International Journal of Environmental Science and Technology (2023) 20:10153–10162

https://doi.org/10.1007/s13762-022-04692-w

ORIGINAL PAPER

Ecotoxicity of selected carbon‑based nanomaterials

F. Gamoń1 · A. Ziembińska‑Buczyńska1 · D. Łukowiec2 · M. Tomaszewski1

Received: 23 August 2021 / Revised: 15 November 2021 / Accepted: 23 November 2022 / Published online: 30 November 2022
© The Author(s) 2022

Abstract
The widespread use of the nanomaterials increases the emission of nano-pollutants into the water. Carbon nanomaterials are
particularly interesting. They are characterized by relatively stable structure, which makes them able to migrate and accumu-
late in the environment. Therefore, the aim of this study was to determine the potential toxicity at the different trophic levels
of four selected carbon nanostructures: graphene oxide (GO), reduced graphene oxide (rGO), multi-walled carbon nanotubes
(MWCNTs) and oxidized multi-walled carbon nanotubes (f-MWCNTs) on indicators at three trophic levels. Producers was
represented by Lemna minor in growth inhibition test. The ecotoxicological effect for consumers was estimated by acute tests
on Artemia franciscana, Brachionus calyciflorus and Thamnocephalus platyurus, while the acute toxicity on decomposers
was studied on bacteria Escherichia coli. Results show that the short-term exposure on MWCNTs, f-MWCNTs, GO and rGO
can be toxic at three trophic levels. The influence of the tested materials was much higher for the consumers, than for the
producers. The lowest toxicity from all researched carbon-based nanomaterials was presented by GO. Moreover, generation
of high reactive form of oxygen, mechanical damage of cell wall and membrane is one of the main toxicity mechanism; thus,
the toxicity depends heavily of the dose and the shape of the nanomaterials.

Keywords Nanomaterials · Ecotoxicity · Graphene · Carbon nanotubes

Introduction these nanomaterials are widely studied, and hold immerse to

The application of nanomaterials allows to change the fun-
damental chemical and physics properties of conventional
materials. Changing material properties is possible as their
size reduced to the nanoscale, what offering new materi-
als with unique optical, electrical and mechanical prop-
erties. Nanomaterials are increasingly used in such fields
as biomedicine, electronics, mechanical engineers, pho-
tonics, energy generation and storage (Basiuk et al. 2011;
Novoselov et al. 2012; Montagner et al. 2016). Among all
the nanomaterials, special attention should be paid to the
carbon-based nanomaterials such as the carbon nanotubes
(CNTs) and graphene (Montagner et al. 2016). Currently,
Editorial responsibility: S. Mirkia.
* F. Gamoń
filip.gamon@polsl.pl
1
Environmental Biotechnology Department, Silesian
University of Technology, Akademicka 2, Gliwice, Poland
2
Institute of Engineering Materials and Biomaterials, Silesian
University of Technology, Konarskiego 18a, 44‑100 Gliwice,
Poland
impact several scientific areas (Lalwani et al. 2016).
Graphene is a 2D layer sheet of ­sp2 hybridization carbon
atoms. Basic graphene is a polar and hydrophobic, so to
improve its dispersibility in aqueous media its need to be
oxidized for example via chemical oxidation (Lalwani et al.
2016). Currently, the production of graphene-based nanoma-
terials is not as important for the global economy as carbon
nanotubes (CNTs). However, investment in graphene-based
nanomaterials is expected to reach $ 400 million by 2025
(Montagner et al. 2016). Such a large production and indus-
trial application of both graphene and CNTs nanomaterials
would transfer these nanomaterials into the environment
where they may have potential deleterious impact on the
flora and fauna as well as microorganisms.
Current studies of the toxicological effect of carbon-based
nanomaterials are mainly focused on the animal and human
cells, while the studies considering their effect on the devel-
opment and physiology of plants, algae, fungi and bacteria
are limited (Boros and Ostafe 2020; Penga et al. 2020). In
this study Lemna minor (duckweed), Artemia franciscana,
Brachionus calyciflorus (rotifer), Thamnocephalus platyu-
rus (shrimp) and Escherichia coli were used to evaluate

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10154 International Journal of Environmental Science and Technology (2023) 20:10153–10162
the ecotoxic effect of four carbon nanomaterials (graphene
oxide—GO, reduced graphene oxide—rGO, multi-walled
carbon nanotubes—MWCNTs, functionalized multi-
walled carbon nanotubes—f-MWCNTs). The test organ-
isms selected for the study are characterized by fast growth,
small size, ease in culturing and cost effectivity as well as
are sensitivity to toxic substances (Gamoń et al. 2019), that
make them good indicators.
Khodakovskaya et al. (2011) investigated the genotoxicity
of several carbon nanomaterials, such as activated carbon,
graphene, SWCNTs and MWCNTs on the germination of
tomato seedlings. Results of the research over leaves and
roots show that graphene induced the lowest activation of
stress-related LeAqp2 gene encoding tomato water-channel
protein, while the highest activation stress was observed for
CNTs. Tabei et al. (2019) studied the toxicity of MWCNTs
and showed that it has very high level of phagocytic activity
for undifferentiated HL-60 cells and a low level of cytotoxic-
ity for differentiated HL-60 cells. MWCNTs affect the repair
mechanism of DNA, showing genotoxic activity. Moreover,
the molecular larval studies at aquatic levels conducted by
Martinez-Paz et al. (2019) showed that MWCNTs affect the
transcription of genes involved in apoptosis. On the other
hand, important factors influencing the MWCNTs toxicity
are also their length, diameter and structure. Stronger toxic-
ity is revealed by the longer tubes (Shi et al. 2017).
Antifungal effects of reduced graphene oxide against
Aspergillus niger, Aspergillus oryzea and Fusarium oxyspo-
rium were investigated by Sawangphruk et al. (2012) via
quantifying mycelial growth inhibition. This research indi-
cated a good antifungal activity of rGO reaching ­IC50 value
between 50 and 100 µg/mL against all three fungi. Antimi-
crobial activity of the graphene oxide nanowall and reduced
graphene oxide nanowall was reported by Akhavan et al.
(2013). These results showed that rGO nanowalls were more
toxic against Escherichia coli and Staphylococcus aureus
than the GO nanowalls because of effective charge transfer
between bacteria cells and the edges of nanomaterials during
direct contact. However, E. coli demonstrated lower impact
than S. aureus as effect of cell wall structures. Gram-positive
S. aureus doesn’t include cell membrane compered to gram-
negative E. coli making them less resistant (Akhavan et al.
2013; Lalwani et al. 2016). Chen et al. (2013) compared
antimicrobial activity of the GO and rGO against gram-neg-
ative Xanthomonas oryzae pv. oryzae. However, these results
showed grater antibacterial activity of the GO in comparison
to the rGO. Graphene phytotoxicity was evaluated by Begum
et al. (2011) on roots as effect of shoot growth and shape,
cell death and biomass by incubating seedlings of cabbage,
tomatoes, red spinach and lettuce with 500–2000 mg/L for
20 days. The physiological and morphological analysis
showed that graphene significantly inhibited plant growth
and biomass production. Nanomaterials toxicity was also
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investigated in some model marine organisms. Artemia
salina was not affected by graphene concentration in range
0.675–5 mg/L (Pretti et al. 2014), while in concentration
between 0 and 700 mg/L acute mortality was observed by
Mesarič et al. (2015). Graphene-related materials also have
an effect on the lysosomal function and metabolic activity of
fish cells and the degree of graphitization is highly related to
the toxicity—they are inversely proportional (Kalman et al.
2019). Analysis provided by Evariste et al. (2020) indicated
that communities and organisms from the sediment are
impacted more than those from the water column due to
sedimentation of the CNTs.
Development of nanotechnology including carbon nano-
material bring potential risk for environment. Especially
water environment is regarded to be more influenced by
all types of nanoparticles, as these compounds are washed
out from other parts of the environment and directed to
water from soil and air. Despite an increasing number of
studies examining the toxicity of carbon nanomaterials,
current knowledge is still insufficient, and it is extremely
important to research their potentially hazardous effects
on the ecosystem (Ghadimi et al. 2020). Therefore, in this
study the ecotoxic effect of four carbon nanomaterial at
different trophic levels was investigated to present the full
spectrum of the results of these selected types of carbon
nanomaterials. This knowledge enables to understand the
mechanisms of the carbon nanomaterials influence on vari-
ety of organisms in the environment.
Materials and methods
Carbon nanomaterials characteristics
Commercially available GO, rGO (Nano Carbon, Poland)
and MWCNTs (Nanocyl NC7000™, Belgium) were used.
MWCNTs were functionalized according to Kolanowska
et al. (2019) by oxidative treatments with a mixture of
nitric acid and sulfuric acid solution to obtain f-MWC-
NTs. Nanomaterials samples were analysed by transmis-
sion electron microscope using a S/TEM TITAN 80–300
(FEI Company). The samples were prepared by dispers-
ing the nanoparticles in ethanol (99.8%) using an ultra-
sound washer, and then depositing one or two drops of
the dispersion on a TEM copper 200 mesh grids coated
with a carbon film. The samples were air-dried at room
temperature. Nanomaterials solutions for toxicity assays
were prepared in test media. Each test was provided with
the same concentration of nanomaterials—25, 50, 100,
150 and 200 mg/L for GO and rGO, while MWCNTs and
f-MWCNTs concentrations were 125, 250, 500, 750 and
1000 mg/L.
International Journal of Environmental Science and Technology (2023) 20:10153–10162
Phytotoxicity
The phytotoxicity was evaluated using growth inhibition
tests on Lemna minor, according OECD Test No. 221
(2006). L. minor plants, with at least 12 leaves, were placed
in plastic Petri dishes containing 20 mL of the test solu-
tion and incubated in a thermostatic cabinet, at 24 ± 1 °C
under 16 h photoperiod (16 h of light: 8 h of darkness). After
14 days of incubation, the number of leaves was counted,
and the percentage of the growth inhibition was determined.
Standard growth medium for macrophytes was used as a
negative control and to prepare nanomaterials solutions.
Tests were performed in triplicate and ­IC50 values were cal-
culated with a probit model.
Artoxkit M
The acute toxicity test—Artoxkit M kit was obtained from
MicroBioTests Inc. (Belgium). It is based on the mortality
of Artemia franciscana. Test was developed by the research
team at the State University of Ghent (Belgium) (Van
Steertegem and Persoone 1993) and is based on the ASTM
Standard Guide E1440-91 (2012). Assay was performed
according to the standard procedure (Artoxkit 2020). The
standard seawater medium with a salinity of 35 ppt was pre-
pared with deionized water and NaCl. It was used to prepare
nanomaterials dilutions and as a negative control. The bioas-
say was conducted in a multi-well test plate with 24 (6 × 4)
test wells. Instar II-III larvae of the brine shrimp Artemia
franciscana were hatched from cysts 30 h before the start of
the toxicity test. The test was based on one control and five
nanomaterials concentrations, each with 3 replicates of 10
animals. The test plates were covered with a strip of parafilm
and incubated at 25 °C in darkness for 24 h. I­ C50 values were
calculated with a probit model.
Rotoxkit F
The acute toxicity was assessed based on the mortality of
Brachionus calyciflorus after 24 h exposure in accordance
with ASTM Standard Guide E1440-91 (2012). Thamno-
toxkit F kit was obtained from MicroBioTests Inc. (Bel-
gium). Assay was performed according to the standard pro-
cedure (Rotoxkit 2020). The standard freshwater medium
consisted of N­ aHCO3 (96 mg/L), C ­ aSO4·2H2O (120 mg/L),
­MgSO4·7H2O (123 mg/L), KCl (4 mg/L) was used to pre-
pare nanomaterials dilutions and as a negative control. Lar-
vae of Brachionus calyciflorus hatched from cysts after 18 h
of incubation were placed to the multi-well plates with one
control solution and five toxicant concentrations, each with
6 replicates of 5 animals. The test plates were covered with a
strip of parafilm and incubated at 25 °C in darkness for 24 h.
­IC50 values were calculated with a probit model.
10155
Thamnotoxkit F
The acute toxicity test—Thamnotoxkit F kit was obtained
from MicroBioTests, Inc. (Belgium). It is based on the
mortality of Thamnocephalus platyurus. Test was devel-
oped by the research team by the research team of Prof.
Dr. G. Persooneat at the State University of Ghent (Bel-
gium) and is based on the standards of ISO 14380 (2011).
Assay was performed according to the standard procedure
(Thamnotoxkit 2020). The standard freshwater medium
consisted of N­ aHCO3 (96 mg/L), C ­ aSO4·2H2O (120 mg/L),
­MgSO4·7H2O (123 mg/L), KCl (4 mg/L) was used to pre-
pare nanomaterials dilutions and as a negative control. Lar-
vae of Thamnocephalus platyurus hatched from cysts after
24 h of incubation were placed to the multi-well plates with
one control and five nanomaterials concentrations, each with
3 replicates of 10 animals. The test plates were covered with
a strip of parafilm and incubated at 25 °C in darkness for
24 h. ­IC50 values were calculated with a probit model.
Toxi‑ChromoPad™
The acute sediment toxicity test is based on the ability
of nanomaterials to reduce the synthesis of an inducible
enzyme—β-galactosidase—in a highly permeable mutant
of the bacterium E. coli. Toxi-ChromoPad™ was obtained
from Environmental Biodetection Products, Inc. (Canada).
Test was performed according to the standard procedure.
Bacteria-sediment mixture solution was placed to the test
tubes with one control and five nanomaterials concentra-
tions. All tubes were incubated for two hours at 37 °C. After
this time test tubes were hand-mixed vigorously and a small
drop from each tube was transferred onto the chromoge-
nic pad. Then, samples were incubated overnight at 37 °C
before results reading. Blue colour intensity is correlated
with enzyme synthesis. Toxicity reduces enzyme synthesis
and colour development. Blue colour intensity was measured
based on image histogram.
Results and discussion
Nanomaterials morphology was observed using TEM and
Figs. 1 and 2 present representative images of the observed
samples. MWCNTs are elongated hollow nanotubes formed
by curled multi-layer graphite sheets. MWCNTs function-
alized by acid treatment contains carboxyl and hydroxyl
groups at the walls (Sezer and Koç 2019). Both types of car-
bon nanotubes were characterized by an average inner diam-
eter of 2–6 nm, outer diameter of 5–20 nm and lengths in the
microns. In turn, GO is a single layer of carbon atoms (gra-
phene), comprising carboxyl and hydroxyl groups. Reduc-
tion of GO removes some of these oxygen-containing groups
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10156 International Journal of Environmental Science and Technology (2023) 20:10153–10162
Fig. 1  Representative transmission electron microscopy (TEM)
images of A multi-walled carbon nanotubes (MWCNTs), B function-
alized multi-walled carbon nanotubes (f-MWCNTs) used in the eco-
toxicological study
Fig. 2  Representative transmission electron microscopy (TEM)
images of A graphene oxide (GO) and B reduced graphene oxide
(rGO) used in the ecotoxicological study
and leads to the formation of rGO. Both of them occur in
the water environment in the form of few-layer flakes and
Fig. 2 illustrates much higher disintegration of the carbon
basal plane in the rGO sample.
As it has been previously reported that even a small
change in the physical–chemical characteristics of the
nanomaterial can be a reason of its toxicity change (Man-
shian et al. 2013; Parise et al. 2014). However, the detailed
mechanism of CNMs toxicity is still unclear and requires
further elucidation, but three main phenomena are currently
considered: (1) mechanical damage to the microorganism’s
cell wall and membrane; (2) reactive oxygen species (ROS)
production, which can damage membranes, proteins, and
DNA; (3) other physical–chemical properties related, i.e.
with their functionalization or electron transport and multi-
electron accepting ability (Chen et al. 2019).
The ecotoxicological effect of the chosen carbon-based
nanomaterials was estimated at three trophic levels in order
to present the wide spectrum of these compounds influence
on living organisms. For this experiment graphene oxide,
reduced graphene oxide, multi-walled carbon nanotubes and
their functionalized form were used. rGO and MWCNTs
13
are hydrophobic, while GO and f-MWCNTs are hydro-
philic (Liu et al. 2014), thus more toxic towards bacteria
(Nguyen and Rodrigues 2018; Santos et al. 2012). These
features should be taken into consideration while discussing
their influence on living organisms. Moreover, much con-
centrated graphene-based nanomaterials aggregate in larger
complexes, becoming less available for microorganisms and
limiting their toxic activity. Their features differ; thus, it
should be expected that the mechanism of their influence
on various organisms will differ as well.
Lemna minor was a representative of produces for eco-
toxicological tests. According to the results obtained in
this experiment the increasing concertation of GO, rGO in
range of 25–200 mg/L and the range of 125–1000 mg/L for
MWCNTs is successfully inhibiting the producer’s growth.
In case of all used GO concentrations, as well as rGO at
concentration 100, 150 and 200 mg/L and MWCNTs at
concentration of 250, 500, 750 and 1000 mg/L the results
are statistically significant. Interestingly for functionalized
form of MWCNTs the increasing inhibition was observed for
concentration of 125, 250 and 500 mg/L, while above this
value the inhibition was decreasing for ca. 4% and 6% for
750 and 1000 mg/L, respectively. The results obtained for
125–750 mg/L were statistically significant (Fig. 3).
The 50% lethal concentration ­(LC50) is comparable for
GO and MWCNTs (543 and 431 mg/L, respectively). For
rGO ­LC50 is lower—144 mg/L. No observed effects con-
centration (NOEC) was estimated for rGO and MWCNTs at
level of 50 and 125 mg/L, while for GO it was not possible.
The highest value for the lowest observed effects concentra-
tion (LOEC) was also for MWCNTs, while for GO and rGO
it was 25 and 100 mg/L. For functionalized MWCNTs it was
impossible to estimate ­LC50 as well as NOEC and LOEC
values (Table 1).
The ecotoxicological effect for consumers was estimated
on the basis of the tests for Artemia franciscana, Brachionus
calyciflorus and Thamnocephalus platyurus. In case of A.
franciscana no inhibition effect was estimated for GO at
concertation of 25 mg/L and for MWCNTs at concertation
of 125 mg/L. For rGO and f-MWCNTs all researched con-
centrations were inhibiting test organisms. It is also impor-
tant to notice that the influence of the tested substances was
far higher for consumers, than for producers. It could be
explained with the presence of plant cell wall preventing
the cell from the carbon-based nanomaterials penetration
(Khodakhowskaya et al. 2011).
In case of L. minor, the highest observed inhibition value
(ca. 18%) was for MWCNTs at concertation of 1000 mg/L,
while for producers only the lowers concentrations values
for all tested substances gave the mortality result lower than
20% (Fig. 4). The highest used concentrations of GO and
rGO caused higher mortality of A. franciscana (95% and
90%, respectively) than the highest used concentrations of
International Journal of Environmental Science and Technology (2023) 20:10153–10162 10157

Fig. 3  Growth inhibition of

Lemna minor after 14 days of
exposure on A graphene oxide
(GO), B reduced graphene
oxide (rGO), C multi-walled
carbon nanotubes (MWCNTs)
and D functionalized multi-
walled carbon nanotubes
(f-MWCNTs). Bars represent
standard error; *a statistically
significant difference with con-
trol at p < 0.05 (t-test)

Table 1  The results of Lemna minor Inhibitory Concentration, 10%
­(IC10); No Observed Effects Concentration (NOEC) and Lowest
Observed Effects Concentration (LOEC) induced by graphene oxide
(GO), reduced graphene oxide (rGO), multi-walled carbon nano-
tubes (MWCNTs) and functionalized multi-walled carbon nanotubes
(f-MWCNTs)
(mg/L) GO rGO MWCNT f-MWCNT
IC10 543 144 431 – that GO presents the lowest toxicity from all carbon-based
NOEC – 50 125 – nanomaterials used in this study (Talukdar et al. 2014; Yuan
LOEC 25 100 250 – et al. 2014). It was also stated that the toxicity of these mate-
MWCNTs and f-MWCNTs (90% and 66%, respectively)
(Fig. 4). The values of L­ C50, NOEC and LOEC were com-
parable for rGO and GO. ­LC50 for MWCNTs was slightly
lower (568 mg/L) than for f-MWCNTs (631 mg/L). In case
of NOEC and LOEC the valued obtained for MWCNTs and
f-MWCNTs were twice higher (250 and 125 mg/L for NOEC
and 500 and 250 mg/L for LOEC, respectively (Table 2).
B. calyciflorus appeared to be more sensitive towards
the tested compounds in their lower concentrations. Only
25 mg/L GO did not caused mortality for this testing organ-
ism. For GO, rGO and MWCNTs their increasing concerta-
tion caused increasing mortality with the highest value of 98
and 95%, respectively, for GO and rGO at concentration of
200 mg/L and 75% mortality for 1000 mg/L of MWCNTs.
It is important to point that the mortality for the increasing
concentration of functionalized MWCNTs was the highest
for 500 mg/L, but for 750 and 1000 mg/L the mortality per
cent dropped from over 80% to 32 and 35%, respectively
(Fig. 5). It could be explained by the fact that more oxidized
carbon-based nanomaterials are more cytocompatible thus
less harmful for animal cells (Sasidharan et al. 2011; Lal-
wani et al. 2016).
As in many animal-based studies these results also show
rials depends heavily on dose and the shape of the structure
(Akhavan et al. 2013; Lawani et al. 2016).
It was also impossible to estimate ­LC50 for f-MWCNTs.
NOEC and LOEC values for this compound were 125 and
250 mg/L, respectively. ­LC50 for GO, rGO and MWCNTs
were 87, 65 and 712 mg/L, respectively. The value of NOEC
and LOEC for rGO were lower than for GO (25 and 50 mg/L
for NOEC and 50 and 100 mg/L for LOEC, respectively).
The NOEC and LOEC values for MWCNTs were 125 and
250 mg/L, respectively (Table 3).
For T. platyurus only in case of rGO the lowest concerta-
tion used (25 mg/L) did not caused mortality effect for this
testing organism and values of 50 and 100 mg/L have similar
mortality effect (ca. 25%; Fig. 6). GO low concentrations (25
and 50 mg/L) have similar mortality effect on T. platyurus
while concentration 100 mg/L and above caused increas-
ing mortality reaching 92% for 200 mg/L. Interestingly,
MWCNTs influence seems to be very strong. This substance

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10158 International Journal of Environmental Science and Technology (2023) 20:10153–10162

Fig. 4  Mortality of Artemia

franciscana after 24 h of expo-
sure on A graphene oxide (GO),
B reduced graphene oxide
(rGO), C multi-walled carbon
nanotubes (MWCNTs) and D
functionalized multi-walled
carbon nanotubes (f-MWCNTs).
Bars represent standard error;
*a statistically significant dif-
ference with control at p < 0.05
(t-test)

Table 2  The results of Artemia franciscana Lethal Concentration, 500 mg/L, respectively. While for MWCNTs these values
50% ­(LC50); No Observed Effects Concentration (NOEC) and Lowest were 404, 125 and 250 mg/L, respectively (Table 4).
Observed Effects Concentration (LOEC) induced by graphene oxide
The Toxi-ChromoPad™ tests the toxic influence of the
(GO), reduced graphene oxide (rGO), multi-walled carbon nano-
tubes (MWCNTs) and functionalized multi-walled carbon nanotubes compounds studied on decomposers—bacteria Escherichia
(f-MWCNTs) coli producing—β-galactosidase. The increase in the toxic-
(mg/L) GO rGO MWCNT f-MWCNT
ity causes the decrease in enzyme production and the colour
development. According to the results obtained in this test it
LC50 88 96 568 631 could be stated that GO and rGO in concentration from 25
NOEC 50 50 250 125 to 200 mg/L are less toxic than MWCNTs and f-MWCNTs
LOEC 100 100 500 250 used in the concertation between 125 and 1000 mg/L. For
GO/rGO the values of colour intensity are estimated at level
for 4–6 a.u. (arbitrary unit), while for MWCNTs/f-MWCNTs
concentration at 125 mg/L caused 18% mortality and this at level of ca. 4 a.u (Fig. 7). These results let to suspect
rate is increasing heavily and reaching 82% for 1000 mg/L. that in case of carbon nanomaterials the more complex and
Functionalized MWCNTs also influence T. platyurus more sharper the structure is, more harmful it is to bacterial cell
heavily than rGO and GO, but the concentrations between wall. As it has been already studied in case of GO and rGO,
125 and 750 mg/L did not give such a strong effect as non- the latter is found its sharper structure and its better charge
functionalized MWCNTs (mortality at level of 10–42%). transfer ability (Yang et al. 2013). Considering mechanical
However, the mortality for the highest concentration of damages of cell membranes caused by nanoparticles, TEM
f-MWCNTs unexpectedly reached almost 100% (Fig. 6). images demonstrate that rGO is more irregularly shaped than
The values of NOEC and LOEC for GO and rGO pre- GO, with sharp folds and edges. It is also possible that in
sented similar trend as for B. calyciflorus: 50 and 25 mg/L case of MWCNTs and f-MWCNTs there are far more points
for GO and 100 and 50 mg/L for rGO, respectively. The L­ C50 of the direct contact for the bacterial cells which direct to
for these compounds was at the comparative level (94 and their damage as it has been presented by Kurantowicz et al.
99 mg/L, respectively). ­LC50 as well as NOEC and LOEC (2015) who hypothesized that the presence of the oxidative
values for f-MWCNTs were twice higher than for MWCNTs. functional groups on graphene—based nanomaterial can
­LC50, NOEC and LOEC for f-MWCNTs were 894, 250 and act as an attractant for bacterial cells enabling the direct

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International Journal of Environmental Science and Technology (2023) 20:10153–10162 10159

Fig. 5  Mortality of Brachionus

calyciflorus after 24 h of expo-
sure on A graphene oxide (GO),
B reduced graphene oxide
(rGO), C multi-walled carbon
nanotubes (MWCNTs) and D
functionalized multi-walled
carbon nanotubes (f-MWCNTs).
Bars represent standard error;
*a statistically significant dif-
ference with control at p < 0.05
(t-test)

Table 3  The results of Brachionus calyciflorus Lethal Concentration, against various types of cells inducing damage to proteins
50% ­(LC50); No Observed Effects Concentration (NOEC) and Lowest and DNA which finally leads to cell death via apoptotic or
Observed Effects Concentration (LOEC) induced by graphene oxide
necrotic pathways (Li et al. 2012). Previous studies have
(GO), reduced graphene oxide (rGO), multi-walled carbon nano-
tubes (MWCNTs) and functionalized multi-walled carbon nanotubes also reported that directly contact of sharp edges of carbon
(f-MWCNTs) nanomaterials (graphene, CNTs) with cells lead to loss of
(mg/L) GO rGO MWCNT f-MWCNT
cell integrity as well as may cause DNA fragmentation and
chromosomal aberration (Akhavan et al. 2013). Moreover,
LC50 87 65 712 – it was observed that long sheet of graphene nanomaterials
NOEC 50 25 125 125 can wrap around bacterial cell leading to growth inhibition
LOEC 100 50 250 250 (Carpio et al. 2012). The strong cytotoxicity effect may be
the result of integration between graphene-based nanoma-
terials and phospholipids from the cell membrane, which
contact and cell impairment as its consequence. Moreover, are extract from the membrane structures (Tu et al. 2013).
according to Saleemi et al. (2020) functionalized CNTs Thus, it is difficult to demonstrate the evidential mechanism
can destroy cell membrane and affect microbial activity. As of toxicity of nanomaterials on cells. It is suggested that the
illustrated in Fig. 1B, f-MWCNTs have sharp ends, with an toxic mechanism is the result of the simultaneous action of
average diameter between 5 and 20 nm, which could easily nanomaterials on different cellular structures.
damage membrane integrity.

Toxicity mechanism Conclusion

The toxicity mechanism of carbon nanomaterials depend-
ent mostly on the type of cells on which it acts (prokaryotic
or eukaryotic), as well as the physicochemical properties
of the nanomaterials as shape, size, types and density of
functional group and charge transfer abilities (Lalwani et al.
2016). The generation of reactive oxygen species (ROS) is
mentioned as the main toxicity mechanism of nanomaterials
Presented work investigates the ecotoxicity of four carbon
nanomaterials at different trophic levels. Results show that
the short-term exposure on MWCNTs, f-MWCNTs, GO and
rGO can be toxic at all trophic levels. However, the influence
of the tested materials was much higher for consumers, than
for producers, what can be explained with the presence of
plant cell wall preventing the cell from the carbon-based

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10160 International Journal of Environmental Science and Technology (2023) 20:10153–10162

Fig. 6  Mortality of Thamno-

cephalus platyurus after 24 h of
exposure on A graphene oxide
(GO), B reduced graphene
oxide (rGO), C multi-walled
carbon nanotubes (MWCNTs)
and D functionalized multi-
walled carbon nanotubes
(f-MWCNTs). Bars represent
standard error; *a statistically
significant difference with con-
trol at p < 0.05 (t-test)

nanomaterials penetration. The toxicity of these materials one of the main toxicity mechanism. Furthermore, GO pre-
depends heavily of dose and the sharpness of the structure, sents the lowest toxicity from all the carbon-based nanoma-
because mechanical damage of cell wall and membrane is terials used in this study. Considering mechanical damages,
Fig. 7  Blue colour development
induced by β-galactosidase in
E. coli after exposure on A gra-
phene oxide (GO), B reduced
graphene oxide (rGO), C
multi-walled carbon nanotubes
(MWCNTs) and D function-
alized multi-walled carbon
nanotubes (f-MWCNTs). a.u.
arbitrary unit

13
International Journal of Environmental Science and Technology (2023) 20:10153–10162 10161

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