TEM, AFM, DLS analysis, Zeta potential, FT-IR
spectra, XPS, UV–Vis spectroscopy and Raman spectroscopy
(Jastrzębska and Olszyna, 2015). Table 3 summarizes all the characteri-
zation techniques used in each of the described ecotoxicity study.
SEM is a type of electron microscope that produces images of a sam-
ple by scanning the surface with a focused beam of electrons and gener-
ally is a technique used to give information concerning the morphology-
average shape and size-of NPs crystallites (De Marchi et al., 2017a). In
contrast to optical microscopy, which uses light sources and glass lenses
to illuminate specimens to produce magnified images, electron micros-
copy (EM) uses beams of accelerated electrons and electrostatic or elec-
tromagnetic lenses to generate images of much higher resolution,
because of the much shorter wavelengths of electrons than visible
light photons (Lin et al., 2014). The size distribution and shape of
nanomaterials can be directly acquired from SEM, however, the process
of drying and contrasting samples may cause shrinkage of the specimen
and alter the characteristics of the nanomaterials. Moreover, because a
cryogenic freezing method is often required in EM to image surface
groups attached to NPs, the size of nanomaterials cannot be investigated
in physiological conditions (Hall et al., 2007) with an exception for en-
vironmental SEM (ESEM), through which samples can be imaged in
their natural state without modification or preparation. Also, TEM is a
type of electron microscope that gives information to generate particle
size data for individual particles, number-based size distributions for
the entire dispersion and various shape and morphological parameters.
In the TEM mode, an incident electron beam is transmitted through a
very thin foil specimen, during which the incident electrons interacting
with the specimen are transformed to unscattered electrons, elastically
scattered electrons or inelastically scattered electrons and the magnifi-
cation of TEM is mainly determined by the ratio of the distance between
objective lens and the specimen and the distance between objective
lens and its image plane (Lin et al., 2014). However, the primary
Table 4
Physicochemical characteristics of nanomaterials and suitable evaluation modalities (Y.
Lin et al., 2013; P.C. Lin et al., 2013).
Nanomaterial Techniques
characteristics
Size/size DLS, FCS, RS, NSOM, SEM, TEM, STM, AFM, NMR, TOF-MS, XRD,
distribution SAXS, FS, UV–visible, AUC, GE, CE, FFF
Surface charge Zeta potential (ELS), ATR-FTIR, GE, CE
Shape NSOM, SEM, TEM, STM, AFM, XRD, SAXS, AUC
Structure TERS, CD, MS, IR, STM, AFM, RS, NMR, XRD, SAXS, FS, DSC, AUC
Composition MS, NMR
Purity MS, NMR, HPLC, HDC
Stability Zeta potential measurement, CD, TGA, DSC, ITC, thermophoresis,
HPLC, HDC
Dispersion ESEM, TEM, STM, AFM
Surface CD coupled with an enzyme-linked immunosorbent assay,
properties time-of-flight secondary ion MS, ATR–FTIR, modified AFM, X-ray
photoelectron spectroscopy
1451
difference in data output between the two techniques is the way in
which the nanoparticle images are resolved. SEM produces accurate
3D images of particles in the dispersion while TEM produces 2D images
that require further interpretation and, although the images rendered
are two dimensional, TEM systems are capable of delivering much
greater resolution (Hassellöv et al., 2008). Like SEM and TEM tech-
niques, AFM can be used for investigating the size, shape, structure,
sorption, dispersion and aggregation of nanomaterials. The different
scanning modes employed in AFM studies include noncontact mode
(also called static mode), contact mode and intermittent sample contact
mode (also called dynamic mode and tapping mode) (Lin et al., 2014).
In addition, AFM is capable of characterizing dynamics between
nanomaterials in biological situations, such as observing the interaction
of nanomaterials with supported lipid bilayers in real time, which is not
achievable with current EM techniques. AFM is gaining importance due
to its capability for imaging biomaterials without causing appreciable
damage to many types of native surfaces (Parot et al., 2007). The main
strength of AFM is its capability to image a variety of biomaterials at
the sub-nanometer scale in aqueous fluids (Parot et al., 2007). However,
a major drawback is that the size of the cantilever tip is generally larger
than the dimensions of the nanomaterials examined, leading to unfa-
vorable overestimation of the lateral dimensions of the samples (Hoo
et al., 2008). Several physicochemical characteristics of nanomaterials
including hydrodynamic size, shape, structure, aggregation state and
biomolecular conformation can be explored using radiation scattering
techniques and DLS is one of the most popular light scattering modali-
ties (Dzakpasu and Axelrod, 2004). DLS is one of the most sensitive
techniques for the detection of subvisible particles, with the maximum
light scattering efficiency per unit of weight occurring between 0.1 and
1 μm (Mach et al., 2012; De Marchi et al., 2017a, 2017b, 2017c, 2017d).
DLS is commonly used and referred to as photon correlation spectros-
copy (PCS), which is a particle suspended in a liquid solvent that un-
dergoes a random Brownian motion (Hoo et al., 2008), however, the
functions of DLS are impacted by several disadvantages, such as diffi-
culty in correlating size fractions with a particular composition when
certain amounts of aggregates are present, dust particles interfering in
the scattering intensity, and a relatively small range of particle or mole-
cule size (1 nm–3 μm), although the scale limitation is not really a pitfall
for characterization of nanomaterials (Bootz et al., 2004; Brar and
Verma, 2011; Filipe et al., 2010). In addition, DLS has limited utility for
analysis of samples with heterogeneous size distributions and resolving
the dimensions of a mixed sample population varying in size less than a
factor of three; moreover, DLS is unsuited to accurately measure the size
of non-spherical nanomaterials because the spherical nature of particles
is already assumed in the analysis (Lin et al., 2014). Zeta Potential anal-
ysis is a technique for determining the surface charge of nanoparticles in
solution (colloids). The electric potential at the boundary of the double
layer of nanoparticles is known as the Zeta potential and has values that
typically range from +100 mV to −100 mV (Clogston and Patri, 2011).
This technique is an important tool for understanding the state of the
nanoparticle surface and predicting the long-term stability of the nano-
particle (Clogston and Patri, 2011). However, it still suffers the electro-
osmotic effect that reduces precision and reproducibility of the mea-
surement, a property also sensitive to environmental changes including
pH and ionic strength (Lin et al., 2014). XPS, also known as X-ray photo-
emission spectroscopy, is a very powerful surface technique commonly
used to understand electronic structure and elemental compositions of
nanoparticles, including the oxidation state of an element. Specifically,
the ability of XPS is to provide information on the local bonding envi-
ronment of a given species makes it an essential tool for understanding
the surface chemistry of the nanomaterial (Sarma et al., 2013). FT-IR
spectra is techniques for the study of nanomaterial surfaces that can
be used in either a qualitative or quantitative way. FT-IR is considered
a method of analysis to probe surface adsorption of ligands on
nanomaterials. The absorption of infrared light, due to the excitation
from the ground vibrational energy level to a higher energy level,
1452 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456
gives information on molecular structure and molecular interactions
(Mudunkotuwa et al., 2014). This spectroscopy can serve as a comple-
ment to UV–Vis, which is a technique able to measure the intensity of
light that passes through the nanoparticle (Tomaszewska et al., 2013).
Although FT-IR spectroscopy can be implemented to study the surface
features of nanomaterials, it is not a very sensitive surface-analysis
method at the nanometer scale because the penetration depth of FT-IR
has the same order of magnitude as the incident IR wavelength (Lin
et al., 2014). Raman spectroscopy (RS) is a spectroscopic technique
used to observe vibrational, rotational, and other low-frequency
modes in nanoparticles but these techniques do not provide any infor-
mation concerning the morphology-average shape and size-of the
nanomaterials (De Marchi et al., 2017a). One of the major advantages
of RS is that it is suitable for studying biological samples in aqueous so-
lution because water molecules tend to be weak Raman scatterers. Fur-
thermore, the detailed molecular information offered by RS can be used
to investigate conformations and concentrations of tissue constituents,
which demonstrates the potential of RS for tissue abnormalities detec-
tion (Kumar, 2012). However, while the conventional RS technique pro-
vides indirect characterization of nanomaterials, such as average size
and size distribution through analysis of the spectral line broadening
and shift, it lacks the spatial resolution necessary to delineate different
domains for application in nanotechnology.
All these detection methods are generally used to develop new syn-
thesis technologies, modify surface properties, and study biological in-
teractions at the individual particle level.
4. Discussion
So far, ecotoxicity of GFMs has been tested on various model and
non-model organisms among which bacteria, algae, invertebrates and
fish (see Section 2.2). There are however many questions that still
need to be addressed. In fact, many challenges are faced in GFMs studies
due to their diversity making difficult to compare the results.
4.1. Environmental safety guidelines
Despite the existing literature on GFMs toxicity, environmental
safety guidelines concerning exposure scenarios of GFMs to aquatic
biota are still undefined, as well as their via of administration, concen-
trations and time of exposure (Khosravi-Katuli et al., 2017). As such,
there are unfortunately no specific standardized protocols or certified
reference materials for GFMs eco-testing probably due to the low sensi-
tivity of the detection method for the low environmental concentra-
tions, or due to the many experimental challenges and issues faced
when assessing the toxicity of nanomaterials. Most of the methods
used for toxicity assessment have been designed without considering
all the different properties of these NPs. As above mentioned, CNMs dis-
play several unique physicochemical properties that can interfere or
pose challenges to the use of classical toxicity assays, and therefore an
incomplete CNM characterization will interfere in the interpretation of
any correlation between biological effects and particle properties. More-
over, the characteristics of commercially available particles that are
specified by the manufacturer sometimes differ from those found by
the researcher and since the facilities in most toxicology laboratories
are not fully equipped, the complete characterization of nanoparticles
is often difficult.
4.2. Levels of exposure concentration
Furthermore, as presented in the Table 3, many studies are done at
high levels of exposure concentration and at present nothing is known
about the subtle physiological and biochemical responses at lower
dose exposures, or the mechanism by which GFMs produce an effect
at these lower concentrations. In order to make the studies the most en-
vironmentally relevant, the concentrations tested should reflect the
expected future releases in the environment. This is related to the ne-
cessity of studies on prolonged timescales, which are so far absent in
the literature. Most of the studies conducted with GFMs relating to
aquatic system, used short term exposures that may not allow organ-
isms to respond to stress or may not allow organisms to adapt to new
conditions. It is necessary to explore the responses of the organisms
considering not only relatively short-term treatment periods but also
continuous or intermittent exposure over a long period of time and con-
sequently along their life cycle trying to obtain results of the toxicity as-
sessments that can be used to estimate the degree of harm to those
organisms.
4.3. Interaction with biological systems
Another important point missing in the cited studies is trying to un-
derstand the possible interaction of GFMs with biological systems. Gen-
erally, GFMs with small size, sharp edges, and rough surfaces easily
internalize into the cell as compared to larger, smooth GFMs (Guo
et al., 2013). The specific surface area and bending stiffness depend on
the number of layers and for biological molecules, the more layers of
GFMs means lower adsorptive capacity (Guo et al., 2013). Also, the lat-
eral dimensions of GFMs, affect cell uptake and many other biological
interactions (Li et al., 2013; Yue et al., 2012). The surface chemistry
varies greatly among the members of GFMs and this determines their
hydrophilicity or hydrophobicity, stability, and dispersibility in physio-
logical conditions (Sanchez et al., 2012). Dose are one of the most im-
portant factors where there is a need for standardizing of the
validation of toxicological methodologies. Standardization will provide
necessary information to researchers for better understanding the phys-
icochemical characteristics and the potential toxicological effects in
cells.
Moreover, graphene NPs when present in physiological fluids such
as plasma or serum redistribute themselves throughout the host tissues
and are transported to different organs due to their material and surface
properties, and how they interact as a consequence of proteins that ad-
sorb on to the surface can cause NP aggregation and cell uptake (Lu
et al., 2017). Although substantial research efforts have been recently
conducted to understand the potential ecological risks from GFMs (see
Table 3), to date, the majority of studies have focused on physiological
responses and less number of studies on biochemical responses or bio-
accumulation by ecological receptors, which particularly important
component of environmental risk assessment.
4.4. Studies in the field
Furthermore, studies in the field are missing; regardless of different
characterization techniques, it is important to take into consideration
different media in which GFMs are dispersed. In the aqueous environ-
ment, the stability of GFMs is determined by the nanoparticle character-
istics (hydrophilicity, surface chemistry, zeta potential, nanoparticle
concentration, size and shape) and the physicochemical parameters of
the aqueous environment (ionic strength, hardness, pH, biochemical
oxygen demand, alkalinity, and presence of organic matter). Changes
in the parameters of the aqueous environment can influence the nano-
particles' stability (Jastrzębska et al., 2012) altering the dispersion and
consequently the detection of NPs in the media: aggregation/disaggre-
gation, adsorption/desorption, sedimentation/resuspension and
dissolution.
5. Conclusion
Nanotechnology is helping to considerably improve and even revo-
lutionize many technology and industry sectors: information technol-
ogy, homeland security, medicine, transportation, energy, food safety.
However, they can cause some hazards for the environment from
their production to their disposal. This emphasizes the need for specific
 L. De Marchi et al. / Science of the Total Environment 631–632 (2018) 1440–1456
guidelines, experimental protocols and study designs in the field of
graphene environmental research considering the unique properties
of nanoscale materials and the associated problems concerning their
toxicological assessment.
For all the above stated reasons, standard proceedings must be as-
sured to enable for an environmentally safe use of these nanotechnol-
ogies, and enable for a sustainable future development, from industrial
applications to environmental and health care approaches.
Acknowledgements
This work was supported by CESAM (UID/AMB/50017/2013) and
TEMA (UID/EMS/00481/2013), by FCT/MEC through national funds
and the co-funding of FEDER, within the PT2020 Partnership Agreement
and Compete 2020. Lucia De Marchi benefited from PhD grant (SFRH/
BD/101273/2014), given by the National Funds through the Portuguese
Science Foundation (FCT), supported by FSE and Programa Operacional
Capital Humano (POCH) e da União Europeia. This work was supported
by the Integrated Programme of SR&TD “Smart Valorization of Endoge-
nous Marine Biological Resources Under a Changing Climate” (reference
Centro-01-0145-FEDER-000018), co-funded by Centro 2020 program,
Portugal 2020, European Union, through the European Regional Devel-
opment Fund. Thanks are also due, for the financial support to CESAM
(UID/AMB/50017), to FCT/MEC through national funds, and the co-
funding by the FEDER, within the PT2020 Partnership Agreement and
Compete 2020. The Authors acknowledge Anthony Moreira for the
English-Language Editing of the review. Paula Marques also acknowl-
edge the FCT/MCTES for a research contract under the Program
Investigador (IF/00917/2013/CP1162/CT0016).
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