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Gondwana Research
journal homepage: www.elsevier.com/locate/gr

Toxicological effects of nanoplastics and phenanthrene to zebrafish

(Danio rerio)
Kaihang Xu a, Wenjie Ai d, Qiuping Wang a, Liyan Tian a, Didi Liu a, Zhuozhan Zhuang a, Jun Wang a,b,c,⇑
a
Joint Laboratory of Guangdong Province and Hong Kong Region on Marine Bioresource Conservation and Exploitation, College of Marine Sciences, South China Agricultural
University, Guangzhou 510642, China
b
Institute of Eco-Environmental Research, Guangxi Academy of Sciences, Nanning 530007, China
c
Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou 510642, China
d
College of Electronic Engineering, College of Artificial Intelligence, South China Agricultural University, Guangzhou 510642, China

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

Article history: Despite the known hazards to wildlife, plastic waste to waterways and oceans continues to increase at
Received 29 March 2021 alarming rates every year. Nanoplastics are a breakdown product of plastic debris in the environment
Revised 6 May 2021 (0.1–100 nm). Their small size renders them bioavailable, and they are likely to accumulate/adsorb per-
Accepted 20 May 2021
sistent organic pollutants, which could increase their toxicity. In this study, we evaluated the effects of
Available online xxxx
Handling Editor: Huang Kai
nanoplastics and phenanthrene on the early life stages of zebrafish. Zebrafish embryos were exposed
to fluorescent nanoparticles and observed with fluorescence microscopy. Polystyrene nanoplastics
(80 nm) attached to the embryonic chorionic membrane and were ingested when zebrafish reached
Keywords:
144 h after fertilization, the point at which they first open their mouth, as observed by the fluorescence
Nanoplastics in their intestines. Exposure alone or in combination with phenanthrene resulted in zebrafish pericardial
Phenanthrene edema, spinal curvature, increased embryonic heart rate, shortened body length, and increased mortality.
Zebrafish embryo Co-exposure to 5 mg/L nanoplastics and 0.1 mg/L phenanthrene caused more negative effects on length,
Embryotoxicity teratogenicity and mortality of zebrafish embryos than the corresponding concentrations of single expo-
sure. This study provides clear evidence that nanoplastics can be ingested by zebrafish larvae, thus future
work to assess the toxicological effects is important to marine wildlife management.
Ó 2021 International Association for Gondwana Research. Published by Elsevier B.V. All rights reserved.

1. Introduction In contrast, the distribution of NPs in the environment remains

Plastic particles smaller than 5 mm are defined as microplastics,
while nano-sized microplastics between 1 and 100 nm are
nanoplastics (NPs) (Frias and Nash, 2019; Lehner et al., 2019).
Microplastics are widely distributed in the environment, but the
abundance varies greatly between regions due to currents, weather
conditions and human activities (Alimi et al., 2018). The abundance
of microplastics tends to be greater in estuarine or urban rivers
(4137.3 ± 2461.5 particles/m3), than in coastal areas (0.167 ± 0.1
38 particles/m3) (Zhao et al., 2014). Microplastics have been
detected in brown shrimp (Crangon crangon) (Devriese et al.,
2015), mussels (Mytilus spp.) (Bråte et al., 2018), Japanese anchovy
(Engraulis japonicus) (Tanaka and Takada, 2016), short-tailed shear-
waters (Puffinus tenuirostris) (Tanaka et al., 2013) and other
species.
⇑ Corresponding author at: College of Marine Sciences, South China Agricultural
University, Guangzhou 510642, China.
E-mail address: wangjun2016@scau.edu.cn (J. Wang).
unclear due to the difficulty of environmental sampling. One rea-
son is that NPs often exist as colloids obfuscating sampling by fil-
tration. Despite this, NPs are considered to be ubiquitous in the
environment through the degradation of large plastic fragments
and microplastics, as well as from the release of nanomaterials
to the environment (Alimi et al., 2018; Oliveira and Almeida,
2019). Nanoplastics are presumed to have more toxic effects than
microplastics. Their small size makes them more bioavailable;
upon entry to tissues and cells, they can cause oxidative stress,
reduce immunity (Li et al., 2020), affect energy metabolism (Li
et al., 2021), cause endocrine disfunction (Brun et al., 2019),
and induce neurotoxicity (Barboza et al., 2018). Their overall
effects lead to abnormal development and reproductive disfunc-
tion (Della Torre et al., 2014; Tallec et al., 2018). Additionally, it
was found that persistent organic pollutants (POPs), such as poly-
cyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls
(PCBs), dichlorodiphenyltrichloroethane (DDT), pharmaceuticals
and others, are readily adsorbed by microplastics through
hydrophobic interactions (Rodrigues et al., 2019; Tourinho et al.,

https://doi.org/10.1016/j.gr.2021.05.012
1751-7311/Ó 2021 International Association for Gondwana Research. Published by Elsevier B.V. All rights reserved.

Please cite this article as: K. Xu, W. Ai, Q. Wang et al., Toxicological effects of nanoplastics and phenanthrene to zebrafish (Danio rerio), Gondwana Research,
https://doi.org/10.1016/j.gr.2021.05.012
K. Xu, W. Ai, Q. Wang et al.
2019). In addition, the diversity of surface properties of nanoplas-
tics would allow for other interactions, for example those with a
surface charge could accumulate heavy metal ions. Thus,
nanoplastics are another route for bioaccumulation of POPs and
other environmental pollutants. It is estimated that their larger
specific surface area relative to microplastics, makes their adsorp-
tion rate one to two orders of magnitude higher than that of
microplastics (Velzeboer et al., 2014; Syberg et al., 2015). There-
fore, it is particularly important to study the toxic effects of NPs
in combination with POPs.
Polycyclic aromatic hydrocarbons (PAHs) are widely distributed
in the atmosphere, water and soil worldwide. The concentration of
PAHs in surface soils of urban and rural areas in Hong Kong ranged
from 7.0 to 410 lg/kg (dry weight) (Zhang et al., 2006), and the
concentration of atmospheric PAHs in Athens, Greece was from
3.5 to 26.0 ng/m3 (Mandalakis et al., 2002). In the Hangzhou Reser-
voir, PAHs were found in surface water (0.989–9.6631 mg/L) and
the sediment (132.7–7343 ng/g [dry weight]) (Chen et al., 2004).
PAHs are not easily degraded and tend to accumulate in living
organisms, inducing genetic mutations that can lead to cancer, ter-
atogenic deformities, and other problems. PAHs come from a wide
range of sources, including forest fires, volcanic eruptions, incom-
plete combustion of fossil fuels, and petrochemical industry pro-
duction activities. Phenanthrene (PHE) is a type of PAH,
containing three benzene rings. It is abundant in aquatic ecosys-
tems and is often used as a model for studying PAHs
(Shirmohammadi et al., 2017).
Zebrafish, an ideal model organism for toxicological studies,
were evaluated during embryonic development, a stage particu-
larly sensitive to environmental pollutants. This study used zebra-
fish embryos to evaluate the combined developmental toxicity of
NPs and PAHs. Uptake of NPs was observed through fluorescence
microscopy. Toxicity was determined by morphological character-
istics and other metrics. This research lays a foundation for study-
ing the mechanism of negative effects exerted by NPs and PHE.
2. Materials and methods
2.1. Materials
Polystyrene nanoplastics microspheres (NPs, 80 nm) were pur-
chased from BaseLine Chromtech Research Centre (Tianjin, China)
and labelled with green fluorescence (excitation wavelength:
470 nm, emission wavelength: 526 nm). The particle size of
nanoplastics was detected under scanning electron microscope
(Supplementary Fig. S1). Phenanthrene (PHE, purity > 99.5%) was
acquired from Aladdin (Shanghai, China) and and pre-prepared as
a 1 g/L and 2 g/L PHE-methanol solution.
2.2. Zebrafish maintenance and embryo collection
Adult wild-type zebrafish (Danio rerio, AB strain) were from
Aquatic Economic Animal Germplasm Resources and Breeding
Engineering Laboratory (College of Marine Sciences, South China
Agricultural University). Zebrafish were housed in a recirculating
water system where the temperature was maintained at 28 °C with
a 14/10 h (light/dark) photoperiod and fed fresh brine shrimp
(Artemia salina) twice a day (Santos et al., 2020).
To obtain embryos, adult zebrafish were paired in a commer-
cial hatchery overnight and spawned under light stimulation the
next morning. The fertilized embryos were selected and col-
lected within 4 h and maintained in E3 medium (Lee et al.,
2019).
2
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2.3. Embryo exposure and morphological parameters
Embryos were randomly allocated into seven groups, with 30
embryos each. Each experimental group has three parallel.
Embryos were exposed after 6 h post fertilization (hpf), divided
into 7 groups: Control (E3 solution), 5 mg/L NPs, 10 mg/L NPs,
0.1 mg/L PHE, 0.2 mg/L PHE, 5 mg/L NPs + 0.1 mg/L PHE, 5 mg/L
NPs + 0.2 mg/L PHE, named as CTR group, 5 NP group, 10 NP group,
0.1 PHE group, 0.2 PHE group, 0.1 PHE + NP group, 0.2 PHE + NP
group, respectively. PHE was added by 1 g/L and 2 g/L of
phenanthrene-methanol solution. Equal amounts of methanol
were added to the exposure group without PHE as well as to the
control group.
Morphological measurements of zebrafish embryos were con-
ducted under a stereomicroscope (Optec SZ680; Optec, Chongqing,
China). Heart rate was measured at 72 hpf, body length was mea-
sured at 144 hpf, hatching rate was 100% at 72 hpf, mortality and
teratogenicity rate was recorded at 168 hpf (Pitt et al., 2018; Qiang
and Cheng, 2019; Le Bihanic et al., 2020). The morphology of zeb-
rafish embryos and larvae were observed every day. Fluorescence
images were taken every day with a Nikon SMZ18 microscope
equipped with a Nikon C-HGF1 fluorescence system.
2.4. Statistical analysis
Data were analyzed by SPSS 25.0, and tested for significance
using one-way ANOVA (One-way ANOVA), p < 0.05 means the dif-
ference is significant.
3. Results
3.1. Uptake of nanoplastics in zebrafish
Fluorescent nanoplastics (NPs) were found on the chorion of
zebrafish embryos in both NP groups at 24 hpf (Fig. 1A), and more
NPs were attached to the chorion at 48 hpf (Fig. 1B). However,
there was no difference in the amount of NPs attached to the
embryonic chorion of the 5 NP group and the 10 NP group at 24
hpf and 48 hpf. The zebrafish hatched at 72 hpf, but the fluores-
cence signal was not detected in the body until 144 hpf. At this
point, NPs were detected in the zebrafish intestine. The fluores-
cence intensity of the 10 NP group was greater than that of the 5
NP group (Fig. 1C-D), and the fluorescence intensity increased with
the prolonged exposure time.
3.2. Morphological changes in zebrafish
Exposure to NPs or PHE alone or in combination caused zebra-
fish embryo malformations, including curvature of the spine and
pericardial edema (Fig. 2). Both types of malformation were
observed in both groups, however, pericardial edema was worse
in NPs than PHE and curvature of the spine was worse in PHE than
NPs.
The number of malformations was used to calculate the terato-
genicity rate (Fig. 3D). No abnormal development of zebrafish was
found in the control group. There was no difference in deformity
rate at the two NPs concentrations (p > 0.05), however, terato-
genicity rate did increase at the higher PHE concentration: in the
0.1 PHE group, the rate was 8.10% ± 0.82% and in the 0.2 PHE group,
the rate was 12.46% ± 0.45% (p < 0.05). The deformity rate in the
zebrafish embryo co-exposed to 0.1 mg/L PHE and 5 mg/L NPs
was nearly twice that of 0.1 mg/L PHE alone. The teratogenic rate
in the 0.2 PHE + NP group was also greater than NPs alone or
PHE alone, though the effect was less pronounced than that of
0.1 mg/L PHE with or without NPs.
K. Xu, W. Ai, Q. Wang et al.
Fig. 1. Uptake and accumulation of nanoplastics by zebrafish embryos. Fluorescent
images of 5 NP group at 24 hpf (A), 48 hpf (B), and 144 hpf (C), and fluorescent
images of the 10 NP group at 144 hpf (D).
3.3. Lethal and sublethal effects on zebrafish embryos
Heart rate at 72 hpf, body length at 144 hpf and mortality rate
at 168 hpf were recorded in each group. The average heart rate of
the control group was 82.25 ± 2.66 beats/min (Fig. 3 A). Changes in
heart rate were only observed for the higher concentrations of PHE
(0.2 mg/L) and NPs (10 mg/L). No difference from control was
observed in the lower concentrations. No additive effect was
observed with co-exposure; co-exposure of PHE and NPs was the
same as PHE alone.
The body length of control group was 3.87 ± 0.07 mm (Fig. 3 B).
Both single and combined exposure to NPs and PHE shortened the
body length of zebrafish larvae. Shorter bodies were observed in
the 10 NP group, but not the 5 NP group, and both concentrations
of PHE resulted in shorter body lengths, but with no difference
between 0.1 PHE and 0.2 PHE groups. The average body length of
zebrafish larvae in the combined exposure 0.1 PHE + NP group
was shorter than that of the single exposure group.
Mortality was higher in the treatment groups than in the con-
trol group and increased with increasing exposure concentration
3
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(Fig. 3C). In the combined exposure group 0.1 PHE + 5 NP, mortality
was higher in than in the corresponding. single exposure groups
While there was no statistically significant difference between
the 0.2 PHE + 5 NP and its corresponding single exposure groups,
the mortality was slightly higher in the combined group compared
to NPs alone, and slightly lower in the combined group than PHE
alone.
4. Discussion
The chorionic membrane is the first barrier protecting embryos
from external aggressions; it selectively allows the passage of oxy-
gen, nutrients and some small organic molecules, but blocks entry
of bacteria, zooplankton and other microorganisms (Stehr and
Hawkes, 1979; Schoots et al., 1982; Fent et al., 2010). This mem-
brane consists of three layers: the granular outermost layer is
extremely permeable, and the middle and inner layers have conical
pores 0.5–0.7 mm in diameter distributed throughout that are par-
tially blocked by granular chunks of the outer layer (Rawson et al.,
2000). Fluorescence images indicated the chorionic membrane was
an effective barrier against the 80 nm polystyrene NPs used in this
study. The PS nanoparticles adhered to the chorionic membrane of
zebrafish embryos, and the extent of adherence increased over
time (Fig. 1).
Previous studies have confirmed that some nanoparticles can
permeate the chorionic membrane. For example, individual Ag
nanoparticles (5–46 nm) could enter and exit the embryo through
chorion pore canals (CPCs) via non-active transport (Lee et al.,
2007). Graphene oxide (GO) nanosheets (0.8–1 nm thick and
101–258 nm in diameter) can also enter the embryo via CPCs,
and also through endocytosis (Chen et al., 2016). However, this
does not mean that all particles smaller than the pore range can
cross the chorionic membrane. SiO2 nanoparticles at 25 or
115 nm only accumulated on the chorionic membrane of zebrafish
embryos, and no fluorescence signal was detected inside zebrafish
embryos or in zebrafish larvae (Vranic et al., 2019). Au, CdS and
ZnO fluorescent nanoparticles also only attached to the surface of
the embryonic chorion, but after embryo hatching, fluorescent sig-
nals were found in the intestine and cloaca of the larvae, and expo-
sure caused larval death (Lacave et al., 2016).
A recent study found that whether nanoplastics can pass
through the chorion is related to size, and those equal to or less
than 50 nm can pass through the chorionic membrane (Duan
et al., 2020). In accordance with that study, plastic particles of
25 nm and 50 nm accumulated in the eyes of zebrafish embryos,
while those larger than 50 nm were only found in the intestine
and outer epidermis (van Pomeren et al., 2017). However, others
have reported that microplastics of 1–5 mm were observed in the
head and retina at 96 hpf, suggesting they crossed the chorionic
membrane (Santos et al., 2020). A possible explanation for these
conflicting findings is that only when the adsorption capacity of
the chorionic membrane reaches the maximum, the nanoparticles
will pass (van Pomeren et al., 2017). In the present study, no differ-
ence in fluorescence was observed between the 5 NP and 10 NP
groups at 24 and 48 hpf, thus it is possible the maximum adsorp-
tion was reached.
Even when NPs remain adhered to the embryo surface, they can
still exert an effect. Fluorescence analysis tracking of zebrafish
embryos exposed to benzo[a]pyrene (BaP)-coated microplastics,
showed that the microplastic particles were only attached to the
chorionic membrane, but the fluorescent signals from the BaP were
seen in sites such as the yolk and eyes (Batel et al., 2018). The BaP-
coated particles were not observed in the gut of zebrafish larvae
until after 144 hpf. This indicates that microplastics can transfer
their loaded persistent organic pollutants to the attached organ-
K. Xu, W. Ai, Q. Wang et al. Gondwana Research xxx (xxxx) xxx

Fig. 2. Teratogenic effects of nanoplastics and phenanthrene on zebrafish embryos at 168 hpf.

Fig. 3. Lethal and sublethal effects on zebrafish embryos. (A) Heart rate of zebrafish embryos at 72 hpf. (B) Body length of zebrafish larvae at 96 hpf. (C) Mortality of zebrafish
embryos at 168 hpf. (D) Teratogenicity rate of zebrafish embryos at 168 hpf. Vertical lines represent ± SE, and different letters denote significant difference at p < 0.05.

4
K. Xu, W. Ai, Q. Wang et al.
isms during embryo development. We observed that zebrafish lar-
vae opened their mouths 120 hpf or 144 hpf, and at that point the
NPs was ingested and the NPs fluorescent signal was detected only
in the intestine.
Although there is no significant difference, single or joint expo-
sure to NP and PHE has a tendency to increase the heartbeat of zeb-
rafish embryos. A faster embryonic heartbeat upon exposure to
nanoplastics could be related to the reduced gas exchange capacity
of the chorionic membrane littered with attached nanoplastics.
NPs attached to the chorion may block the internal pores, resulting
in a decrease in oxygen content inside the embryo. Previous stud-
ies found that the level of hypoxia is positively correlated with the
concentration of NPs (Malafaia et al., 2020). In that study, prema-
ture hatching of zebrafish embryos exposed to high concentrations
of PE microplastics were suspected to be caused by the low oxygen
environment inside the embryos.
We also found that exposure to PHE elevated the heart rate of
zebrafish embryos. Polycyclic aromatic hydrocarbons are known
to affect heart rate in developing fish: exposure to PHE reduced
heart rate in Oryzia melastigma embryos (Zheng et al., 2020), and
a reduction in heart rate was observed in bony fish larvae exposed
to polycyclic aromatic hydrocarbons (Cherr et al., 2017; Hansen
et al., 2019). Accelerated heart rates have also been observed, for
example in Oryzias latipes embryos exposed to benzo[c]phenan-
threne (BcP), a highly toxic polycyclic aromatic hydrocarbon. In
that study, there was a concomitant acceleration in embryo devel-
opment along with the increased heart rate. Thus, the changes in
heart rate are multifactorial, and may be related to such factors
as the type and concentration of PAHs, as well as the exposed
species.
Exposure to NPs and PHE alone or in combination caused abnor-
mal development of zebrafish embryos, inducing malformation,
shortened body length and increased mortality (Figs. 2 and 3). It
is well established that PHE exposure causes developmental
defects in embryos (Huang et al., 2013; Zheng et al., 2020). How-
ever, studies on the effects of microplastics exposure have been
contradictory. Santos et al. (2020) concluded that 2 mg/L
microplastic exposure did not cause lethal or sublethal effects on
zebrafish embryos while Zhang et al. (2020) did observe lethal
and sublethal effects exposure with 0.05–10 mg/L microplastic.
We believe that this discrepancy may be due to inconsistencies
in the concentration of the microplastics used for exposure. We
noticed that compared with exposure to only PHE or NPs, co-
exposure had a deeper impact on zebrafish’s body length and
deformity rate, but not on heart rate and mortality. Co-exposure
to low concentrations of cadmium and MPs resulted in zebrafish
embryos that were longer than that of microplastics alone
(Zhang et al., 2020). As the concentration of cadmium increased,
the body length of the combined exposure group gradually became
shorter than that of the microplastic group, suggesting that there is
not a simple linear relationship between co-exposure and toxicity.
5. Conclusions
Overall, nanoplastics attached to the chorionic membrane on
zebrafish embryos and were ingested after the zebrafish open its
mouth. Nanoplastics attached to the chorionic membrane can
transfer the adsorbed pollutants to the embryo, and may pass
through the chorionic membrane after reaching the maximum
amount of attachment. Changes in heart rate were related to fac-
tors such as the type and concentration of PAHs. In this study,
exposure to nanoplastics alone or in combination with phenan-
threne increased the heart rate, shortened the body length,
increased mortality, and induced pericardial edema and spinal cur-
vature in embryos. The toxicity may not be linear when co-exposed
5
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and more studies are needed to analyze the combined toxicological
effects.
CRediT authorship contribution statement
Kaihang Xu: Writing - original draft, Methodology, Software.
Wenjie Ai: Conceptualization, Data curation. Qiuping Wang: Con-
ceptualization, Data curation. Liyan Tian: Visualization, Investiga-
tion. Didi Liu: Software, Validation. Zhuozhan Zhuang: Software,
Validation. Jun Wang: Writing - review & editing, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
The research was funded by This study was supported by the
National Natural Science Foundation of China (42077364), Guang-
dong Province Universities and Colleges Pearl River Scholar Funded
Scheme (2018), Key Research Projects of Universities in Guang-
dong Province (2019KZDXM003 and 2020KZDZX1040), the
National Key Research and Development Program of China
(2018YFD0900604). We appreciate the provision of SCAU Wushan
Campus Teaching & Research Base.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.gr.2021.05.012.
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