Received: 24 November 2019 Accepted: 25 November 2019

DOI: 10.1002/jat.3910

REVIEW ARTICLE

Toxicological study of metal and metal oxide nanoparticles in

zebrafish

Changcun Bai | Meng Tang

Key Laboratory of Environmental Medicine

and Engineering, Ministry of Education; School
of Public Health, Southeast University,
Nanjing, People's Republic of China
Correspondence
Meng Tang, Key Laboratory of Environmental
Medicine and Engineering, Ministry of
Education; School of Public Health, Southeast
University, Nanjing 210009, People's Republic
of China.
Email: tm@seu.edu.cn
Abstract
Metal and metal oxide nanoparticles have been widely used in catalytic, electronic
and biomedical fields. It is necessary to investigate their toxicity and potential hazards
to human and aquatic ecosystems. Zebrafish (Danio rerio), as a promising animal
model, has been increasingly utilized to assess the toxicity of nanoparticles. Zebrafish
has numerous characteristics for toxicity evaluation, such as short life cycle and high
fecundity. This review describes the advantages of using zebrafish in the toxicity
assessment of metal and metal oxide nanoparticles. Then we focus on the toxic

Funding information
Provincial Natural Science Funds of Jiangsu,
Grant/Award Number: grant/award no.:
BK20180371).; Fundamental Research Funds
for the Central Universities, Grant/Award
Number: grant no. 2242019K40220; National
Natural Science Funds of China, Grant/Award
Number: nos 31671034, 21876026,
81473003, 81573186, 81673218, 81502783
effects, particularly the acute toxicity and the chronic ones, induced by nanoparticles
in zebrafish. Target organ toxicities are also mentioned, including immunotoxicity,
developmental toxicity, neurotoxicity, reproductive toxicity, cardiovascular toxicity
and hepatotoxicity. The toxic effects of selected metal nanoparticles, including Au,
Ag, Cu, and metal oxide nanoparticles such as TiO2, Al2O3, CuO, NiO and ZnO, as well
as the underlying mechanisms of nanoparticles causing these effects, are also
highlighted and described in detail. Furthermore, we introduce the general factors
that affect nanoparticle‐induced toxicity in zebrafish. The drawbacks and advantages
of using the zebrafish model in nanotoxicity studies are also argued. Finally, we sug-
gest that the application of zebrafish to assess chronic toxicity of metal and metal
oxide nanoparticles and the joint toxicity of metal and metal oxide nanoparticles
and other pollutants could be hot topics in nanotoxicology.

K E Y W OR D S

metal nanoparticles, metal oxide nanoparticles, zebrafish model, nanotoxicity, chronic toxicity

1 | I N T RO D U CT I O N threat to the environment and human health (Ribeiro et al., 2017;

Engineered nanoparticles (ENPs) are a wide variety of nanomaterials,
which are produced using diverse synthesis methods, different core
materials and plenty of capping/coating agents (Matsoukas, Desai, &
Lee, 2015; Singh, 2016). ENPs exhibit diversely unique chemical, elec-
trical, physical and optical properties based on their small size and
large surface‐to‐mass ratios (Biener et al., 2009; Sharma, Pattadar,
Mainali, & Zamborini, 2018; Singh, 2016). ENPs may undergo physical,
chemical and biological transformation when they are discharged into
the environment without or with certain treatments, posing a serious
Karimi, Sadeghi, & Kokini, 2018). One study has indicated that ENPs
can cause lung damage through oxidative damage and inflammatory
responses (Poh et al., 2018). Other studies have also shown that
ENPs can enter the central nervous system through the olfactory
nerve, possibly leading to degenerative diseases such as behavioral
abnormalities through oxidative stress and inflammation (Calderón‐
Garcidueñas et al., 2015).
ENPs consist of metallic‐based nanomaterials, quantum dots,
carbon‐based nanomaterials and polymeric particulate materials in a
variety of forms (Wang & Tang, 2018; Wu & Tang, 2018). Among

J Appl Toxicol. 2019;1–27. wileyonlinelibrary.com/journal/jat © 2019 John Wiley & Sons, Ltd. 1
2 BAI
them, metal NPs and metal oxide NPs account for the largest share in
terms of manufacture and application (Djurisic et al., 2015). With the
rapid development of nanotechnology, metal NPs such as gold
(AuNPs), silver (AgNPs), copper oxide (CuNPs), nickel (NiNPs) and
metal oxide NPs (zinc oxide [ZnO], titanium dioxide [TiO2], iron oxides
[Fe2O3, Fe3O4]), are widely produced and applied in the field of con-
struction, defense, energy, healthcare, transportation, medicine, etc.,
containing ENPs as either the major components or the additives for
better performances (Kessler, 2011; Rudramurthy & Swamy, 2018).
For example, scientists are investigating biologically synthesized
AuNPs, AgNPs and platinum NPs (PtNPs) as a treatment for cancer
(Bendale, Bendale, & Paul, 2017; Ning, Zhu, & Gao, 2017; Yamada,
Foote, & Prow, 2015; Zhang, Liu, Shen, & Gurunathan, 2016). ZnO
and TiO2NPs with the ability to block ultraviolet rays are commonly
used as additives in various sunscreen products. Iron oxide NPs
(IONPs), including Fe3O4, γ‐Fe2O3 and superparamagnetic IONPs,
have been extensively used in drug delivery and magnetic resonance
imaging (Ding & Guo, 2013; Namvar et al., 2014). Meanwhile, their
potential impacts and risks on the environment and human health
have attracted wide attention. Metal and metal oxide NPs can enter
the water environment by various means such as direct discharge,
waste discharge and routine use, and they have good dispersibility
and stability under the action of organic matter in water. Metal and
metal oxide NPs entering the aqueous environment can be enriched
in aquatic organisms such as fish through the food chain. When people
drink water containing these NPs or eat contaminated organisms such
as vegetables, fish and livestock, the metal and metal oxide NPs can
eventually enter and accumulate in the human body, posing a threat
to human health (Baoshan Xing, 2016; Nowack & Bucheli, 2007;
Wang & Wang, 2014). Therefore, to evaluate the toxic risks it is nec-
essary to develop new tools and models that are better able to assess
both exposure levels and toxicity of these nanomaterials.
Nanomaterial toxicity assessments have traditionally been carried
out at two levels: in vitro experiments and in vivo experiments, ranging
from in vitro cell‐based assays to multicellular organisms, such as algae,
protozoa, zooplankton, and advanced higher vertebrate models, such as
rodents, rabbits and the nonhuman primates (Li & Chen, 2011; Schrand
et al., 2010). Primary cultured cells, cell lines and low‐level organisms are
mainly used for cytotoxicity and genotoxicity studies. In contrast, higher
vertebrate models are primarily used to detect complex physiological
interactions. Rodents (mice and rats) and rabbits are the most widely
used higher vertebrate models in toxicological studies, for their rela-
tively inexpensive experimental cost, available extensive information
on normal development, growth, reproduction and toxicology with
relevance to humans (Foote & Carney, 2000; Lee, Inselman, Kanungo,
& Hansen, 2012). However, the embryo developments of rodents and
rabbits are relatively slow and inaccessible. A substantial amount of
material is required for testing due to their relatively large size and their
manipulation, and in vivo whole animal imaging require special tech-
niques and complex manipulations. Nonhuman primates have long life
spans and a relatively small number of offspring. They are rare, expen-
sive and accompanied by ethical concerns about their use. Even the
macaques are not available, let alone orangutans and gibbons.
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In recent years, zebrafish (Danio rerio) as an in vivo model organism
has attracted much interest because of its unique features, including
high fecundity, embryo transparency, fast and well‐characterized devel-
opment, low cost, gene manipulation accessibility, short reproduction
time. The embryo toxicity test of zebrafish has become an important
method for ecotoxicology research, and it is one of the standard
methods for determining the toxicity of single chemicals recognized
by national standards organizations (Fako & Furgeson, 2009).
This review has therefore focused on the current advances of
using zebrafish for toxicity assessment of metal and metal oxide
NPs. Further, we described the different target organ toxicities in
detail. Afterwards, toxicological studies of various metal and metal
oxide NPs conducted using the zebrafish model have been empha-
sized. Finally, we also highlighted the drawbacks and future prospects
of nanotoxicity studies of metal and metal oxide NPs in zebrafish.
2 | Z E B R A F I S H A S A P R O M I SI N G M O D E L
2.1 | Advantages of zebrafish in nanotoxicity
research
More recently, use of the zebrafish model has been considerably
increased in the screening of toxicants (Chun et al., 2017; Da et al.,
2018; Eryilmaz et al., 2018; Sangabathuni et al., 2017; Vicario‐Pares,
Lacave, Reip, Cajaraville, & Orbea, 2018). The most common method
for detecting heavy metal pollutants in the environment is to conduct
toxicological experiments with zebrafish and their embryos
(Holmstrup et al., 2010; Yin et al., 2018). Zebrafish are also widely
used to monitor organic pollutants such as microplastics, pesticides
and antibiotics (Qiao, Lu, Deng, Ren, & Zhang, 2019; Wang et al.,
2018). Numerous characteristics make zebrafish a remarkable alterna-
tive model for toxicological studies of nanomaterials and can be sum-
marized by the following.
First, as a multicellular organism, zebrafish can provide more
complete information in terms of kinetics, migration and transforma-
tion of nanomaterials than in vitro cell culture assays, although
in vitro experiments are commonly used to evaluate potential
toxicological effects of nanomaterials and are acknowledged as
effective for toxicity and genotoxicity studies at the cellular level
(Gad, 2015).
Second, zebrafish are small in size and simple in cultivation, making
it an available model for most of laboratories throughout the world.
Zebrafish have a shorter life cycle and higher fecundity than rodents.
They generally attain reproductive maturity within 3‐6 months
postfertilization in laboratories under appropriate conditions, such as
suitable temperature, food supply and rearing densities (Spence,
Gerlach, Lawrence, & Smith, 2008). Adult female fish produce
embryos on a daily basis and one female can usually produce 100‐
300 embryos at a time (Castranova et al., 2011; Spence & Smith,
2005). The great fecundity of the zebrafish supports high‐throughput
analysis and enhances the statistical power of experiments.
BAI AND TANG 3

Third, the embryogenesis and developmental processes in
zebrafish have been well‐studied (Haffter et al., 1996; Kimmel, Ballard,
Kimmel, Ullmann, & Schilling, 1995). The rapid development of
zebrafish compared with other animal models make it a great advan-
tage for assessing developmental toxicity (Westerfield, 2007) (Lin,
Zhao, Nel, & Lin, 2013). The transparency during embryo stages makes
it easy to observe toxic effects, such as lethality, reproductive toxicity
and teratogenicity (Choi, Kim, Yoon, & Kim, 2016; Ma et al., 2018;
Mesquita et al., 2017; Pecoraro et al., 2017).
Fourth, the zebrafish genomes have been fully sequenced and it is
shown that zebrafish have 26 206 protein‐coding genes (Collins,
White, Searle, & Stemple, 2012; Howe et al., 2013). Approximately
85% of these genes are homologous with their human counterparts
(Renier et al., 2007), making zebrafish an analogous model to investi-
gate developmental toxicity and genotoxicity (Garcia, Noyes, &
Tanguay, 2016; Rizzo et al., 2013; Sarmah & Marrs, 2016; Zhu
et al., 2014). Moreover, there are numerous transgenic zebrafish lines
available (Table 1), which express fluorescent reporter genes such as
green fluorescent protein (GFP) or red fluorescent protein, in specific
cells, tissues or organs allowing for direct toxicity observation in live
fish after nanomaterial exposure. Most of these fish lines can be
found on the Zebrafish Model Organism Database (ZFIN) website
(www.zfin.org). For example, for neurotoxicological studies, some
transgenic fish lines such as huc‐GFP (GFP expressed in most neu-
rons) and islet1‐GFP (GFP expressed in secondary motor neurons
only), can be suitably used.
TABLE 1 Different transgenic zebrafish strains used in the nano-
particle toxicity studies
Transgenic Specific cell/tissue/organ
strains labeling References
2.2 | Application of zebrafish in routine toxicity
evaluation
2.2.1 | Application of zebrafish and its embryos in
acute toxicity experiments
Currently, the acute toxicity of environmental chemicals (including
NPs) to fish is most commonly estimated through a short‐term test
on zebrafish embryos or adults. Because the acute toxicity results of
embryos and adults are very consistent with each other, the fish
embryo toxicity tests have been acknowledged as one of the promis-
ing alternative approaches to classical acute fish toxicity testing for
their advantages such as transparency during embryo stages (Embry
et al., 2010). Specific bioassays are conducted in accordance with
the guidelines for early fish toxicity testing promulgated by the Orga-
nization for Economic Cooperation and Development (OECD TG 236)
(OECD, 2013a; Sobanska et al., 2018).
Kovrižnych et al. compared the acute toxicity of 31 different
nanomaterials (including eight pure metals, 10 metal oxides) in adult
zebrafish and found that metal and metal oxide NPs, such as CuNP,
magnesium oxide (MgONP) and NiNP, caused cumulative mortality.
CuNPs and AgNPs were toxic for zebrafish with median lethal concen-
tration (LC50) values of about 3 mg/L (Kovrižnych et al., 2013). Tang
et al. investigated the toxicities of rutile TiO2NPs in zebrafish embryos
and adults. The results showed that exposure to 100 mg/L TiO2NPs
had no effect on the hatching rate of zebrafish embryos and there
was no clear deformity. On the contrary, in the adult toxicity experi-
ment, TiO2NP exposure gave rise to oxidative damage in liver, intes-
tine and gill tissue. The activity of superoxide dismutase (SOD),
catalase and glutathione‐S transferase expressed highly in these three
organs (Tang, Zhang, & Zhu, 2019). The above examples suggest that
zebrafish is a good model for studying the acute toxicity of NPs.

Hsp70: Heat shock response Asharani, Lianwu, Gong, &

EGFP pathway Valiyaveettil, 2011; George
et al., 2012; Pan et al., 2013
2.2.2 | Application of zebrafish in chronic toxicity
fli‐1/fli‐1a: Blood vessels Chang et al., 2015; Cordeiro
experiments
EGFP et al., 2017; Gao et al.,
2016; Patra et al., 2011
Although the acute toxicity test can reflect the toxic effects of a sub-
HuC‐GFP Neurons Park et al., 2000
stance on organisms in short‐term and high‐dose conditions, most of
β‐actin: Proliferating germ cells Hsiao & Tsai, 2003
environmental chemicals exist only in a low concentration state.
EGFP and female gonads
Therefore, chronic toxicity experiments under low concentration and
ARE Antioxidant response Bar‐Ilan et al., 2012;
long‐term exposure on organisms are necessary. Zebrafish as a model
pathway Tkachenko et al., 2003
of chronic toxicity has a significant advantage: a relatively short life
pdx1 Pancreas Truong et al., 2013
cycle and can perform whole life‐cycle exposure experiments and
mpo Neutrophils Bar‐Ilan et al., 2012
even transgenerational tests to detect long‐term toxic effects of
mmp23b Liver Zhu et al., 2008 nanomaterials. Now there are various guidelines for the chronic toxic-
lysC Macrophages and Browning et al., 2009; ity testing of NPs on zebrafish, such as GB/T 21808‐2008 (Fish,
possible neutrophils Jovanovic, Ji, & Palic, 2011
Prolonged Toxicity Test: 14‐Day Study), OECD TG 229 (Fish Short
krt8 Skin epithelium Bar‐Ilan, Albrecht, Fako, & Term Reproduction Assay) (OECD, 2012), TG 210 (Early‐Life Stage
Furgeson, 2009
Toxicity Test) (OECD, 2013b), TG 234 (Fish Sexual Development Test)
Insulin Endocrine pancreas Truong et al., 2013
(OECD, 2011) and TG 212 (Short‐term Toxicity Test on Embryo and
islet1‐GFP Cranial motor neurons Higashijima, Hotta, & Sac‐Fry Stages) (OECD, 1998; Horie, Yamagishi, Koshio, Iguchi, &
Okamoto, 2000
Tatarazako, 2017; Horie et al., 2017). Zebrafish has multiple toxicity
4 BAI
indicators, which are also convenient for judging the toxic effects and
mechanisms of nanomaterials.
Chen et al. assessed the chronic toxicity of TiO2NPs in adult
zebrafish and found that TiO2NPs could translocate among organs
and penetrate the blood‐brain and the blood‐heart barrier, accumulate
and distribute in gill, liver, heart as well as brain. TiO2NP exposure led
to significant adverse effects on zebrafish, including growth inhibition
and weight ratio decrease in the liver and histopathologic change in
the gills such as thickened edema and the gill lamellae (Chen, Dong,
Xin, & Zhao, 2011). Du et al. studied the joint toxic effects of nano‐
ZnO and perfluorooctanesulfonic acid in the initial generation ( F 0)
of zebrafish (2 hours after incubation) after chronic exposure
(120 days) and examined parental transfer of the transgenerational
effects of these two environmental contaminants on the growth of
first generation ( F 1) larvae. The results revealed that in the F 0
generation co‐exposure led to less growth in body length and body
weight, higher mortality and less spawning, probably because of
the downregulated expression of Vtg1 genes and sex hormone
(testosterone/estradiol) involved in the hypothalamus‐pituitary‐gonad
axis. In the F 1 generation, long‐term joint exposure resulted in
reduced fertilization and hatching, and increased mortality and defor-
mity (Du et al., 2018).
2.3 |Toxicity of metal and metal oxide nanoparticles
on zebrafish organs
As a vertebrate, zebrafish has high homology with humans, and its
development of the central nervous system, cardiovascular system,
visual system and internal organs has much in common with the
human counterparts. The different developmental stages of zebrafish
offer a variety of screening schemes that target various toxicological
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responses to metal and metal oxide NPs, including developmental tox-
icity, immunotoxicity, cardiovascular toxicity, neurotoxicity and repro-
ductive toxicity (Figure 1).
2.3.1 | Developmental toxicity
The developmental toxicity of nanomaterials in zebrafish includes
hatching rate, mortality and teratogenicity. Compared with the toxicity
evaluation of other systems or target organs, the developmental toxic-
ity of embryos using the NP assessment of zebrafish has been rela-
tively mature. Because of its in vitro fertilization and transparency
during embryo stages, it is suitable for image‐based detection and
can record a variety of teratogenic indicators, such as cell movement
during the intestinal phase, brain formation, heartbeat and blood circu-
lation. Embryonic development events can be used as endpoints for
toxicological evaluation (Figure 2). In addition, the zebrafish embryo
teratogenic test cycle is short (usually completed within 1 week) and
suitable for large‐scale gene mutant screening and analysis.
Mortality and hatching delay were observed in AgNP‐treated
embryos. Moreover, AgNP treatments resulted in developmental
toxicity such as body axes abnormality, twisted notochord, slow
blood flow, pericardial edema and arrhythmia (Asharani, Lian, Gong,
& Valiyaveettil, 2008; Shaw, Liddle, Windeatt, & Handy, 2016).
Zebrafish embryos exposed to cetyltrimethylammonium bromide‐
coated gold nanorods were found to induce mortality and delayed
embryonic developments, including tail deformities, pericardial
edema, decreased body length, and delayed eye, head and tail elonga-
tion development (Mesquita et al., 2017). Metal oxide NPs have also
been proved to be effective in causing acute toxicity and develop-
mental toxicity in zebrafish. Copper oxide NP (CuONP) exposure
could induce abnormal phenotypes such as smaller head and eyes
FIGURE 1 Schematic diagram of
nanotoxicological studies relevant to the
different stages of zebrafish development
BAI AND TANG 5

FIGURE 2 Representative nanoparticle‐induced developmental toxicities in zebrafish embryos at 4, 24, 48, 96 and 120 h postfertilization time
points. Embryos were scored for severity of morphological defects, survival and toxic adverse effects. Scores range from 0 to 4, with 0 indicating
no visible deleterious effects and 4 signifying death. Developmental malformations included YND, TM, JM, PE, BS and OY. BS, bent spine; JM, jaw
malformation; OY, opaque yolk; PE, Pericardial edema; TM, tail malformation; YND, yolk not depleted. (Adapted from Bar‐Ilan et al., 2009)

and delayed epiboly in zebrafish (Xu et al., 2017). The toxic effects of
ZnONPs in zebrafish include hatching delay, skin ulceration and high
mortality of zebrafish (Zhu et al., 2008). Samaee et al. evaluated the
TiO2NP toxicity to zebrafish embryos and observed that TiO2NPs
could induce premature hatching and a reduction in the time required
for normal hatching (Samaee, Rabbani, Jovanovic, Mohajeri‐Tehrani,
& Haghpanah, 2015).
2.3.2 | Immunotoxicity
In recent years, the application of zebrafish in the field of immunology
has gradually received attention. The immune system of zebrafish has
both commonality and particularity compared with higher vertebrates.
In mammals, the bone marrow and thymus are central immune organs;
peripheral immune organs include the spleen and lymph nodes. In

FIGURE 3 NP‐induced immunotoxicity in zebrafish at different levels (external barriers, tissue, cellular and subcellular) (Adapted from Torrealba
et al., 2019). NOs, nitric oxides; NPs, nanoparticles; ROS, reactive oxygen species
6 BAI
zebrafish, the head kidney is the main immune organ, similar to human
bone marrow, where T cells, B cells and dendritic cells are found.
Zebrafish also has a bilaterally symmetric thymoid structure, similar
to the mammalian thymus cortex. The head kidney and thymoids
together form the central immune organs of zebrafish. Zebrafish lack
lymph nodes, but the spleen, liver and intestine can replace the lymph
nodes to provide a site for antigen‐presenting cells to exchange infor-
mation with lymphocytes, forming the secondary immune organs.
Immunotoxicity refers to the destructive effects on the functioning
of the immune system when exposed to any toxic substance
(Giannakou et al., 2016; Selgrade, 2007; Torrealba, More‐Bayona,
Wakaruk, & Barreda, 2019) (Figure 3). Exposure of ZnONPs caused
transcriptional changes of proinflammatory cytokines, tumor necrosis
factor‐α and interleukin (IL)‐1β, a downregulation in zebrafish embryos
and a significant upregulation in eleuthero embryos, implying that
ZnONPs result in modulation of the proinflammatory reactions (Brun,
Lenz, Wehrli, & Fent, 2014). A gene expression study was carried out
in liver tissues of adult zebrafish after AgNP exposure. It was shown
that MTF‐1, TLR4, IL1B, CEBP, TRF and TLR22 genes were downregu-
lated, HSP70 and NFKB genes were upregulated, confirming that
AgNPs could lead to oxidative stress and immunotoxicity in adult
zebrafish (Krishnaraj, Harper, & Yun, 2016).
2.3.3 | Cardiovascular toxicity
Zebrafish embryos are particularly suitable for cardiac developmental
toxicological evaluation of NPs. A zebrafish heart resembles the
human embryonic heart and has one atrium and one ventricle with
valves between the atrioventricular compartments. Regular heartbeats
start from 36 hours postfertilization and the microscope can be used
directly to observe the shape and rhythm of the heart, such as heart-
beats, blood vessel morphology and cell activity in blood vessels,
which have greatly facilitated the toxicity evaluation and toxicology
research of NPs. Transgenic zebrafish lines have been successfully
established to monitor and quantify and qualitatively evaluate cardio-
vascular damage caused by specific NPs. Transgenic zebrafish Tg
(nacre/fli1:EGFP) was used to investigate the effects of CuONP expo-
sure on vasculogenesis/angiogenesis (Chang et al., 2015). The results
demonstrated that CuONPs inhibit vasculogenesis via reduction of
vascular endothelial growth factor expression and induction of
apoptosis.
The molecular pathways that regulating the hematopoietic system
of zebrafish is quite conservative. In particular, the early development
of the cardiovascular system is very similar to that of humans. The
long‐term survival of mutants with defective cardiovascular systems
provides favorable conditions for studying the developmental toxicity
of NPs in the cardiovascular system. The toxic effects of AgNPs in
hematopoiesis using a zebrafish model were studied. Microarray anal-
ysis was used to reveal transcriptional responses of zebrafish embryos
to AgNPs at 24 hours postfertilization. Gene ontology analysis showed
that hemoglobin genes were downregulated by AgNPs. The reduction
of hemoglobin expression was further proved by quantitative reverse
transcription‐polymerase chain reaction, whole‐mount in situ
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hybridization and O‐dianisidine staining. Further research such as
mesoderm commitment, hematopoietic progenitor cell specification
and erythroid differentiation were operated at different developmen-
tal stages in AgNP‐exposed embryos. It was found out that inhibition
of erythrogenesis by AgNPs was developmental stage specific and cell
specific, and this inhibition was mostly by AgNPs other than their
releasing ions (Cui et al., 2016).
2.3.4 | Reproductive toxicity
Reproductive toxicity testing of NPs can be carried out by partial life
cycle tests of zebrafish or whole life cycle tests. Commonly used indi-
cators are vitellogenin, histological examination of gonadal glands,
gonadal index and sperm plasma membrane integrity (Rim, 2017).
AgNP exposure gave rise to oxidative stress, caused germ cells apo-
ptosis via mitochondrial‐dependent pathway and finally damaged the
reproductive ability in zebrafish (Ma et al., 2018). Adult female
zebrafish were exposed to AuNPs. The result showed that AuNPs
ranging between 10 and 50 nm could enter ovaries of zebrafish and
give rise to strand breaks in ovarian cells (Dayal et al., 2016). The
effects of TiO2NPs on zebrafish testis were also investigated.
The findings showed that TiO2NP‐induced autophagy and necrosis
at higher doses in Sertoli cells and consequently negatively affected
spermatogenic cells and testicular morphology of zebrafish; for exam-
ple, mitochondrial degeneration with swelling and cristae loss (Kotil,
Akbulut, & Yon, 2017).
2.3.5 | Neurotoxicity
Zebrafish has been proved a sensitive and useful animal model to
assess NP‐induced neurotoxicity. The juveniles can swim freely after
5 days of fertilization and the brain tissues of juveniles have already
differentiated into the structures of the telencephalon, diencephalon,
midbrain, hindbrain and rhomboidal ganglia, which can be used to
evaluate the behavioral toxicity of NPs, such as motion, learning and
memory ability. In addition, biochemical indicators, morphological
changes, necrosis and apoptosis of neurons can also be used to evalu-
ate the neurotoxicity of NPs to zebrafish embryos. The neurotoxicity
of AgNPs to zebrafish exhibited a size‐dependent effect. Powers
et al. exposed developing zebrafish to 10 and 50 nm
polyvinylpyrrolidone‐coated AgNPs (AgNP‐PVP) and evaluated larval
swimming behavior and responses to different lighting conditions
24 hours after the termination of exposure. The results showed that,
under certain light conditions, the smaller AgNP‐PVP (10 nm) caused
decreased locomotor activity, whereas the larger (50 nm) caused
hyperactivity (Powers, Slotkin, Seidler, Badireddy, & Padilla, 2011).
AuNP exposure caused alteration of neurotransmission, showing
increased brain acetylcholine esterase activity (Dedeh, Ciutat,
Treguer‐Delapierre, & Bourdineaud, 2015). CuONP exposure at high
doses (≥12.5 mg/L) resulted in neurotoxicity in zebrafish embryos
and larvae, showing a delay in retinal neurodifferentiation accompa-
nied by reduced locomotor ability (Sun et al., 2016). The toxicity of
dextran‐coated Fe2O3NPs to zebrafish brain was also researched.
BAI AND TANG
The animals exposed to 200 mg/kg and tested 24 hours after admin-
istration of the NPs showed decreased acetylcholinesterase activity,
reduction in the exploratory performance, significantly higher levels
of ferric iron in the brains and induction of casp8, casp9 and jun genes.
Taken together, these findings suggested acute brain toxicity by the
inhibition of acetylcholinesterase and induction of apoptosis (de
Oliveira et al., 2014).
2.3.6 | Hepatotoxicity
The liver is the main metabolic organ and has important functions on
the body. It is highly susceptible to the toxic effects of various
chemical substances and causes functional damage, thus affecting
the normal operation of the body. The response of the liver to
chemicals in the early developmental stages of zebrafish is similar to
that of humans and is an ideal model for studying the hepatotoxicity
of NPs. Studies investigated the effect of CuONPs on zebrafish liver
development and neuronal differentiation. The results reflected that
short‐term exposure to CuONPs at high doses led to hepatotoxicity
and neurotoxicity in zebrafish embryos and larvae, displaying as
hepatic hypoplasia and delayed retinal neurodifferentiation coupled
with reduced locomotor ability (Sun et al., 2016). Xiong et al. studied
the oxidative stress effects and damage of ZnONPs on gill, intestine
and liver of zebrafish, and found that liver tissues were the main target
of oxidative damage. Further study by the same group showed that
ZnONP exposure generated higher ·OH radicals. The malondialdehyde
content was elevated in zebrafish gills and livers, which is one of the
biomarkers of the oxidative effects (Xiong, Fang, Yu, Sima, & Zhu,
2011).
The acute toxicity (after 96 hours exposure) of IONPs in adult
zebrafish were investigated using different endpoints. The results
demonstrated that DNA damage in erythrocytes was observed for
all IONP concentrations (ranging from 4.7 to 74.4 mg/L). Only the
highest IONP concentrations (37.2 and 74.4 mg/L) caused oxidative
stress in liver cells. Liver microarray analysis revealed almost 1000
differentially expressed transcripts (DETs) between control and IONP
treatment groups (37.2 and 74.4 mg/L). Genes in cluster 1
(overexpressed in treated groups) were related to ion binding. Genes
in cluster 2 (downexpressed in treated groups) were associated with
ribosomes and translation (Villacis et al., 2017).
3 | T O X I C O L O G I CA L S TU D I E S O F
D I F F E R E N T M E T A L A N D M E T A L OX I D E
N A N O P A R T I C L E S I N ZE B R A F I S H
3.1 | Gold
AuNPs, due to their unique properties, are widely used in drug deliv-
ery, cellular labeling, and imaging and diagnostics for diseases such
as Alzheimer's, cancer and diabetes (Li & Chen, 2011). In addition, they
might pose potential hazards to living organisms. Several studies have
been done to investigate the toxicity of AuNPs in zebrafish. AuNPs
7
passively diffused into the chorionic space of zebrafish embryos via
chorionic pore canals and then continued their random walk through
the inner mass of embryos. AuNPs were shown to be less toxic to
embryos or adult zebrafish when compared with Ag particles
(Browning et al., 2009; Ramachandran et al., 2018). Other studies also
proved that AuNPs were nontoxic to zebrafish embryos when com-
pared with other NPs such as Ag, Cu and Pt (Asharani et al., 2011;
Bar‐Ilan et al., 2009). On the contrary, some researchers argued that
AuNPs were toxic to zebrafish, and the exposure of AuNPs led to
embryonic lethality, developmental toxicity (Truong, Saili, Miller,
Hutchison, & Tanguay, 2012), neurotoxicity (Kim, Zaikova, Hutchison,
& Tanguay, 2013) and immunotoxicity (Truong, Saili, et al., 2012)
Several factors influence the toxicity of AuNPs in zebrafish, includ-
ing particle shape, surface charge and surface coating. The particle
shape of AuNPs is a critical factor to understand AuNP toxicity.
Sangabathuni et al. systematically examined the toxicity and
biodistribution of modified AuNPs of three different shapes (sphere,
rod and star) in adult zebrafish (Sangabathuni et al., 2017). The results
revealed that rod AuNPs showed increased uptake and clearance. In
contrast, star AuNPs exhibited prolong sequestration compared with
sphere or rod particles. Surface charge and surface coating may also
play an important role in exploring the toxicity of AuNPs. Harper
et al. (2011) assessed the embryonic toxicity of AuNPs of different
sizes and surface charges in zebrafish. The results of this study
showed that the exposure of AuNPs with both positive and negative
charges caused embryonic lethality and developmental toxicity in
zebrafish, with positively charged AuNPs mainly leading to mortality
while negatively charged ones causing malformations. In contrast,
AuNPs with no charge exhibited no adverse impact on biological sys-
tems. In another study, zebrafish embryos were exposed to AuNPs
that possess functional groups with different charges (positive charge,
negative charge and neutral charge). The results showed that AuNPs
with positive and negative charges caused toxic effects such as
hypo‐locomotor activity and high lethality, while AuNPs with neutral
charge coating did not induce any effect (Truong, Saili, et al., 2012).
These results seem to be consistent with Harper et al.'s findings
(Harper et al., 2011). Researchers from the same group further inves-
tigated the mechanism of surface coating and charge influencing
molecular responses (Zhu et al., 2008). The results indicated that
exposure to both positively charged and negatively charged AuNPs
disturbed pathways associated with the inflammation and immune
response and perturbed transport mechanisms implying that surface
charge and surface coating influences many molecular pathways.
Kim et al. investigated the developmental toxicity of AuNPs with pos-
itive charges (coated with N,N,N‐trimethylammoniumethanethiol) in
zebrafish embryos and found that exposure to positively charged
AuNPs caused abnormalities in eye development and pigmentation,
and led to neurotoxic effects and behavioral changes (Kim, Zaikova,
et al., 2013). Taken together, these results suggest that there is a close
association between surface charge and surface coating of AuNPs and
their toxicities in zebrafish.
As for AuNPs, dose‐dependent toxic effects are still debatable.
One experiment showed that the amount of AuNPs accumulated in
8 BAI AND TANG

embryos was dose‐dependent, showing higher accumulation coupled 3.2 | Silver

with increasing concentration, but the effects on embryonic develop-
ment were not proportionally related to the concentration (Browning
et al., 2009). On the contrary, another group investigated the impact
of AuNPs (two sizes, 12 and 50 nm) on zebrafish at very low doses
(from 36 to 106 ng gold/fish/day). The results showed that AuNP
exposure caused brain and muscle mitochondrial dysfunctions in adult
zebrafish and these toxic effects were dependent on size, concentra-
tion and exposure time (Geffroy et al., 2012). The influence of surfac-
tants on AuNP toxicity in embryonic zebrafish were also investigated.
Ligand‐stabilized AuNPs and surfactant (Polysorbate 20) were mixed
together and the investigation showed that these mixtures elicited
increased uptake and toxicity in zebrafish (Ginzburg, Truong, Tanguay,
& Hutchison, 2018) (Table 2).
AgNPs are extensively used in consumer products such as clothing,
bactericidal applications, antimicrobial agents and medical imaging
(Ahamed, AlSalhi, & Siddiqui, 2010). The toxicological studies of
AgNPs in zebrafish are still in the exploration stage, focusing on acute
and chronic exposure experiments. Griffitt et al. measured the toxicity
of metallic NPs and soluble metals in zebrafish using 48‐hour static
bioassays. The results indicated that the 48‐hour LC50 of AgNPs
(26.6 nm) were 7.07 and 7.20 mg/L in adult and juvenile zebrafish
respectively (Griffitt, Luo, Gao, Bonzongo, & Barber, 2008). The 48‐
hour LC50 of AgNPs (18 nm) in deionized water on 3‐day‐old
zebrafish‐hatched embryos was 0.94 mg/L (Olasagasti et al., 2014).
Bilberg et al. (Bilberg, Hovgaard, Besenbacher, & Baatrup, 2012)

TABLE 2 Toxic effects of AuNPs on zebrafish

Concentration and
Size (nm) Surface coating exposure duration Stage Results References

1.5 TMAT 0.08, 0.4, 2, 10 and Embryos Abnormally small and under Kim, Zaikova, et al., 2013
50 mg/L, 6‐120 hpf pigmented eyes in the
developing zebrafish
0.8, 1.5 and 15 TMAT, MES, MEE and 0.016, 0.08, 0.4, 2, 10, Embryos TMAT‐AuNPs primarily Harper et al., 2011
MEEE 50 and 250 mg/L, 8‐ caused mortality. Exposure
120 hpf to MES‐AuNPs generally
resulted in malformations.
MEEE‐AuNPs did not elicit
any adverse effects
1.5 TMAT, MES and MEEE 50 mg/L, exposed from Embryos and Acute exposure to MES‐ and Truong, Saili, et al., 2012
6 to 120 hpf and then adults TMAT‐AuNPs during
raised in fresh water embryonic development
to adulthood results in larval behavioral
(122 dpf) abnormalities that persist
into adulthood.
Developmental exposure to
both NPs resulted in larger
adults, and exposure to
MES‐AuNPs at 50 μg/mL
resulted in reduced adult
survivorship
1.2 MPA 0.08, 0.4, 2, 10 and Embryos AuNPs dispersed in low ionic Truong, Zaikova, Richman,
50 mg/L, 4‐120 hpf media‐induced morbidity Hutchison, & Tanguay,
and mortality 2012
1.5 TMAT, MES and MEEE 0.016, 0.08, 0.4, 2, 10, Embryos Pathways involved in Truong et al., 2013
50 and 250 mg/L, 4‐ inflammation and immune
120 hpf response were perturbed
and transport mechanisms
were misregulated after
exposure to TMAT‐ and
MES‐AuNPs
3, 10, 50 and TPPMS 50, 5, 0.5 and 0.05 mg/ Embryos Minimal sublethal toxic effects Bar‐Ilan et al., 2009
100 L, 2‐120 hpf
14 Citrate 0.25 and 0.8 μg/L, Adults Gene expression level Dedeh et al., 2015
20 days modifications, DNA
alterations and AChE
activity variation
BAI AND TANG
investigated acute toxicity of PVP‐coated AgNPs (81 nm) to zebrafish
and found that the AgNP 48‐hour LC50 value was 84 μg/L. The differ-
ent 48‐hour LC50 values of AgNPs in these studies are mainly due to
different nanomaterial characteristics of nanosilver, including NP mor-
phology (size or shape), surface characteristics (e.g., organic surface
coating), composition or crystallinity, solution chemistries such as
pH, ionic strength, valence of electrolyte ions and environmental
parameters (e.g., temperature and light irradiations).
Many studies have focused on the relationship between particle
size and toxic effects of nanosilver. Lee et al. observed size‐dependent
toxic effects of the NPs on zebrafish embryonic development. Single
AgNPs (30‐72 nm) passively entered zebrafish embryos through cho-
rionic pores via random Brownian motion. At the same molar concen-
tration, the larger AgNPs were more toxic than the smaller ones (Choi
Lee et al., 2012; Lee, Nallathamby, Browning, Osgood, & Xu, 2007). In
contrast, Bar‐Ilan et al. found that AgNP toxicity was size‐dependent
at certain concentrations and time points with smaller NPs being most
toxic (Bar‐Ilan et al., 2009). In another study, Xin et al. found a size‐
dependent uptake of AgNPs. In their experiment, the 4 nm AgNPs
were taken up more efficiently compared with the 10 nm sized ones
and the head area of zebrafish accumulated more AgNPs than the
trunk area. These findings were in accordance with Park et al.'s view
(Park, Tuttle, Sinche, & Harper, 2013). They evaluated the stability of
citrate‐capped AgNPs and the embryonic developmental toxicity in
the fish test water and found that the large sized aggregates of AgNPs
were low toxic compared with the nano‐sized AgNPs. This point of
view was also supported by Kim et al.'s research. In their study, the
stability of AgNPs was significantly higher in 62.5 μM CaCl2 and ultra-
pure water than that in standard zebrafish embryo medium. AgNPs
suspended in ultrapure water and CaCl2 were more toxic to zebrafish
embryos than that in the embryo medium. Twenty‐nanometer AgNPs
possessed higher toxicity than 110 nm AgNPs (Kim, Truong et al.,
2013). In addition, the neurotoxicity of nanosilver to zebrafish also
exhibited a size‐dependent effect. Powers et al. exposed developing
zebrafish to 10 and 50 nm AgNP‐PVP, and evaluated larval swimming
behavior and responses to different lighting conditions 24 hours after
the termination of exposure. The results showed that, under certain
light conditions, the smaller AgNP‐PVP (10 nm) caused decreased
locomotor activity, whereas the larger (50 nm) caused hyperactivity
(Powers, Slotkin, et al., 2011). AgNPs of several different shapes were
all shown to induce oxidative stress, with plate‐shaped AgNPs being
more toxic than spherical and wire‐like shaped forms (Abramenko
et al., 2018; George et al., 2012).
Researchers have also studied the relationship between nanosilver
surface characteristics and toxicity (Abramenko et al., 2019). Wang
et al. examined the toxicity of a bare AgNP, a PVP‐coated AgNP and
a monodispersed AgNP with a dispersant (DIS‐AgNP) to three aquatic
organisms, including zebrafish and found that the toxicity of the AgNP
in the form of colloids decreased in the order DIS‐AgNP>PVP‐
AgNP>Bare‐AgNP for all three trophic aquatic organisms (Wang,
Chen, Li, Shao, & Peijnenburg, 2012). Liu, H et al. also investigated
the toxicities of AgNPs with different surface coatings (sodium citrate
and PVP) and found that sodium citrate‐coated AgNPs were more
9
toxic than the PVP‐coated ones (Liu, Wang, et al., 2019). Powers
et al. examined developmental neurotoxicity of AgNPs in zebrafish
and argued that the neurobehavioral effects of AgNPs were highly
dependent on particle coating and size, and distinct patterns of devel-
opmental neurotoxicity were produced by different AgNP formula-
tions (Powers, Slotkin, et al., 2011).
AgNPs could passively diffuse into the zebrafish embryos through
chorionic pores and stayed inside the embryos, thereby affecting
embryonic development and producing specific phenotypes (Lee
et al., 2007; Lee, Browning, et al., 2012; Lee, Browning, Nallathamby,
Osgood, & Xu, 2013), including abnormal body axes, twisted noto-
chord, slow blood flow, pericardial edema and cardiac arrhythmia
(Asharani et al., 2008; Bar‐Ilan et al., 2009; Bowman, Bailey, Elrod‐
Erickson, Neigh, & Otter, 2012; Yeo & Kang, 2008). AgNPs were dis-
tributed in the gills, brain, heart, yolk and blood of embryos (Asharani
et al., 2008; Griffitt, Lavelle, Kane, Denslow, & Barber, 2013). Dose‐
dependent toxic effects were confirmed in zebrafish in studies using
AgNPs, ZnONPs and CuNPs (Bai et al., 2010; Lee, Browning, et al.,
2012; Zhu et al., 2008). In their research, Griffitt et al. discovered that
AgNPs accumulated in zebrafish gill tissues and eviscerated carcass
tissues and both tissue accumulations were dose‐dependent (Griffitt
et al., 2013). The respiratory toxicity of zebrafish was observed after
nanosilver exposure with symptoms such as an increased rate of oper-
culum movement and surface respiration (Bilberg et al., 2012). AgNPs
could also produce neurotoxicities after exposure to zebrafish. When
exposed to nanosilver under different lighting conditions, zebrafish
appeared to be hypoactive or hyperactive compared with the control
group (Powers, Slotkin, et al., 2011). In vitro experiments also verified
that AgNPs could impede the DNA synthesis and differentiation of
PC12 cells, and impair neurodevelopment (Powers, Badireddy, Ryde,
Seidler, & Slotkin, 2011).
Oxidative stress is an important mechanism for the toxicity of
nanomaterial to organisms (Nel, Xia, Madler, & Li, 2006; Wise et al.,
2010). Massarsky et al. characterized the effects of AgNPs on
zebrafish development. AgNP exposure led to death and delayed
hatching in surviving embryos and depleted glutathione levels
(Massarsky et al., 2013). Christen et al. investigated the effects of
AgNPs on zebrafish and found that AgNPs induced all endoplasmic
reticulum stress responses in vitro in zebrafish liver cells and weak
endoplasmic reticulum stress in zebrafish embryos. AgNPs could also
induce oxidative stress and transcripts of pro‐apoptosis genes such
as p53 and Bax (Khan et al., 2019). In zebrafish liver cells, the expres-
sion of p53 gene was downregulated. Activation of p53 target genes
led to cell cycle arrest and DNA repair processes, but prolonged acti-
vation of p53 resulted in the induction of apoptosis (Christen, Capelle,
& Fent, 2013). The p53‐related pro‐apoptotic genes Bax, Noxa and
p21 were upregulated after treatment with AgNPs to zebrafish liver
tissues. The expression of one pro‐survival marker gene Blp1 was
not significantly altered by AgNP exposure in the liver of adult
zebrafish (Choi et al., 2010). These results inferred that AgNPs could
induce oxidative damage and apoptosis in zebrafish.
Some previous studies have found that the toxicity of AgNPs was
due to the NPs themselves, while others have found that Ag+ ions
10 BAI AND TANG

released by AgNPs played an important role. In their study, van Aerle
et al. declared that the toxicity caused by AgNPs was principally asso-
ciated with bioavailable Ag+ ions in exposed zebrafish embryos (van
Aerle et al., 2013). Wang et al. evaluated the toxicity of AgNPs and
Ag+ to three aquatic organisms including zebrafish larva. Their results
demonstrated that free Ag+ could not be neglected in explaining the
toxicity of AgNP colloids, corroborating the findings of the previous
work in van Aerle et al.'s study (Wang et al., 2012).
Other studies showed that the toxicity of AgNPs might be more
than the role of Ag+ ions. Powers et al. exposed zebrafish to Ag+ or
citrate‐coated AgNP and discovered that fish in the Ag+ group were
extremely sensitive to changes in light conditions. In contrast, the
citrate‐coated AgNP group had no discernible effect on swimming
behavior, just like the controls (Powers, Slotkin, et al., 2011). Similarly,
Ribeiro et al. investigated the toxicity of AgNP and AgNO3 by
assessing different biological endpoints and exposure periods in three
species including zebrafish. The results demonstrated that AgNPs and
AgNO3 induced dissimilar abnormalities on zebrafish embryo develop-
ment, implying that the toxicity of AgNO3 and AgNPs differed dramat-
ically in zebrafish (Ribeiro et al., 2014). Therefore, it is still
controversial whether the toxicity of AgNPs is due to its own effects
or the release of silver ions (Table 3).
3.3 | Titanium dioxide
TiO2NPs are one of the most widely produced and commercially
applied nanomaterials. Applications of TiO2NPs range from pigments
in sunscreens to components of soaps, shampoos, toothpastes, etc.
(Noman, Ashraf, & Ali, 2018). Size‐dependent toxicity was observed

TABLE 3 Toxic effects of AgNPs on zebrafish

Concentration and exposure

Size (nm) duration Stage Results References

5‐20 5‐100 μg/mL, 72 h Embryos Dose‐dependent toxicity in embryos. AgNPs distributed in brain, Asharani et al.,
heart, yolk and blood of embryos 2008
20‐30 7.2 mg/L, every 12 h Embryos Cytotoxicity Griffitt et al., 2008
and
adults
20‐30 1 mg/L, 24 h, 48 h Adults Alteration of gene expression Griffitt, Hyndman,
Denslow, &
Barber, 2009
5‐20 30, 60, 120 mg/L, 24 h Adults Increase in γ‐H2AX and dose‐dependent increase in p53 mRNA Choi et al., 2010
5‐35 – Embryos Phenotypic changes starting from 25 mg/mL Asharani et al.,
2011
41.6 ± 9.1 0, 5, 12, 24, 48, 95, 119 and Embryos Dose‐ and size‐dependent toxic effects of the AgNPs on embryonic Lee et al., 2012
166 μg/mL, 0‐120 hpf development
1‐20 0.001‐100 mg/L, 12, 24 and 48 hpf Embryos A toxic effect and a death risk, inhibition of AChE and Myrzakhanova
pseudocholinesterase activity, and impaired recruitment of T et al., 2013
lymphocytes (CD41+ cells)
3.10 ± 2.23 5, 15, 25 and 50 μg/L, 7, 14, 21 Adults AgNP accumulation in zebrafish gill and carcass tissue, dramatic Griffitt et al., 2013
and 28 days alterations in global gene transcription profiles in exposed gills
4 and 10 0.481, 0.963, 1.925, 3.850, 7.700, Embryos AgNPs could affect the neural development of zebrafish embryos Xin, Rotchell,
11.550 and 23.100 mg/L, from 4 Cheng, Yi, &
to 96 hpf Zhang, 2015
10 and 40 0.2, 0.4, 0.75, 1 mg/L, from 4 to Embryos Developmental stage‐specific and cell‐specific erythrogenesis Cui et al., 2016
24 hpf suppression
40 0.4 mg/L Embryos AgNP‐induced hypopigmentation in zebrafish embryos and Xu et al., 2017
suppressed transcription of melanophore and xanthophone genes
20 0.01, 0.1, 0.5, 1 and 10 mg/L Larva AgNP exposure led to an accumulation of Ag in the blood vessels in Cambier et al.,
the liver with a disturbance of the expression of genes involved in 2018
the photoreceptor, in the feedback‐loop of the circadian rhythm,
and the cardiovascular system regulation
49 10, 33 and 100 μg/L, 5 weeks Adults AgNP exposure caused oxidative stress, induced germ cells apoptosis Ma et al., 2018
via mitochondrial‐dependent pathway, and impaired the
reproduction in zebrafish
81 0.084 mg/L, 48 h Adults Excess mucus production in gills Bilberg et al., 2012
20‐30 20 mg/L, 2, 5, 8, 22, 27, 32, 48 and Embryos Lots of genes involved in apoptosis, endocytosis and immune Park & Yeo, 2013
72 hpf responses were differently expressed after exposure to AgNPs
BAI AND TANG
for TiO2NPs to zebrafish embryos, with the 120‐hour LC50 of
TiO2NPs (5.7 and 12.4 nm) to be 23.4 and 610 mg/L, respectively,
but the value was over 1000 mg/L for 15 nm sized TiO2NPs (Kim et al.,
2014). In contrast, another study showed no impact of TiO2NPs (size
<20 nm) and bulk TiO2 on zebrafish embryos hatching, even when the
concentration reached as high as 500 mg/L (Zhu et al., 2008). Several
groups demonstrated that TiO2NPs could cause premature hatching in
a dose‐dependent manner (Clemente, Castro, Moura, Jonsson, &
Fraceto, 2014; Samaee et al., 2015). A low dose (1 mg/L) of TiO2NPs
failed to induce major developmental malformations in zebrafish
embryos (Wang et al., 2014), while higher doses could lead to
embryonic malformation and even death (Chakraborty et al., 2009).
Chronic exposure of TiO2NPs at low concentrations (<4 mg/L) for
6 months caused low toxicity in adult zebrafish. However, at higher
concentrations these NPs were found to accumulate in different
parts of the fish, including the gill, liver, heart and brain (Chen, Dong,
et al., 2011). High‐level exposure could also cause genotoxic effects
(Rocco et al., 2015).
3.3.1 | Phototoxic effects of titanium dioxide
nanoparticles
Zebrafish have routinely been used to evaluate the phototoxic
effects of TiO2NPs (Clemente et al., 2014). Xiong et al. studied the
acute toxicity of TiO2NPs in zebrafish and explored the underlying
mechanisms. ·OH radicals produced by TiO2NP exposure and oxida-
tive effects biomarkers such as SOD, catalase activities and
malondialdehyde were investigated. The result showed that the pho-
totoxic effect of TiO2NPs in zebrafish was mediated via oxidative
damage (Xiong et al., 2011).
Several other studies have confirmed the phototoxic effect of
TiO2NPs in zebrafish. Bar‐Ilan et al. exposed TiO2NPs to developing
zebrafish embryos and discovered that TiO2NP exposure led to the
photo‐dependent production of reactive oxygen species (ROS)
in vivo and subsequent malformation and death of zebrafish (Bar‐Ilan
et al., 2012). Further study demonstrated the photoactivation of
TiO2NPs and the subsequent production of ROS were able to initiate
cumulative damage to zebrafish larvae, such as significant mortality,
stunted growth, delayed metamorphosis, malformations, organ pathol-
ogy and DNA damage (Bar‐Ilan et al., 2013, 2012). Another study
showed that phototoxicity of TiO2NPs in zebrafish was life‐stage
dependent. Among the four life stages tested (embryos, yolk‐sac lar-
vae, free‐swimming larvae, juvenile), yolk‐sac larvae were found to
be the most sensitive (Ma & Diamond, 2013).
A simulated solar radiation (SSR) apparatus was used in a number
of TiO2 phototoxicity studies. Faria et al. examined sublethal oxidative
stress effects of three different types of TiO2NPs to zebrafish
embryos without and with SSR. The results evidenced that under
SSR radiation the TiO2NPs P25 influenced antioxidant and oxidative
stress responses. P25 was phototoxic due to the production of ROS
(Faria, Navas, Ráldua, Soares, & Barata, 2014). The toxicity of TiO2NPs
to a fish cell line (BF‐2) and zebrafish embryos under dark and simu-
lated solar light (SSL) exposure conditions were also investigated.
11
The results showed that the oxidative stress‐dependent cytotoxicity
and embryonic toxicity of TiO2NPs were significantly increased upon
exposure to SSL. TiO2NPs generated abiotic ROS during SSL exposure
and exposure to SSL in turn induced the physicochemical transforma-
tion of TiO2NPs (George et al., 2014). The protective ability of Pro‐
NP™ (biodegradable NPs containing the antioxidant enzymes SOD
and catalase) against TiO2NP‐induced photo‐oxidative stress was also
evaluated in zebrafish mode. Co‐treatment TiO2NP with Pro‐NP™ not
only increased zebrafish survival in a dose‐dependent manner but also
improved the percentage of embryos hatching and resulted in normal
growth of zebrafish (Kim, Stees, et al., 2017).
3.3.2 | Toxic effects of titanium dioxide nanoparti-
cles on zebrafish behavior and nervous system
Swimming ability is vital to the survival of fish. Abnormal swimming
ability has a negative impact on predation, migration and reproduction,
reducing the environmental adaptability of fish. Zebrafish embryos
were exposed to TiO2NPs from fertilization to the free swimming
stage. Hatchability, survival and malformation rate of zebrafish were
not affected by TiO2NP exposure. On the contrary, larval swimming
parameters such as average velocity, maximum velocity were signifi-
cantly affected, demonstrating that the behavioral endpoints were
more sensitive than the others were (e.g., hatchability and survival)
(Bar‐Ilan et al., 2012; Chen, Lin, & Tseng, 2011).
TiO2NPs could overcome the blood‐brain barrier and enter into
the brain of fish and inhibit Na+,K+‐ATPase activity, making the brain
a target for TiO2NPs (Chakraborty et al., 2009; Ramsden, Smith, Shaw,
& Handy, 2009). Neurotoxicity in adult zebrafish after subchronic
exposure to TiO2NPs was examined. After low‐dose exposures for
45 days, decreased spatial recognition memory, reduced levels of
neurotransmitters such as norepinephrine, dopamine and 5‐
hydroxytryptamine, histopathological changes in the zebrafish brain,
for example, proliferation of glial cells, neuron apoptosis as well as
changes in gene expression profile were discovered (Sheng et al.,
2016). These results implied that low‐dose exposures of TiO2NPs
may result in brain damage in zebrafish. The relationship between
TiO2NPs and neurodegenerative disease such as Parkinson's disease
were also studied. Some researchers selected zebrafish embryos and
PC12 cell lines to study neurotoxicity of TiO2NPs. The results showed
that TiO2NP accumulation in the brain of zebrafish larvae led to ROS
generation and cell death of the hypothalamus. TiO2NP exposure
could also activate expression of the pink1, parkin, alpha‐syn and uchl1
genes, which are related to the formation of Lewy bodies. Loss of
dopaminergic neurons in vivo and in vitro indicate that TiO2NP expo-
sure may be a significant risk factor for the development of
Parkinson's disease (Hu, Guo, Zhao, & Fu, 2017).
3.3.3 | Toxic effects of titanium dioxide nanoparti-
cles on zebrafish reproductive system
Chronic exposure to low concentrations of TiO2NPs was proved to
be toxic to the zebrafish reproductive system. A reduction in the
12
cumulative number of zebrafish eggs was discovered after 13 weeks
of TiO2NP exposure. It was proposed that TiO2NPs might act on pri-
mary follicles directly and/or subsequently interfere with vitellogenin
synthesis (Wang et al., 2011). Another study showed that zebrafish
could maintain their reproductive behaviors and produce viable
embryos during 1 mg/L exposures to TiO2NPs or bulk TiO2 for
14 days. However, the cumulative production of viable embryos was
lower when exposed to 1.0 mg/L TiO2 (both NPs and bulk) by the
end of the 21‐day recovery period (Ramsden, Henry, & Handy,
2013). The effects of TiO2NPs on zebrafish testis were also investi-
gated. The findings showed that TiO2NPs induced autophagy and
necrosis at higher doses in Sertoli cells and consequently negatively
affected spermatogenic cells and testicular morphology of zebrafish;
for example, mitochondrial degeneration with swelling and cristae loss
(Kotil et al., 2017).
3.3.4 | Genotoxicity of titanium dioxide nanoparti-
cles on zebrafish
The genotoxic and cytotoxic potential of TiO2NPs on fish cells in vitro
has been investigated. Elevations in DNA damage and significantly
increased levels of DNA strand breaks were observed only in combi-
nation with ultraviolet A radiation, implying that genotoxicity was
photo‐dependent (Vevers & Jha, 2008). Another study also showed
that genotoxicity was dose‐ and exposure time‐dependent. DNA
damage induced by TiO2NPs appeared only in the highest concentra-
tion tested (10 mg/L) and only for 14 and 21 days of treatment
(Rocco et al., 2015). Other researchers investigated the gene expres-
sion pattern of zebrafish embryos exposed to TiO2NPs. The results
showed that the TiO2NP treatment could downregulate genes
involved in the circadian rhythm, kinase activity, vesicular transport
and immune response (Jovanovic et al., 2011).
3.3.5 | Interactions between dissolved organic
matter/organic toxicants and titanium dioxide nano-
particles in zebrafish
Interactions between dissolved organic matters/organic toxicants and
NPs in the aquatic environment may modify toxicant bioavailability
and consequently alter the environmental fate and toxicity of the tox-
icant. Recently, interactions between TiO2NPs and organic dissolved
organic matter have been studied using zebrafish as a model (Fang,
Yu, Zhang, & Bao, 2015; López‐Serrano, Muñoz‐Olivas, Sanz, Rainieri,
& Cámara, 2015; Yang et al., 2013). The effects of humic acid (HA) on
the toxicity of TiO2NPs to developing zebrafish were investigated in
the dark and under simulated sunlight illumination. The results indi-
cated that adsorption of HA increased suspension stability and
decreased TiO2NP exposure. TiO2NPs were more toxic in the pres-
ence of HA than applied alone. The lethality of zebrafish was signifi-
cantly increased when exposed to mixtures of HA and TiO2NPs
under simulated sunlight (Yang et al., 2013). Studies also evaluated
the effect of HA and ionic strength (by adding NaCl) on the
BAI AND TANG
aggregation and sedimentation of TiO2NP suspension (Fang, Yu,
et al., 2015). The results showed that the formation of aggregations
and hydrodynamic diameters of TiO2NPs in suspensions were not crit-
ical factors to determine toxicity, but they could accelerate sedimenta-
tion of TiO2NPs in suspensions and partly prevent the induction of
toxicity. Both ionic strength and HA could protect zebrafish from the
oxidative stress of TiO2NP exposure through encouraging sediment
deposition.
Interactions between organic toxicants and TiO2NPs in zebrafish
were also examined (Boran, Boyle, Altinok, Patsiou, & Henry, 2016;
Fang et al., 2016; Jhamtani, Shukla, Sivaperumal, Dahiya, & Agarwal,
2018; Li, Wu, Wang, Xiang, & Zhu, 2018; Miao et al., 2015; Pavagadhi,
Sathishkumar, & Balasubramanian, 2014; Qiang, Pan, Zhu, Fang, &
Tian, 2016; Wang et al., 2014; Yan et al., 2014). Many studies have
paid special attention to investigate the combined toxicological effects
associated with TiO2NPs and bisphenol A (BPA) in zebrafish. Yan et al.
tested the combined toxicity of TiO2NPs and BPA in zebrafish
embryos. The observations reflected that adsorptive interactions
exist between the two chemicals and increased toxicity effects includ-
ing an advanced toxicological effect time, decreased survival,
increased morphological abnormalities and delayed embryo hatching
(Yan et al., 2014). TiO2NPs were carriers for BPA and enhanced its
bioconcentration in zebrafish. BPA and TiO2NPs located in the liver,
brain and gonad tissues of zebrafish, and increased the burdens of
these tissues. Co‐exposure of BPA and TiO2NPs caused a reduction
of sex hormone in plasma concentrations, including estradiol, testos-
terone, follicle‐stimulating hormone and luteinizing hormone
(Fang et al., 2016).
Co‐exposure of TiO2NPs and other organic toxicants including
deca‐BDE (BDE‐209; a polybrominated diphenyl ether congener),
cypermethrin, aldrin, pentachlorophenol (PCP) in zebrafish were also
examined (Fang et al., 2015; Jhamtani et al., 2018; Li et al., 2018;
Wang, Chen, et al., 2014). In these experiments, TiO2NPs were
proved able to increase the uptake of toxicants and accelerate the
bioaccumulation of these toxicants in zebrafish (Li et al., 2018;
Wang, Chen, et al., 2014). Co‐exposure of TiO2NPs and toxicants
enhanced the induction of ROS generation, leading to lipid peroxida-
tion and DNA damage (Fang, Shi, et al., 2015; Jhamtani et al., 2018;
Li et al., 2018; Wang, Chen, et al., 2014). A decrease in locomotor
activity was also observed in zebrafish larvae after co‐exposure
(Li et al., 2018; Wang, Chen, et al., 2014). Co‐exposure of TiO2NPs
and BDE‐209 increased thyroid hormone, thyroxine, content and
upregulated several gene transcriptions such as tsh beta, tg and
dio2 in the hypothalamic‐pituitary‐thyroid axis, leading to thyroid
endocrine disruption. Downregulation of specific genes and proteins
involved in the central nervous system were also observed, implying
that TiO2NPs enhanced BDE‐209 developmental neurotoxicity in
zebrafish (Wang, Chen, et al., 2014). The effects of TiO2NPs on
the toxicity of PCP in zebrafish larvae showed that, unlike other
co‐exposure experiments, PCP content was not increased in the
presence of TiO2NPs, but the metabolism of PCP to
tetrachlorohydroquinone was enhanced in zebrafish larvae (Fang,
Shi, et al., 2015) (Table 4).
BAI AND TANG 13

TABLE 4 Toxic effects of titanium dioxide nanoparticles on zebrafish

Size (nm) Concentration and exposure duration Stage Results References

20.5 ± 6.7 1 mg/L, 48 h Adults Alteration of gene expression Griffitt et al.,

2009
14.10 ± 1.52 10 ppm, 14 days Adults An increase in alpha‐helical structure and a decrease in Palaniappan
beta‐sheet structure in fish gill tissue proteins &
Pramod,
2010
240‐280 0.1 mg/L, 13 weeks Adults Mortality, organ injury, behavior alteration and organ Chen, Dong,
distribution at higher concentration et al.,
2011
20‐70 0, 10, 50, 100, 150, 200 and 300 mg/L, 96 h Adults Acute toxicity, extracellular ·OH generation and oxidative Xiong et al.,
damage on the cell membranes of gill tissue 2011
20 10 ppm, 14 days Adults Alterations on the major biochemical constituents such as Palaniappan
proteins, lipids and nucleic acids in the brain tissues &
Pramod,
2011
27.74 ± 4.09 0.1, 0.5, 1, 5, 10 mg/L for the embryos 1 mg/L for Embryos No effect on hatchability, survival, and malformation rate Chen, Lin, &
larvae Examined every 12 h until 120 hpf for and but larval swimming parameters, including average and Tseng,
the embryos, 5 dpf for larvae larvae maximum velocity and activity level were affected 2011
24.1 ± 2.8 1 mg/L, 14 days Adults Limited oxidative stress and organ pathology but lower Ramsden
cumulative number of viable embryos produced et al.,
2013
5‐10, 21 0.01‐10 000 ng/mL 0‐23 dpf Embryos Significant mortality at 1 ng/mL, stunted growth, delayed Bar‐Ilan
and metamorphosis, malformations, organ pathology and et al.,
larvae DNA damage 2013
21 1 mg/L, 0‐72 hpf Embryos No delayed embryonic development or retinal neurotoxicity Wang, He,
et al.,
2014
21 1 and 10 μg/L, 5, 7, 14, 21 and 28 days Adults DNA damage at 10 μg/L after 14 and 21 days exposure Rocco et al.,
2015
6.5 5, 10, 20 and 40 μg/L, 45 consecutive days Adults Brain injury, reductions of spatial recognition memory Sheng et al.,
2016
<150 1, 2 and 4 mg/L, 5 days Adults Autophagy and necrosis at higher doses in Sertoli cells, Kotil et al.,
spermatogenic cells and testicular morphology alteration 2017

3.4 | Copper and copper oxide
CuNPs are commonly used as gel propellants, combustion active
agents, catalysts, adsorbents for water purification, sintering active
agents, petroleum lubricants and other consumer products of the
pharmaceutical industry (Adeleye, Oranu, Tao, & Keller, 2016;
Dankovich & Smith, 2014; Lee et al., 2016). Studies have been per-
formed to reveal the toxic effects of CuNPs in zebrafish. It was
reported that CuNPs could cause acute toxicity in zebrafish with a
48‐hour LC50 value of 1.5 mg/L. The gill was found to be the primary
target organ for CuNPs with different gill morphologies and global
gene expression patterns generated by CuNP exposure (Griffitt
et al., 2009, 2007). CuNP exposure could also lead to cumulative mor-
talities in zebrafish with LC50 values of about 3 mg/L (Kovrižnych et al.,
2013). The analysis of the potential toxicity of CuNP exposure on
zebrafish embryos showed that CuNPs generated ROS in a
concentration‐dependent manner (Denluck, Wu, Crandon, Harper, &
Harper, 2018). CuNPs delayed embryo hatching time and caused ter-
atogenicity of the larvae. CuNP exposure caused mortality in zebrafish
embryos in a dose‐dependent manner with the gastrula‐stage
zebrafish embryos being killed by CuNPs at a higher concentration
(Bai, Tian, et al., 2010). The toxicity of CuNPs on the lateral line sys-
tem and behavior in zebrafish larvae were examined. CuNP exposure
reduced the number of lateral line neuromasts modestly, but even
low concentrations of CuNPs could significantly reduce the rheotaxis
behavior of zebrafish (McNeil, Boyle, Henry, Handy, & Sloman,
2014). CuNPs were also shown to inhibit the specification and forma-
tion of three layers of the swim bladder in zebrafish and impair its
inflation in a stage‐specific manner by downregulating Wnt signaling
in embryos. Wnt agonist 6‐bromoindirubin‐3′‐oxime was found to
neutralize the inhibiting effects of CuNPs on zebrafish swim bladder
development (Xu et al., 2017).
The molecular roles of CuNPs and dissolved Cu2+ ions from CuNPs
during zebrafish embryogenesis and the potential mechanisms were
also investigated. The toxicity of CuNP is either its own role or the
release of Cu2+ ions, are still controversial. Bai et al. found that CuNPs
had a similar effect on zebrafish embryos as Cu2+ (Bai, Tian, et al.,
2010). Some other studies compared CuNPs with soluble Cu2+ ions
14 BAI AND TANG

in zebrafish and revealed that the main toxic effects on zebrafish
embryos were from the particle form of Cu (CuNPs) rather than ions
(Griffitt et al., 2009; Hua, Vijver, Ahmad, Richardson, & Peijnenburg,
2014). In contrast, Song et al. assessed the acute toxicity of spherical
50 nm CuNPs in zebrafish and discovered that soluble Cu2+ ions
were the main factors of CuNP toxicities (Song, Vijver, Peijnenburg,
Galloway, & Tyler, 2015).
CuONPs are extensively used in a variety of applications such as
high‐temperature superconductors, antimicrobial agent, batteries,
agricultural biocides, photocatalyst, energy transfer fluids and gas
sensors (Batley, Kirby, & McLaughlin, 2013; Hou, Wang, Hayat, &
Wang, 2017; Kim, Lee, & Lee, 2012; Llorens, Lloret, Picouet,
Trbojevich, & Fernandez, 2012). The expanding production and wide-
spread utilization of CuONPs may pose risks to individual organisms
and ecosystems.
Analysis of the potential toxicity of CuONPs in zebrafish embryos
and larvae (Bai, Tian, et al., 2010) revealed that CuONPs interfered
with embryo hatching in a dose‐dependent manner and caused
increased expression of the heat shock protein 70 in zebrafish larvae
when a higher concentration was used (Lin et al., 2011). The develop-
mental toxicity of CuONPs was studied. After studying the sublethal
acute exposure effects on the developing zebrafish embryos, it was
proved that CuONP exposure could cause oxidative stress followed
by apoptosis and developmental anomalies in zebrafish embryos
(Ganesan, Anaimalai, Raghunath, Vijayakumar, & Perumal, 2016).
Transgenic zebrafish Tg(nacre/fli1:EGFP) was used to investigate the
effects of CuONP exposure on vasculogenesis/angiogenesis (Chang
et al., 2015). The results demonstrated that CuONPs inhibit
vasculogenesis via reduction of vascular endothelial growth factor
expression and induction of apoptosis. Studies also investigated the
effect of CuONPs on zebrafish liver development and neuronal differ-
entiation. The results reflected that short‐term exposure to CuONPs
at high doses led to hepatotoxicity and neurotoxicity in zebrafish
embryos and larvae, displaying as hepatic hypoplasia and delayed ret-
inal neurodifferentiation coupled with reduced locomotor ability (Sun
et al., 2016). Further studies from other groups revealed that CuONP
exposure could elicit abnormal phenotypes of a smaller head and
smaller eyes, affect dorsoventral patterning, disturb cell migration of
gastrulation, decrease the sizes of several structures in the neural sys-
tem and prevent looping of the heart tube during cardiogenesis (Xu, J.
Zhang, Q. Li, X. et al., 2017). Some studies have found that the toxicity
of CuONP exposure on zebrafish is due to Cu2+ ions released by
CuONPs (Lin et al., 2011). While others have found that CuONP tox-
icities could not solely be explained by dissolution of CuONPs into the
exposure medium, CuONP particles themselves may play a more
important role (Muller, Lin, & Nisbet, 2015; Thit, Skjolding, Selck, &
Sturve, 2017; Vicario‐Pares et al., 2018) (Table 5).
3.5 | Zinc oxide
The ZnONPs, due to their antimicrobial property and other special
properties such as high isoelectric point, transparency, photocatalytic
efficiency and biocompatibility, are commonly used in sunscreens, cos-
metics, electrical appliances, photonics and ceramics (Mirzaei &
Darroudi, 2017). Exposures of ZnONPs generated toxic effects on

TABLE 5 Toxic effects of CuNPs and CuONPs on zebrafish

NP Concentration and
type Size (nm) exposure duration Stage Results References

Cu 80 0.25–1.5 mg/L, 48 h Adults Biochemical, histopathological changes, and Griffitt et al., 2007
alterations in gene expression
Cu 26.7 ± 7.1 100 μg/L,0, 2, 24, 48 h Adults Nanocopper increased mean gill filament width by Griffitt et al., 2009
three‐ to fourfold between 24 and 48 h and
produced a distinct gene expression profile at
both 24 and 48 h
Cu 20 ± 9 50 and 225 μg/L,exposed for 4 h at Embryos A reduction in the number of lateral line neuromasts McNeil et al., 2014
96 hpf and performance of positive rheotaxis
Cu 25, 50, 0.25, 0.5, 1, 2, 4 and 8 mg/L, from 24 Embryos CuNP exposure inhibited hatching, altered Hua, Vijver, Ahmad,
100 to 120 hpf behavioral responses and increased the incidence et al., 2014
of malformations
Cu 40‐60 0.25 mg/L, 24, 36, 60, 96, 120 hpf Embryos CuNP impaired zebrafish swimbladder development Xu, Zhang, Zhang, et al.,
by downregulating Wnt signaling 2017
CuO 51 5, 20, 40, 60 and 80 ppm, 48 hpf Embryos Induction of oxidative stress, developmental Ganesan et al., 2016
anomalies and apoptosis in the exposed embryos
CuO 50‐60 1, 6.25, 12.5, 25 and 50 mg/L Embryos and CuONP exposure at high doses (≥12.5 mg/L) Sun et al., 2016
larvae activates xenobiotics‐metabolizing enzymes,
induces an inflammatory response and shows
developmental toxicity on the zebrafish liver and
central nervous system
CuO 40 100 mg/L, 5 days Embryos Reduction of the number of subintestinal vessels Chang et al., 2015
and the expression of vegfAa, vegfr1
BAI AND TANG 15

zebrafish embryos and larvae, including retarded hatching, tissue
damage, tail malformations, reduction in body length of the larvae at
lower concentrations and embryonic mortality at higher concentra-
tions (Bai et al., 2010; Kteeba et al., 2017; Zhu, Wang, Zhang, Chang, &
Chen, 2009; Zhu et al., 2008). Particle shape and surface coating
appear to be important factors responsible for the observed toxicity
of ZnONPs. In one study, the toxicity of differently shaped ZnONPs,
such as nanospheres and nanosticks, exposed to zebrafish embryos
were assessed. The ZnO nanosticks were found to be more toxic
than any other NP forms with respect to hatching retardation and
mortality endpoints (Hua, Vijver, Richardson, Ahmad, & Peijnenburg,
2014). The toxic effects of ZnONPs, ZnONPs coated with chitosan
(ZnO‐CTS) and ZnONPs coated with polyethylene glycol (ZnO‐PEG)
on zebrafish embryos were also examined (Girigoswami, Viswanathan,
Murugesan, & Girigoswami, 2015). Compared with ZnONP‐treated
ones, ZnO‐CTS and ZnO‐PEG NPs showed reduced deposition atop
the zebrafish eggs and ZnO‐CTS NPs also led to a higher survival rate
of zebrafish embryos. Another study also confirmed that surface‐
modified ZnONPs, no matter what the type was, caused higher
mortality compared with uncoated particles (Zhou, Son, Harper, Zhou,
& Harper, 2015).
Other factors affecting the toxicity of ZnONPs are size and
agglomeration of the particles. Comparison of bulk and nano‐sized
ZnONP toxicities on developing zebrafish embryos and larvae were
conducted. The results showed that exposure of ZnONPs produced
the same toxic effects on zebrafish embryos and larvae as their bulk
counterparts did and ZnONP aggregates were proved to exert toxicity
in a dose‐dependent manner (Xiong et al., 2011; Zhu et al., 2008).
Zn2+ ions released from the NP are considered to influence the
toxic effects of ZnONPs but their roles are still debatable. Some
researchers declared that Zn2+ ions were only partially responsible
for the ZnONP toxicities, the main toxic contribution was due to
ZnONPs themselves (Bai, Zhang, et al., 2010; Chen, Lin, & Meng,
2014; Hua, Vijver, Richardson, et al., 2014; Kim, Choi, Kim, & Kim,
2017; Kteeba et al., 2017; Ong et al., 2014; Yu, Fang, Xiong, Zhu, &
Sima, 2011; Zhao, Wang, Wu, You, & Lv, 2013; Zhu et al., 2009). On
the contrary, others drew the conclusion that the toxic effects of
ZnONPs were primarily related to the release of Zn2+ (Boran & Ulutas,
2016; Brun et al., 2014). More studies are still needed to draw a gen-
eralized conclusion.
ROS generated by ZnONP exposure on zebrafish is thought to be
another potential explanation of the toxic effects. Xiong et al. studied
the oxidative stress effects and damage of ZnONPs on gill, intestine
and liver of zebrafish, and found that liver tissues were the main target
of oxidative damage. Oxidative stress affected gill and intestine but
caused no oxidative damage on gill and intestine. Further study
showed that ZnONP exposure generated higher ·OH radicals. The
malondialdehyde content was elevated in zebrafish gills and
livers, which was one of the biomarkers of oxidative effects (Xiong
et al., 2011). ZnONPs were also found to generate developmental
toxicity, oxidative stress and DNA damage on zebrafish embryos
(Zhao et al., 2013). ROS generation and DNA damage were highly

TABLE 6 Toxic effects of ZnONPs on zebrafish

Concentration and exposure

Size (nm) duration Stage Results References

50‐70 0.1, 0.5, 1, 5, 10, 50 mg/L Embryos Developmental toxicity, hatching retardation and mortality Zhu et al., 2008
and
larvae
30 1, 5, 10, 25, 50 and 100 mg/L, Embryos Nano‐ZnO killed zebrafish embryos (50 and 100 mg/L), retarded the Bai, Zhang, et al.,
8,24, 32, 48‐96 hpf embryo hatching (1–25 mg/L), reduced the body length of larvae, 2010
and caused tail malformation after the 96 hpf exposure
30 2, 5, 10, 30 and 50 mg/L Adults Acute toxicity, oxidative stress and oxidative damage Xiong et al., 2011
50‐100 1, 5, 10, 20, 50 and 100 mg/L, 0‐ Embryos Developmental toxicity, oxidative stress and DNA damage Zhao et al., 2013
144 hpf and
larvae
20‐30 24‐120 hpf Embryos 20‐nm spherical ZnONPs causes a higher mortality and Niyaghi, Haapala,
malformations when compared with the formulation without NPs Harper, &
Weismiller, 2014
79.41 ± 6.17 0.1, 0.5, 1, 5 and 10 mg/L, 0‐ Embryos Developmental and behavioral toxicity Chen et al., 2014
144 hpf and
larvae
20‐30 0.01, 0.1, 1 and 10 mg/L, 24, 48, Embryos ZnONPs may affect genes related to inflammation and the immune Choi et al., 2016
72 and 96 hpf system, resulting in yolk‐sac edema and pericardia edema in
embryonic/larval developmental stages
<100 10, 30, 60, 90 and 120 mg/L, Embryos Nano‐ZnO induce an excessive production of ROS, activate the Zhao et al., 2016
96 hpf apoptosis pathway mediated by mitochondria and caspases
10‐30 0.153, 0.307, 1.228, 2.457, 4.914, Larva ZnONPs slightly induced cell differentiation and pathways Kim, Choi, et al.,
9.827, 12.284 and associated with the immune system and activated several key 2017
122.838 μmol/L, 72 h genes involved in cancer cell signaling
16
increased on ZnONP exposure in a dose‐dependent manner. Genes
related to oxidative damage, ROS production and DNA damage, such
as Bcl‐2, Nqo1 and Gstp2, were highly downregulated on ZnONP
exposure. Another study showed that zebrafish embryos exposed to
ZnONPs increased ROS production, followed by p53 upregulation,
bcl‐2/bax ratio reduction, mitochondrial membrane potential
reduction, the release of cytochrome c into the cytosolic fraction
and activation of caspases 9 and 3, implying that ZnONPs induced
an higher ROS production, which then activated the apoptosis
pathway regulated by mitochondria and caspases (Zhao, Ren, Zhu,
Luo, & Ren, 2016) (Table 6).
3.6 | Nickel and nickel oxide
NiNPs have recently been adopted in a broad range of applications
including catalytic sensing, electrochemical supercapacitors and elec-
tronics (Imran Din & Rani, 2016; Kong, Hu, Lu, Cheng, & Tang,
2019). Exposing zebrafish embryos to NiNPs demonstrated that
particle shape appeared to play an important role in NiNP toxicities
as aggregated particle clusters with a dendritic structure were more
toxic than all three NiNPs at 30, 60 and 100 nm. The toxicity of NiNPs
was less than or equal to that of soluble nickel. It was proposed that
gut was the major target for NiNP exposure. NiNP exposure to
zebrafish also caused skeletal muscle abnormalities and head defects
(Ispas et al., 2009). Other research showed that NiNPs caused cumu-
lative mortality in zebrafish (Kovrižnych et al., 2013). Some studies
also investigated NiNP toxicity in zebrafish larvae to clarify that
toxic contribution was due to NiNPs themselves or Ni 2+
ions released
from NPs. The results revealed that the toxicity of NiNPs in zebrafish
larvae was primarily attributable to Ni 2+
(Boran & Saffak, 2018). The
toxicity of NiONPs to adult zebrafish was examined. The results
showed that the acute toxicity of NiONPs was low but chronic expo-
sure of NiONPs could lead to the accumulation and increase in toxicity
in zebrafish tissue (Kovrižnych, Sotníková, Zeljenková, Rollerová, &
Szabová 2014).
3.7 | Platinum
Exposing zebrafish embryos to PtNPs led to retarded hatching as well
as a dose‐dependent drop in heart rate and axis curvatures. The accu-
mulation of PtNPs in embryos was found at lower concentrations
compared with AgNPs and AuNPs, probably due to their small size
resulting in little retention in the embryos (Asharani et al., 2011).
Another study confirmed that PtNPs coated with Bacopa monnieri
phytochemicals had a protective ability in 1‐methyl‐4‐phenyl‐1,2,3,6‐
tetrahydropyridine‐induced neurotoxicity in the zebrafish model of
Parkinson's disease via its dual functions as mitochondrial complex I
and antioxidant activity (SOD and catalase mimic activities) (Nellore,
Pauline, & Amarnath, 2013).
BAI AND TANG
3.8 | Magnesium oxide
MgONPs are used in anticancer therapy, medicine, manufacturing and
antibacterial agents in the food industries. The widespread use of
these NPs in our daily lives results in the inevitable release and envi-
ronmental exposure. MgONPs caused cumulative mortality in
zebrafish (Kovrižnych et al., 2013). Further research showed that
MgONP‐induced cellular apoptosis and ROS at different concentra-
tions. The hatching rate and survival rate of embryos decreased in a
dose‐dependent manner and different types of malformation were
also observed in exposed embryos (Ghobadian, Nabiuni, Parivar, Fathi,
& Pazooki, 2015).
3.9 | Iron oxide
IONPs are being utilized in biomedical applications such as cell label-
ing, targeting drug delivery, gene delivery and magnetic resonance
imaging (Caro et al., 2019). The toxic effects of IONPs during the early
life stages of the zebrafish were studied. The results showed that
≥10 mg/L of IONPs instigated developmental toxicity in these
embryos, causing mortality, hatching delay and malformation (Zhu,
Tian, & Cai, 2012). The toxicity of dextran‐coated IONPs to zebrafish
brain was researched. The animals exposed to 200 mg/kg and tested
24 hours after administration of the NPs have shown decreased ace-
tylcholinesterase activity, reduction in the exploratory performance,
significantly higher levels of ferric iron in the brains and induction of
casp8, casp9 and jun genes. Taken together, these findings suggest
acute brain toxicity by the inhibition of acetylcholinesterase and
induction of apoptosis (de Oliveira et al., 2014). The accumulation
and elimination profiles of two FeO nanomaterials (nano‐Fe2O3 and
nano‐Fe3O4) in zebrafish were investigated. The results indicated that
the two kinds of FeO nanomaterials can be accumulated in zebrafish,
and can be cleared with a high elimination rate by 24 days post‐
exposure. The FeO nanomaterials entering zebrafish may be adsorbed
within the gastrointestinal tract, and reserved for more than 12 days
(Zhang, Zhu, Zhou, & Chen, 2015). The acute toxicity (after 96 hours
exposure) of IONPs in adult zebrafish were investigated using differ-
ent endpoints. The results demonstrated that DNA damage in erythro-
cytes was observed for all IONP concentrations (ranging from 4.7 to
74.4 mg/L). Only the highest IONP concentrations (37.2 and
74.4 mg/L) caused oxidative stress in liver cells. Liver microarray
analysis revealed almost 1000 DETs between the control and IONP
treatment groups (37.2 and 74.4 mg/L). Genes in cluster 1
(overexpressed in treated groups) were related to ion binding. Genes
in cluster 2 (downexpressed in treated groups) were associated with
ribosomes and translation (Villacis et al., 2017).
3.10 | Aluminum and aluminum oxide
Aluminum nanoparticules (AlNPs) and Al2O3NPs have been widely
applied in the electronics and optoelectronics industry, drug
delivery systems and biomedical products. Al2O3NPs and Al2O3 bulk
BAI AND TANG 17

TABLE 7 Toxic effects of selected NPs on zebrafish

Concentration and exposure

NP type Size (nm) duration Stage Results References

α‐Fe2O3 30 0.1, 0.5, 1, 5, 10, 50, 100 mg/L, 6, Embryos ≥10 mg/L of iron oxide Zhu et al., 2012
12, 24, 36, 48, 60, 72,84, 96, 120, nanoparticles cause mortality,
144 and 168 hpf hatching delay and malformation
γ‐Fe2O3 5.7 4.7, 9.3, 18.6, 37.2 and 74.4 mg/L, Adults Genotoxicity Villacis et al., 2017
96 h
NiO <50 6.25, 12.5, 25, 50, 100 mg/L, Adults 30‐day LC50 value was 45.0 mg/L, Kovrižnych et al., 2014
30 days LC100 was 100.0 mg/L, and LC0
was 6.25 mg/L
MgO 20 50, 100, 200 and 400 mg/L, 4‐ Embryos and larvae MgONPs induced cellular apoptosis Ghobadian et al., 2015
144 hpf and intracellular reactive oxygen
species. Hatching rate and survival
of embryos decreased in a dose‐
dependent manner
Al 51 0.5, 2.5, 12.5mg/L, 48 h Adults Decreased Na+,K+‐ATPase activity in Griffitt et al., 2011
gills and changes in expression of
two genes

showed no toxicity to zebrafish embryos and larvae (Griffitt et al.,
2008; Zhu et al., 2008). AlNP has little acute toxicity for zebrafish
(Griffitt et al., 2011) (Table 7).
4 | F A C TO R S I N F L U E NC I N G
NANOTOXICOLOGICAL EFFECTS
There are numerous factors that can affect the toxicity of NPs,
such as chemical composition, particle size, shape, surface coating/
modification, solubility characteristics and other physical and chemical
properties (Truong et al., 2019; Liu, Dumitrescu, et al., 2019). (Figure 4).
Single AgNPs (30‐72 nm) passively entered zebrafish embryos through
chorionic pores via random Brownian motion. At the same molar con-
centration, the larger AgNPs were shown to be more toxic than the
smaller ones (Lee, Browning, et al., 2012; Lee et al., 2007). In contrast,
Bar‐Ilan et al. found that AgNP toxicity was size‐dependent at certain
concentrations and time points with smaller NPs being most toxic
(Bar‐Ilan et al., 2009). These contradictory results of particle size in
zebrafish might be explained by distinct NPs used. Therefore, size is
seemed not as important for toxicity as the chemical composition of
the NPs used.

4.3 | Particle shape

4.1 | Chemical composition
Chemical composition is critical for determining the toxic effects of
NPs. For example, comparison of AgNPs and AuNPs revealed that
AgNPs caused 100% mortality while AuNPs caused only 3% mortality
under the same experimental conditions (Bar‐Ilan et al., 2009). AgNPs
in sublethal concentrations also generated a variety of developmental
defects, including smaller heads and caudal fins, larger yolk sac and
larger eyes while AuNPs only exhibited a small sublethal toxic effect
(Asharani et al., 2011; Bar‐Ilan et al., 2009).
Shape is one of the properties of NPs and an important factor respon-
sible for NP toxicity. NiNP exposure to zebrafish embryos showed
that particle shape played a key role in NiNP toxicity. Aggregated par-
ticle clusters with a dendritic structure were proved more toxic than
30, 60 and 100 nm NiNPs (Ispas et al., 2009). The particle shape of
AuNPs was also proved a critical factor influencing AuNP toxicity.

4.2 | Particle size

Size‐dependent toxicity was observed for many kinds of metal and

metal oxide NPs in zebrafish. TiO2NP exposure in zebrafish embryos
showed that the 120‐hour LC50 of 5.7 and 12.4 nm TiO2NPs was
23.4 and 610 mg/L respectively, but for 15 nm TiO2NPs the value
was 1000 mg/L (Kim et al., 2014). Particle size was also shown to be
one of the factors affecting AgNP toxicity but its effects were diamet-
rically opposed in different studies. Lee et al. observed size‐dependent FIGURE 4 Schematic diagram of factors influencing
toxic effects of the AgNPs on zebrafish embryonic development. nanotoxicological effects. NP, nanoparticle
18
Research on the toxicity and biodistribution of AuNPs with different
shapes (sphere, rod and star) in adult zebrafish revealed that rod
AuNPs showed increased uptake and clearance while star AuNPs
exhibited prolong sequestration compared with sphere or rod particles
(Sangabathuni et al., 2017).
4.4 | Surface coating/modification
The surface properties of NPs are the significant determinant for their
toxic potential. Surface modifications can give rise to surface
functionalization and affect their toxicity. For example, research on
the toxicity of ZnONPs, ZnO‐CTS and ZnO‐PEG in zebrafish embryos
showed that compared with ZnONP‐treated ones, ZnO‐CTS and ZnO‐
PEG NPs showed reduced deposition on the top of the zebrafish eggs,
and ZnO‐CTS NPs resulted in a higher survival rate of zebrafish
embryos (Girigoswami et al., 2015). Another study also confirmed that
surface‐modified ZnONPs, regardless of what the type was, led to
higher mortality than uncoated particles (Zhou et al., 2015).
4.5 | Effects of ionic solutes
The metal and metal oxide NPs, which release ions in physiological
fluids, can also influence the toxic potential. The exposure of
these NPs is actually a mixture of nanomaterials and metal ions
(Cunningham, Brennan‐Fournet, Ledwith, Byrnes, & Joshi, 2013;
Griffitt et al., 2009). Ispas et al. compared NiNP with soluble Ni in
zebrafish and found out that the gut was the major target, and intes-
tinal defects only occurred with NiNP exposure (Ispas et al., 2009).
On the contrary, another study revealed that NiNP toxicity in
zebrafish larvae was primarily attributable to Ni2+ ions released from
NPs (Boran & Saffak, 2018). Considerably more work will need to be
done to determine whether the toxic effects of NPs on zebrafish are
their own or the release of ions.
5 | CONCLUSION AND PROSPECTIVE
The reviewed findings suggest that metal and metal oxide NPs have
potential developmental, immunological, cardiovascular, reproductive,
neural and liver toxicity in zebrafish. The data also show that the
chemical composition, particle size, shape, surface coating and modifi-
cation of NPs are all determinants that may give rise to toxicity. Sev-
eral studies have indicated that oxidative stress and inflammation
may be the underlying mechanisms for the toxicity of metal and metal
oxide NPs in zebrafish (Torrealba et al., 2019). More data are still
needed to study the in vivo toxicity of metal and metal oxide NPs
because of their wide variety.
In modern society, NPs are used intensively in many applications
and are becoming a common part of daily life. Meanwhile, their toxic-
ity to organisms, particularly to human beings cannot be neglected and
it is necessary to develop effective toxicity testing models for these
NPs. Zebrafish is a relatively “young” model organism, but in the last
10 years it has played an important role in nanotoxicology research
BAI AND TANG
(Kaur, Khatri, & Puri, 2019). Zebrafish represent a prominent verte-
brate model for toxicological studies due to many features, including
small size, simple husbandry requirements, high fecundity and short
generation cycle. The use of genetically modified zebrafish for
nanotoxicology research is faster, more sensitive and more convenient
than wild‐type zebrafish. A variety of transgenic zebrafish eggs with
different response elements can be arranged in a microplate like a
gene chip, which can accurately detect different types of NPs in the
water environment, and can estimate the concentration of these
NPs according to the fluorescence intensity.
Zebrafish has 25 pairs of chromosomes, and its more than 30 000
genes are highly homologous to human genes (Kettleborough et al.,
2013). Many of these genes have been cloned and found to be func-
tionally similar with their human counterparts and belong to
orthologous genes. The main developmental processes and physiolog-
ical functions of zebrafish are also well conserved. The key systems
and organs of zebrafish, such as the immune, digestive and nervous
systems, are also similar to that of humans. Multiple mutants of
zebrafish have also been shown to be consistent with human disease
phenotypes (Noyes, Garcia, & Tanguay, 2016). The toxic effects of
zebrafish and mammals after exposure to toxicants are basically con-
sistent with the signaling pathways, indicating that zebrafish are an
effective tool for predicting the toxicity of chemical toxicants. At pres-
ent, zebrafish has been widely used in the research of genetic and
developmental toxicology, environmental toxicology, pathological tox-
icology and other toxicology fields.
The application of metal and metal oxide NPs in various fields is
gradually increasing, but the harmfulness to the human body and the
environment and their toxicological mechanisms are not fully under-
stood. In recent years, a series of studies have been carried out on the
toxic effects induced by metal and metal oxide NPs in zebrafish, partic-
ularly for AgNPs, TiO2NPs, ZnONPs, AuNPs and CuNPs. The existing
results show that metal and metal oxide NPs can translocate among
organs and penetrate the blood‐brain barrier, accumulating in many
organs such as gill, liver, heart and brain, and bringing damage to multi-
ple systems of the zebrafish, including the digestive, reproductive and
nervous systems (van Pomeren, Peijnenburg, Vlieg, van Noort, & Vijver,
2019). Most studies on the toxicity of metal and metal oxide NPs in
zebrafish are focused on acute toxicity, and chronic toxicity studies
are conducted relatively less. However, in most cases the concentration
of metals and metal oxide NPs in the real natural environment (particu-
larly in the water environment) is quite low, far from high‐dose condi-
tions that acute toxicity tests required. Therefore, in zebrafish, more
chronic toxicity studies of metal and metal oxide NPs are needed.
The physical and chemical properties of metal and metal oxide NPs
may also have toxic effects. A large number of experiments in
zebrafish have focused on these factors influencing toxic effects,
including chemical composition, particle size, shape, surface
coating/modification and solubility characteristics, and have achieved
many results (Bar‐Ilan et al., 2009). However, the experimental results
are still insufficient and some conclusions are contradictory; for
example, the influence of particle size and effects of ionic solutes.
Considerably more work will need to be done to determine the toxic
BAI AND TANG
effects of physical and chemical properties of metal and metal oxide
NPs on zebrafish.
Nanotoxicology is different from traditional toxicology in the study
of chemicals, which need to consider more practical significance, such
as the toxic effects of NPs and other pollutants in the mixed state or
interactions with other NPs, because this is more in line with NP
exposure under natural conditions. Co‐exposure of pollutants such as
persistent organic pollutants, perfluoro compounds, polycyclic aromatic
hydrocarbons and pesticides, and metal and metal oxide NPs at
biochemical and molecular levels in zebrafish has been carried out
(Guo et al., 2019). However, there is still no systematic understanding
of the transport, distribution and deeper toxicological mechanisms of
complex pollutants in zebrafish. In addition, the current research on
the toxic effects of metal and metal oxide NPs in zebrafish mainly
focuses on the analysis of pathological research such as body distribu-
tion, cell damage and organ damage. The studies on toxic mechanisms
of metal and metal oxide NPs in zebrafish remain insufficient. It is nec-
essary to explore the mechanism of the toxic effects of metal and metal
oxides in zebrafish as soon as possible, which will promote the further
application and development of metal and metal oxide NPs and lay
the foundation for the establishment of the risk assessment of metal
and metal oxide NP products. In recent years, the high‐throughput
screening assays and omics have been exploited for NP toxicity studies,
in particular, the applications of transcriptome sequencing, proteomics
and metabolomics technology have been rapidly developed, which will
be more conducive to study the toxic biomarkers and the underlying
mechanisms of NPs in zebrafish (Huang et al., 2019).
ACKNOWLEDGMENTS
This work was Supported by National Natural Science Funds of China
(nos 31671034, 21876026, 81473003, 81573186, 81673218,
81502783); Fundamental Research Funds for the Central Universities
(grant no. 2242019K40220); Provincial Natural Science Funds of
Jiangsu (grant/award no.: BK20180371).
CONF LICT OF INT E RE ST
The authors declare no conflict of interest.
ORCID
Changcun Bai https://orcid.org/0000-0003-4650-4741
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How to cite this article: Bai C, Tang M. Toxicological study of
metal and metal oxide nanoparticles in zebrafish. J Appl Toxicol.
2019;1–27. https://doi.org/10.1002/jat.3910