http://dx.doi.org/10.5772/intechopen.74834

**Abstract**

**1. Introduction**

#### **Chapter 7 Provisional chapter**

#### **Zebrafish or** *Danio rerio***: A New Model in Nanotoxicology Study Zebrafish or** *Danio rerio***: A New Model in Nanotoxicology Study**

Maria Violetta Brundo and Antonio Salvaggio Maria Violetta Brundo and Antonio Salvaggio

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74834

#### **Abstract**

Nanotoxicology represents a new research area in toxicology that allows to evaluate the toxicological properties of nanoparticles in order to determine whether and to what extent they represent an environmental threat. Behavior, fate, transport, and toxicity of nanoparticles are influenced to their particular properties and of several environmental factors. The mechanisms underlying the toxicity of nanomaterials have recently been studied specially in aquatic organisms. In particular, in recent years, the use of *Danio rerio* or zebrafish as an animal model system for nanoparticle toxicity assay increased exponentially. In this review, we compare the recent researches employing zebrafish, adults or embryos, for different nanoparticles' toxicity assessment.

DOI: 10.5772/intechopen.74834

**Keywords:** *Danio rerio*, nanoparticles, nanomaterials, biomarkers, ZFET

### **1. Introduction**

Nanotechnology has advanced exponentially over the past decade, and nanoscale materials are being exploited in several applications [1]. Between 2011 and 2015, there has been a 30-fold increase in the production of nanoproducts [2].

Engineered nanodevices are finding a new range of applications for the possibility of modifications of their shape, size, surface, and chemical properties. These characteristics are not present in their bulk counterparts [1]. For example, they have very high specific surface areas that give rise to enhanced reactivity and solubility, reduced melting and sintering temperatures, as well as altered crystal structures [1].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nowadays, we are using a wide variety of commercially available nanoparticles [1]. Metal and carbon nanoparticles (NPs) represent the largest and fastest growing group of NPs [2].

Ballard [27] coined the term "pharyngula" (24–48 h) to refer to the embryo that has developed to the phylotypic stage, when it possesses the classic vertebrate bauplan. This is the time of development when one can most readily compare the morphologies of embryos of diverse

Zebrafish or *Danio rerio*: A New Model in Nanotoxicology Study

http://dx.doi.org/10.5772/intechopen.74834

123

During hatching period (48–72 h), they are called "embryos" until the end of the third day and

There are several advantages for using zebrafish as a model species in nanotoxicological studies. Main benefit regards its size. Zebrafish adult is approximately 5 cm long, so it can be

The tiny size of the larval and adult zebrafish allows to reduce quantities the dosing of experimental solutions and thereby creates limited volumes of waste to disposal and minimizes

Small embryos allow reasonable sample sizes to be tested together using a multiwell plate or series of Petri dishes to provide several experimental replicates at one time. From the egg stage, zebrafish embryos can survive for several days through the absorption of yolk and can

The rapid maturation of zebrafish (sexual maturation occurs around 100 days) allows easy experimentation for transgenerational endpoints required for mutagenesis screening and

This species shows high fecundity (single female can lay up to 200 eggs per week) and transparent embryos. The eggs hatch rapidly and organogenesis occurs quickly. As a result, the

Zebrafish eggs remain transparent from fertilization to when the tissues become dense and pigmentation is initiated (at approximately 30–72 h post fertilization (hpf)); this allows unobstructed observations of the main morphological changes up to and beyond pharyngulation. Therefore, using little magnification, adverse effects of chemical exposure on development of the brain, notochord, heart, jaw, trunk segmentation, and measurements of size can be

Their optical clarity allows for identification of phenotypic traits during mutagenesis screening and assessment of endpoints of toxicity during toxicity testing. This proves even more valuable when immunochemistry (IHC) techniques are used. There are a vast amount of immunohistochemical markers available, allowing assessments of aberrant morphology or activation of certain signaling pathways by toxicants through the staining of specific tissues

The zebrafish research community has developed a range of resources very useful to the toxicologists, including mutant strains, cDNA clone collections, and whole genome that has been sequenced a few years ago. Highly conserved signaling pathways are found both in zebrafish

and mammals with a high level of genomic homology [28].

major organs are developed within few days post fertilization (dpf) in larvae.

handled without any difficulty and reduces housing space and husbandry costs.

afterward "larvae," whether they have hatched or not [25].

quantities of lab ware and chemicals.

be visually assessed for malformation [26].

assessing chemicals for teratogenicity.

assessed quantitatively.

and cells types.

vertebrates.

Hence, environmental contamination is already occurring and is predicted to increase dramatically. This growth of nanotechnology has not advanced without concerns regarding their potential adverse environmental impacts. Several studies of nanotoxicology have been made in fact to evaluate the toxicity of various NPs [3–5]. However, there is much to do for evaluating whether the NPs may be an environmental threat. In fact, there are an increasing number of studies where the toxicity of a several engineered nanoparticles or nanomaterials such as fullerenes, graphene metal nanoparticles, metal oxides nanoparticles, crystalline materials, amorphous materials, and nano-sized polymers has been evaluated [6]. For toxicity assays several model organisms are used, such as *Daphnia magna* [7–9], *Paracentrotus lividus* [10, 11], *Mytilus galloprovincialis* [12, 13] or *Mytilus edulis* [14, 15], *Artemia salina* [16, 17], *Danio rerio* [18**–**21], and other animal models like mice [22, 23]. Each and every year, the number of engineered nanomaterials and their products is continuously increasing, which is necessary to have a representative model organism, able to assess nanotoxicity accurately. In this regard, zebrafish as model organism has attracted scientific interest. In this review, we focused on nanotoxicological studies conducted using as model zebrafish, adults or embryos, with different assessment methods

### **2. Zebrafish: a perfect experimental animal model**

*Danio rerio* (Hamilton and Buchanan, 1822), commonly known as zebrafish, is a small tropical freshwater fish which lives in river basins of India, Northern Pakistan, Nepal, Bhutan, and South Asia. It belongs to the family of Cyprinidae, within the order of the Cypriniformes. Zebrafish adults are 4–5 cm long, and so they can be easily managed in large numbers in the laboratory.

Zebrafish can tolerate a temperature range of 24.5–28.5°C [24]; however, the growth speed of zebrafish embryos varies according to temperature [25].

One of the reasons why zebrafish is an excellent laboratory model is for its ability to spawn huge amounts of eggs the whole year.

Zebrafish embryonic development has been well characterized to [25]. The embryos themselves are transparent during the first few days of their lives because chorion is transparent. Pigmentation in the embryos starts about 30–72 h post fertilization [26]. Fertilization activates cytoplasmic movements, easily evident within about 10 min. The first cleavage of the newly fertilized egg occurs about 40 min after fertilization. The cytoplasmic divisions are meroblastic, and at the end of them, a blastodisc forms.

Blastula of zebrafish is a "stereoblastula" because blastocoel is not present. The blastula and gastrula stage of zebrafish at 28°C is equivalent to 2.25–5.25 h post fertilization (hpf) and 5.25–10 hpf, respectively [25].

Ballard [27] coined the term "pharyngula" (24–48 h) to refer to the embryo that has developed to the phylotypic stage, when it possesses the classic vertebrate bauplan. This is the time of development when one can most readily compare the morphologies of embryos of diverse vertebrates.

Nowadays, we are using a wide variety of commercially available nanoparticles [1]. Metal and carbon nanoparticles (NPs) represent the largest and fastest growing group of NPs [2]. Hence, environmental contamination is already occurring and is predicted to increase dramatically. This growth of nanotechnology has not advanced without concerns regarding their potential adverse environmental impacts. Several studies of nanotoxicology have been made in fact to evaluate the toxicity of various NPs [3–5]. However, there is much to do for evaluating whether the NPs may be an environmental threat. In fact, there are an increasing number of studies where the toxicity of a several engineered nanoparticles or nanomaterials such as fullerenes, graphene metal nanoparticles, metal oxides nanoparticles, crystalline materials, amorphous materials, and nano-sized polymers has been evaluated [6]. For toxicity assays several model organisms are used, such as *Daphnia magna* [7–9], *Paracentrotus lividus* [10, 11], *Mytilus galloprovincialis* [12, 13] or *Mytilus edulis* [14, 15], *Artemia salina* [16, 17], *Danio rerio* [18**–**21], and other animal models like mice [22, 23]. Each and every year, the number of engineered nanomaterials and their products is continuously increasing, which is necessary to have a representative model organism, able to assess nanotoxicity accurately. In this regard, zebrafish as model organism has attracted scientific interest. In this review, we focused on nanotoxicological studies conducted using as model zebrafish, adults or embryos, with dif-

*Danio rerio* (Hamilton and Buchanan, 1822), commonly known as zebrafish, is a small tropical freshwater fish which lives in river basins of India, Northern Pakistan, Nepal, Bhutan, and South Asia. It belongs to the family of Cyprinidae, within the order of the Cypriniformes. Zebrafish adults are 4–5 cm long, and so they can be easily managed in large numbers in the

Zebrafish can tolerate a temperature range of 24.5–28.5°C [24]; however, the growth speed of

One of the reasons why zebrafish is an excellent laboratory model is for its ability to spawn

Zebrafish embryonic development has been well characterized to [25]. The embryos themselves are transparent during the first few days of their lives because chorion is transparent. Pigmentation in the embryos starts about 30–72 h post fertilization [26]. Fertilization activates cytoplasmic movements, easily evident within about 10 min. The first cleavage of the newly fertilized egg occurs about 40 min after fertilization. The cytoplasmic divisions are meroblas-

Blastula of zebrafish is a "stereoblastula" because blastocoel is not present. The blastula and gastrula stage of zebrafish at 28°C is equivalent to 2.25–5.25 h post fertilization (hpf) and

ferent assessment methods

122 Recent Advances in Zebrafish Researches

laboratory.

**2. Zebrafish: a perfect experimental animal model**

zebrafish embryos varies according to temperature [25].

huge amounts of eggs the whole year.

tic, and at the end of them, a blastodisc forms.

5.25–10 hpf, respectively [25].

During hatching period (48–72 h), they are called "embryos" until the end of the third day and afterward "larvae," whether they have hatched or not [25].

There are several advantages for using zebrafish as a model species in nanotoxicological studies. Main benefit regards its size. Zebrafish adult is approximately 5 cm long, so it can be handled without any difficulty and reduces housing space and husbandry costs.

The tiny size of the larval and adult zebrafish allows to reduce quantities the dosing of experimental solutions and thereby creates limited volumes of waste to disposal and minimizes quantities of lab ware and chemicals.

Small embryos allow reasonable sample sizes to be tested together using a multiwell plate or series of Petri dishes to provide several experimental replicates at one time. From the egg stage, zebrafish embryos can survive for several days through the absorption of yolk and can be visually assessed for malformation [26].

The rapid maturation of zebrafish (sexual maturation occurs around 100 days) allows easy experimentation for transgenerational endpoints required for mutagenesis screening and assessing chemicals for teratogenicity.

This species shows high fecundity (single female can lay up to 200 eggs per week) and transparent embryos. The eggs hatch rapidly and organogenesis occurs quickly. As a result, the major organs are developed within few days post fertilization (dpf) in larvae.

Zebrafish eggs remain transparent from fertilization to when the tissues become dense and pigmentation is initiated (at approximately 30–72 h post fertilization (hpf)); this allows unobstructed observations of the main morphological changes up to and beyond pharyngulation. Therefore, using little magnification, adverse effects of chemical exposure on development of the brain, notochord, heart, jaw, trunk segmentation, and measurements of size can be assessed quantitatively.

Their optical clarity allows for identification of phenotypic traits during mutagenesis screening and assessment of endpoints of toxicity during toxicity testing. This proves even more valuable when immunochemistry (IHC) techniques are used. There are a vast amount of immunohistochemical markers available, allowing assessments of aberrant morphology or activation of certain signaling pathways by toxicants through the staining of specific tissues and cells types.

The zebrafish research community has developed a range of resources very useful to the toxicologists, including mutant strains, cDNA clone collections, and whole genome that has been sequenced a few years ago. Highly conserved signaling pathways are found both in zebrafish and mammals with a high level of genomic homology [28].

In recent years, the use of zebrafish as an established animal model system for nanoparticle toxicity assay is growing exponentially. Different types of parameters are used to evaluate nanoparticle toxicity such as hatching achievement rate, developmental malformation of organs, damage in gills and skin, abnormal behavior (movement impairment), reproduction toxicity, and finally mortality. In fact, there are an increasing number of literatures that document the concern over toxicity for broad range of engineered nanoparticles or nanomaterials. In this regard, zebrafish as an in vivo model organism has attracted scientific interest because of its unique features abovementioned.

toxicity indicating that functionalization affects the toxicity profile by influencing penetrance

ing the success in hatching in relationship with hours postexposure. Zebrafish embryos 4 h

Ong et al. [37] have used silicon, cadmium selenide, silver, and zinc NPs as well as singlewalled carbon nanotubes to assess NP effects on zebrafish hatch. They have reported complete inhibition of hatching and embryo death within chorion upon nanoparticle exposure, because the nanoparticles interact with the hatching enzymes, and they concluded that the observed effects arose from the NPs themselves and not their dissolved metal components.

Zebrafish embryo toxicity test (ZFET) is a modern nonanimal test, and it represents an alternative approach to acute toxicity testing, since with the same sensitivity and specificity, it is possible to find more simplification, economicity, and speedy of execution, as well as suggested by the European Community in order to decrease the impact of the experimental tests on live animals [20, 38]. Fish Embryo Toxicity Test is included in the guidelines to perform toxicity test about FDA and ICH for the pharmaceutical products and about EPA and OECD

Fish embryo-larval assays provide a screening and investigative tool able of testing a larger number of nanoparticles, and this model has become increasingly common for investigation of developmental toxicity mechanisms [18–20, 40, 41]. ZFET is not a suitable test if you want to evaluate the developmental malformations after the 96 hours post fertilization (hpf), such as the skeletal anomalies, because calcification process in zebrafish starts the seventh day of development.

Usenko et al. [42] have evaluated carbon fullerene (C60, C70, and C60(OH)24) toxicity in zebrafish embryos. They observed caudal fin malformation at the concentrations of 200 ppb of C60 and C70 and yolk sac edema, pericardial edema, and pectoral fin malformations over the concentrations of 2500 ppb of C60(OH)24. Additionally, they also observed swelling of zebrafish

Brundo et al. [18] tested the nanomaterials that were synthesized proposing a groundbreak-

nanoparticles. The system was synthesized by wrapping Au nanoparticles with a thin layer of

the expression of inducible metallothioneins. The results obtained by toxicity test showed

. The nontoxicity of the nano-system was established by testing the effect of the material on *Danio rerio* larvae. Zebrafish larvae were exposed to different concentrations of nanopar-

and Au and to new nanomaterials. Authors evaluated as biomarkers of exposure

embryos and delay in development upon exposure to 5000 ppb of C60(OH)24.

ing approach by an upside-down vision of the Au/TiO2

ing rate (HR) was calculated for each concentration tested. It was observed that TiO2

ticles can cause premature hatching in zebrafish embryos, dose dependently.

**2.2. Developmental disorder analysis of zebrafish embryos: zebrafish embryo**

toxicity to zebrafish embryos through evaluat-

Zebrafish or *Danio rerio*: A New Model in Nanotoxicology Study

http://dx.doi.org/10.5772/intechopen.74834

nano-system to avoid the release of

nanopar-

125

(0, 0.01, 10, and 1000 μg mL(−1)) for 130 h. The hatch-

through biological barriers.

**toxicity test**

TiO2

ticles of TiO2

Samaee et al. [36] have studied the nano-TiO2

post fertilization were exposed to nTiO2

for the chemical substances [39].

Interestingly, zebrafish behavioral response is also a sensitive indicator for abnormal change in toxicity. An experiment performed by [29] has also shown that TiO2 nanoparticles affect larval swimming parameters, including velocity and activity level.

The disruption of gills, skin, and endocrine system by nanoparticles is another parameter to understand nanoparticle-induced toxicity. It has been reported that silver ions (Ag+ ) generated by AgNPs exert acute toxicity, mainly due to their interaction with the gills. In the gills, ionic Ag+ inhibits Na+ /K+ -ATPase action and the enzymes related to Na+ and Cl− uptake, finally affecting osmoregulation [30].

Nanoparticle affects male and female reproductivity and fetal development. Wang et al. [31] assessed the disturbance in zebrafish reproduction after the chronic exposure of TiO2 nanoparticles.

Using this model organism, several specific protocols have been used for the toxicity screening. The correlation of successful hatching efficiency and embryo toxicity is an important parameter to evaluate the nanotoxicity.

### **2.1. Hatching analysis**

The hatching-related parameters may be one of the endpoints that have been underestimated in the several studies. There are conflicting results about the endpoint in the same and different species [32, 33], because the results are not easy to interpret. Consequently, hatchingrelated parameters do not seem to be able to show the toxicity of a nanoparticle especially at environmental-relevant concentrations. In fact, many papers associate different endpoints related to hatching and embryo development [34].

Paatero et al. [35] have used *Danio rerio* embryos to study toxicity profiles of differently surface-functionalized mesoporous silica nanoparticles (MSNs). Embryos with the chorion membrane intact, or dechorionated embryos, were incubated or microinjected with amino (NH2-MSNs), polyethyleneimine (PEI-MSNs), succinic acid (SUCC-MSNs), or polyethylene glycol (PEG-MSNs)-functionalized MSNs. Toxicity was assessed by viability and cardiovascular function. Typically cardiovascular toxicity was evident prior to lethality. Confocal microscopy revealed that PEI-MSNs penetrated into the embryos, whereas PEG− , NH2 − , and SUCC-MSNs remained aggregated on the skin surface. Direct exposure of inner organs by microinjecting NH2 -MSNs and PEI-MSNs demonstrated that the particles displayed similar toxicity indicating that functionalization affects the toxicity profile by influencing penetrance through biological barriers.

In recent years, the use of zebrafish as an established animal model system for nanoparticle toxicity assay is growing exponentially. Different types of parameters are used to evaluate nanoparticle toxicity such as hatching achievement rate, developmental malformation of organs, damage in gills and skin, abnormal behavior (movement impairment), reproduction toxicity, and finally mortality. In fact, there are an increasing number of literatures that document the concern over toxicity for broad range of engineered nanoparticles or nanomaterials. In this regard, zebrafish as an in vivo model organism has attracted scientific interest because

Interestingly, zebrafish behavioral response is also a sensitive indicator for abnormal change

The disruption of gills, skin, and endocrine system by nanoparticles is another parameter to

ated by AgNPs exert acute toxicity, mainly due to their interaction with the gills. In the gills,

Nanoparticle affects male and female reproductivity and fetal development. Wang et al. [31] assessed the disturbance in zebrafish reproduction after the chronic exposure of TiO2

Using this model organism, several specific protocols have been used for the toxicity screening. The correlation of successful hatching efficiency and embryo toxicity is an important

The hatching-related parameters may be one of the endpoints that have been underestimated in the several studies. There are conflicting results about the endpoint in the same and different species [32, 33], because the results are not easy to interpret. Consequently, hatchingrelated parameters do not seem to be able to show the toxicity of a nanoparticle especially at environmental-relevant concentrations. In fact, many papers associate different endpoints

Paatero et al. [35] have used *Danio rerio* embryos to study toxicity profiles of differently surface-functionalized mesoporous silica nanoparticles (MSNs). Embryos with the chorion membrane intact, or dechorionated embryos, were incubated or microinjected with amino (NH2-MSNs), polyethyleneimine (PEI-MSNs), succinic acid (SUCC-MSNs), or polyethylene glycol (PEG-MSNs)-functionalized MSNs. Toxicity was assessed by viability and cardiovascular function. Typically cardiovascular toxicity was evident prior to lethality. Confocal

SUCC-MSNs remained aggregated on the skin surface. Direct exposure of inner organs by

microscopy revealed that PEI-MSNs penetrated into the embryos, whereas PEG−

understand nanoparticle-induced toxicity. It has been reported that silver ions (Ag+

nanoparticles affect

and Cl−

) gener-

uptake, finally

, NH2 − , and

in toxicity. An experiment performed by [29] has also shown that TiO2

larval swimming parameters, including velocity and activity level.

of its unique features abovementioned.

124 Recent Advances in Zebrafish Researches

/K+

parameter to evaluate the nanotoxicity.

related to hatching and embryo development [34].

ionic Ag+ inhibits Na+

**2.1. Hatching analysis**

microinjecting NH2

nanoparticles.

affecting osmoregulation [30].

Samaee et al. [36] have studied the nano-TiO2 toxicity to zebrafish embryos through evaluating the success in hatching in relationship with hours postexposure. Zebrafish embryos 4 h post fertilization were exposed to nTiO2 (0, 0.01, 10, and 1000 μg mL(−1)) for 130 h. The hatching rate (HR) was calculated for each concentration tested. It was observed that TiO2 nanoparticles can cause premature hatching in zebrafish embryos, dose dependently.

Ong et al. [37] have used silicon, cadmium selenide, silver, and zinc NPs as well as singlewalled carbon nanotubes to assess NP effects on zebrafish hatch. They have reported complete inhibition of hatching and embryo death within chorion upon nanoparticle exposure, because the nanoparticles interact with the hatching enzymes, and they concluded that the observed effects arose from the NPs themselves and not their dissolved metal components.

### **2.2. Developmental disorder analysis of zebrafish embryos: zebrafish embryo toxicity test**

Zebrafish embryo toxicity test (ZFET) is a modern nonanimal test, and it represents an alternative approach to acute toxicity testing, since with the same sensitivity and specificity, it is possible to find more simplification, economicity, and speedy of execution, as well as suggested by the European Community in order to decrease the impact of the experimental tests on live animals [20, 38]. Fish Embryo Toxicity Test is included in the guidelines to perform toxicity test about FDA and ICH for the pharmaceutical products and about EPA and OECD for the chemical substances [39].

Fish embryo-larval assays provide a screening and investigative tool able of testing a larger number of nanoparticles, and this model has become increasingly common for investigation of developmental toxicity mechanisms [18–20, 40, 41]. ZFET is not a suitable test if you want to evaluate the developmental malformations after the 96 hours post fertilization (hpf), such as the skeletal anomalies, because calcification process in zebrafish starts the seventh day of development.

Usenko et al. [42] have evaluated carbon fullerene (C60, C70, and C60(OH)24) toxicity in zebrafish embryos. They observed caudal fin malformation at the concentrations of 200 ppb of C60 and C70 and yolk sac edema, pericardial edema, and pectoral fin malformations over the concentrations of 2500 ppb of C60(OH)24. Additionally, they also observed swelling of zebrafish embryos and delay in development upon exposure to 5000 ppb of C60(OH)24.

Brundo et al. [18] tested the nanomaterials that were synthesized proposing a groundbreaking approach by an upside-down vision of the Au/TiO2 nano-system to avoid the release of nanoparticles. The system was synthesized by wrapping Au nanoparticles with a thin layer of TiO2 . The nontoxicity of the nano-system was established by testing the effect of the material on *Danio rerio* larvae. Zebrafish larvae were exposed to different concentrations of nanoparticles of TiO2 and Au and to new nanomaterials. Authors evaluated as biomarkers of exposure the expression of inducible metallothioneins. The results obtained by toxicity test showed that neither mortality nor sublethal effects were induced by the different nanomaterials and free nanoparticles tested. However, only zebrafish larvae exposed to free Au nanoparticles showed a different response to anti-MT antibody. In fact, the immunolocalization analysis highlighted an increase synthesis of the inducible metallothioneins.

Pecoraro et al. [21] did a study on adverse effects of AgNPs in adult of *Danio rerio*. Fishes exposed to increasing concentrations (8, 45, 70 μg/l) of silver nanoparticles (AgNPs, 25 nm in average diameter) and after treatment for 30 days were quickly euthanized in MS-222. Authors have evaluated bioaccumulation of AgNPs using ICP-MS and analyzed histological changes, biomarkers of oxidative damage, and gene expression in the gut, liver, and gill tissues of AgNPs-treated zebrafish. The histological analysis showed lesions of secondary lamellae of the gills with different degrees of toxicity such as hyperplasia, lamellar fusion, subepithelial edema, and even in some cases telangiectasia. Huge necrosis of the intestinal villi was found in the gut. No lesion was detected in the liver. The analysis revealed a high expression of metallothioneins 1 (MTs 1) in animals exposed to AgNPs compared to the control group. The ICP-MS analysis shows that the amount of particles absorbed in all treated samples is almost the same. They affirm that AgNP toxicity is linked more to their size and state of aggregation than to their concentrations. Silver nanoparticles can damage gills and gut because they are able to pass through the mucosal barrier thanks to their small size. However, the damage is

Zebrafish or *Danio rerio*: A New Model in Nanotoxicology Study

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127

The toxicology of engineered NMs is a relatively new and evolving field, and although their applications are increasing, there are many concerns about their environmental and health impacts [44]. A large number of studies carried out on several nanoparticles have produced conflicting results. In fact, despite continuous attempts to establish a correlation between structure of the particles and their interactions with biological systems, we are still far from elucidating with certainty the toxicological profile of NPs [45]. Among these investigations, a large numbers of authors, for example, have confirmed the nontoxicity of AuNPs [46–50];

tumor formation after 2 years in rats [56]. Moreover, the International Agency for Research on

For this reason, same researches are developing an innovative nanomaterial that could help to overcome problems related to the toxic effects of NPs, being able to exploit all their qualities

The use of zebrafish as animal model is recommended in several of these researches because it is an inexpensive, quick, and easy model to assess the nanoparticle toxicity [58], and it can offer many advantages for toxicological research [59, 60]. In particular, ZFET, an alternative approach to acute toxicity testing, is important in order to decrease the impact of the experimental tests on live animals as well as suggested by the European Community. Therefore, the use of zebrafish model can be proposed for screening the toxicity profile of nanomaterials and

NPs [54, 55], studies

particles was able to induce lung

as a possibly carcinogenic to human (Group 2B carcinogen) [57].

still reversible because it is not documented injury to the basal membrane.

conversely, others have observed the toxicity of AuNPs [51–53].

demonstrated that exposure to high concentrations of TiO2

Cancer (IARC) has classified TiO2

their rapid feedback [61].

[12, 18, 20].

Despite some authors showed low toxicity of other particles such as TiO2

**3. Discussion**

Xu et al. [41] evaluated the effect of CuO-NPs on early zebrafish development. The results reveal that CuO-NPs can induce abnormal phenotypes of a smaller head and eyes and delayed epiboly. The gene expression pattern shows that CuO-NPs spatially narrow the expression of dorsal genes chordin and goosecoid and alter the expression of dlx3, ntl, and hgg which are related to the cell migration of gastrulation. The decreased expression of pax2 and pax6 involved in neural differentiation was accordant with the decreased sizes of neural structures. Cmlc2 expression suggests that CuO-NPs prevented looping of the heart tube during cardiogenesis. Furthermore, quantitative RT-PCR results suggest that the CuO-NPs could increase the canonical Wnt signaling pathway to narrow the expression of chordin and goosecoid in dorsoventral patterning as well as decrease the transcription of Wnt5 and Wnt11 to result in slower, less directed movements and an abnormal cell shape. These findings indicated the CuO-NPs exert developmental toxicity.

Pecoraro et al. [20] have tested nanocomposite membranes prepared using Nafion polymer combined with various fillers, such as anatase-type TiO2 nanoparticles and graphene oxide. The nontoxicity of these nanocomposites, already shown to be effective for water purification applications [19], was recognized by testing the effect of these different materials on zebrafish embryos. They evaluated as biomarkers of exposure the expression of heme-oxygenase 1 and inducible nitric oxide synthases. Embryo toxicity test showed that neither mortality nor sublethal effects were caused by the different nanoparticles and nanosystems tested. Only zebrafish larvae exposed to free nanoparticles have shown a different response to antibodies anti-heme-oxygenase 1 and anti-inducible nitric oxide synthases. The immunolocalization analysis in fact has highlighted an increase in the synthesis of these biomarkers.

### **2.3. Pathologies analysis in organs of zebrafish embryos and adults**

As no authorization is required, many authors prefer the ZFET, and few toxicity studies on nanoparticles are conducted with embryos after 96 hpf. Developmental malformation of zebrafish embryos was studied to several authors, and they can relate incomplete organ development, deformity of body parts, or lack of pigmentation. Zhu et al. [43] did one of the first studies on developmental toxicity in fish caused by iron oxide nanoparticles in aquatic environments. To study the ecological effects of iron oxide nanoparticles on aquatic organisms, they used early life stages of the *Danio rerio* to examine such effects on embryonic development in this species. The results showed that ≥10 mg/L of iron oxide nanoparticles instigated developmental toxicity in these embryos, causing mortality, hatching delay, and malformation. Moreover, an early life stage test using zebrafish embryos/larvae is also discussed and recommended in this study as an effective protocol for assessing the potential toxicity of nanoparticles.

Pecoraro et al. [21] did a study on adverse effects of AgNPs in adult of *Danio rerio*. Fishes exposed to increasing concentrations (8, 45, 70 μg/l) of silver nanoparticles (AgNPs, 25 nm in average diameter) and after treatment for 30 days were quickly euthanized in MS-222. Authors have evaluated bioaccumulation of AgNPs using ICP-MS and analyzed histological changes, biomarkers of oxidative damage, and gene expression in the gut, liver, and gill tissues of AgNPs-treated zebrafish. The histological analysis showed lesions of secondary lamellae of the gills with different degrees of toxicity such as hyperplasia, lamellar fusion, subepithelial edema, and even in some cases telangiectasia. Huge necrosis of the intestinal villi was found in the gut. No lesion was detected in the liver. The analysis revealed a high expression of metallothioneins 1 (MTs 1) in animals exposed to AgNPs compared to the control group. The ICP-MS analysis shows that the amount of particles absorbed in all treated samples is almost the same. They affirm that AgNP toxicity is linked more to their size and state of aggregation than to their concentrations. Silver nanoparticles can damage gills and gut because they are able to pass through the mucosal barrier thanks to their small size. However, the damage is still reversible because it is not documented injury to the basal membrane.

### **3. Discussion**

that neither mortality nor sublethal effects were induced by the different nanomaterials and free nanoparticles tested. However, only zebrafish larvae exposed to free Au nanoparticles showed a different response to anti-MT antibody. In fact, the immunolocalization analysis

Xu et al. [41] evaluated the effect of CuO-NPs on early zebrafish development. The results reveal that CuO-NPs can induce abnormal phenotypes of a smaller head and eyes and delayed epiboly. The gene expression pattern shows that CuO-NPs spatially narrow the expression of dorsal genes chordin and goosecoid and alter the expression of dlx3, ntl, and hgg which are related to the cell migration of gastrulation. The decreased expression of pax2 and pax6 involved in neural differentiation was accordant with the decreased sizes of neural structures.

 expression suggests that CuO-NPs prevented looping of the heart tube during cardiogenesis. Furthermore, quantitative RT-PCR results suggest that the CuO-NPs could increase the canonical Wnt signaling pathway to narrow the expression of chordin and goosecoid in dorsoventral patterning as well as decrease the transcription of Wnt5 and Wnt11 to result in slower, less directed movements and an abnormal cell shape. These findings indicated the

Pecoraro et al. [20] have tested nanocomposite membranes prepared using Nafion poly-

oxide. The nontoxicity of these nanocomposites, already shown to be effective for water purification applications [19], was recognized by testing the effect of these different materials on zebrafish embryos. They evaluated as biomarkers of exposure the expression of heme-oxygenase 1 and inducible nitric oxide synthases. Embryo toxicity test showed that neither mortality nor sublethal effects were caused by the different nanoparticles and nanosystems tested. Only zebrafish larvae exposed to free nanoparticles have shown a different response to antibodies anti-heme-oxygenase 1 and anti-inducible nitric oxide synthases. The immunolocalization analysis in fact has highlighted an increase in the synthesis of

As no authorization is required, many authors prefer the ZFET, and few toxicity studies on nanoparticles are conducted with embryos after 96 hpf. Developmental malformation of zebrafish embryos was studied to several authors, and they can relate incomplete organ development, deformity of body parts, or lack of pigmentation. Zhu et al. [43] did one of the first studies on developmental toxicity in fish caused by iron oxide nanoparticles in aquatic environments. To study the ecological effects of iron oxide nanoparticles on aquatic organisms, they used early life stages of the *Danio rerio* to examine such effects on embryonic development in this species. The results showed that ≥10 mg/L of iron oxide nanoparticles instigated developmental toxicity in these embryos, causing mortality, hatching delay, and malformation. Moreover, an early life stage test using zebrafish embryos/larvae is also discussed and recommended in this study as an effective protocol for assessing the potential

nanoparticles and graphene

highlighted an increase synthesis of the inducible metallothioneins.

mer combined with various fillers, such as anatase-type TiO2

**2.3. Pathologies analysis in organs of zebrafish embryos and adults**

Cmlc2

these biomarkers.

toxicity of nanoparticles.

CuO-NPs exert developmental toxicity.

126 Recent Advances in Zebrafish Researches

The toxicology of engineered NMs is a relatively new and evolving field, and although their applications are increasing, there are many concerns about their environmental and health impacts [44]. A large number of studies carried out on several nanoparticles have produced conflicting results. In fact, despite continuous attempts to establish a correlation between structure of the particles and their interactions with biological systems, we are still far from elucidating with certainty the toxicological profile of NPs [45]. Among these investigations, a large numbers of authors, for example, have confirmed the nontoxicity of AuNPs [46–50]; conversely, others have observed the toxicity of AuNPs [51–53].

Despite some authors showed low toxicity of other particles such as TiO2 NPs [54, 55], studies demonstrated that exposure to high concentrations of TiO2 particles was able to induce lung tumor formation after 2 years in rats [56]. Moreover, the International Agency for Research on Cancer (IARC) has classified TiO2 as a possibly carcinogenic to human (Group 2B carcinogen) [57].

For this reason, same researches are developing an innovative nanomaterial that could help to overcome problems related to the toxic effects of NPs, being able to exploit all their qualities [12, 18, 20].

The use of zebrafish as animal model is recommended in several of these researches because it is an inexpensive, quick, and easy model to assess the nanoparticle toxicity [58], and it can offer many advantages for toxicological research [59, 60]. In particular, ZFET, an alternative approach to acute toxicity testing, is important in order to decrease the impact of the experimental tests on live animals as well as suggested by the European Community. Therefore, the use of zebrafish model can be proposed for screening the toxicity profile of nanomaterials and their rapid feedback [61].

### **Author details**

Maria Violetta Brundo1 \* and Antonio Salvaggio2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Biological, Geological and Environmental Science, University of Catania, Catania, Italy

[10] Šiller L, Lemloh ML, Piticharoenphun S, Mendis BG, Horrocks BR, Brümmer F, Medaković D. Silver nanoparticle toxicity in sea urchin *Paracentrotus lividus*. Environmental

[11] Kanold JM, Wang J, Brümmer F, Šiller L. Metallic nickel nanoparticles and their effect on the embryonic development of the sea urchin *Paracentrotus lividus*. Environmental

[12] Scuderi V, Impellizzeri G, Romano L, Scuderi M, Brundo MV, Bergum K, Zimbone M, Sanz R, Buccheri MA, Simone F, Nicotra G, Svensson BG, Grimaldi MG, Privitera V. An

[13] Gornati R, Longo A, Rossi F, Maisano M, Sabatino G, Mauceri A, Bernardini G, Fasulo S. Effects of titanium dioxide nanoparticle exposure in *Mytilus galloprovincialis* gills and digestive gland. Nanotoxicology. 2016;**10**(6):807-817. DOI: 10.3109/17435390.2015.1132348

[14] Tedesco S, Doyle H, Iacopino D, O'Donovan I, Keane S, Sheehan D. Gold nanoparticles and oxidative stress in the blue mussel, *Mytilus edulis*. Methods in Molecular Biology.

[15] Hu W, Culloty S, Darmody G, Lynch S, Davenport J, Ramirez-Garcia S, Dawson KA, Lynch I, Blasco J, Sheehan D. Toxicity of copper oxide nanoparticles in the blue mussel, *Mytilus edulis*: A redox proteomic investigation. Chemosphere. 2014;**108**:289-299. DOI:

[16] Arulvasu C, Jennifer SM, Prabhu D, Chandhirasekar D. Toxicity effect of silver nanoparticles in brine shrimp *Artemia*. Scientific World Journal. 2014;**2014**:10. DOI: 10.1155/

*salina* cysts and three stages of larvae. Science of the Total Environment. 2017;**598**:847-855.

[18] Brundo MV, Pecoraro R, Marino F, Salvaggio A, Tibullo D, Saccone S, Bramanti V, Buccheri MA, Impellizzeri G, Scuderi V, Zimbone M, Privitera V. Toxicity evaluation of new engineered nanomaterials in zebrafish. Frontiers in Physiology. 2016;**130**. DOI:

[19] Buccheri MA, D'Angelo D, Scalese S, Spano SF, Filice S, Fazio E, Compagnini G, Zimbone M, Brundo MV, Pecoraro R, Alba A, Sinatra F, Rappazzo G, Privitera V. Modification of graphene oxide by laser irradiation: A new route to enhance antibacterial activity.

[20] Pecoraro R, D'Angelo D, Filice S, Scalese S, Capparucci F, Marino F, Iaria C, Guerriero G, Tibullo D, Scalisi EM, Salvaggio A, Nicotera I, Brundo MV. Toxicity evaluation of graphene oxide and titania loaded nafion membranes in zebrafish. Frontiers in Physiology.

Nanotechnology. 2016;**27**:245704-245712. DOI: 10.1088/0957-4484/27/24/245704

eco-friendly water applications. Nanoscale. 2014;**6**:11189-11195. DOI: 10.1039/c4nr02820a

wrapping of Au nanoparticles for

Zebrafish or *Danio rerio*: A New Model in Nanotoxicology Study

http://dx.doi.org/10.5772/intechopen.74834

129

O3

nanoparticles to *Artemia*

Pollution. 2013;**178**:498-502. DOI: doi.org/10.1016/j.envpol.2013.03.010

Pollution. 2016;**212**:224-229. DOI: doi.org/10.1016/j.envpol.2016.01.050

enhanced photocatalytic response of nanometric TiO2

2013;**1028**:197-203. DOI: 10.1007/978-1-62703-475-3\_12

[17] Wang C, Jia H, Zhu L, Zhang H, Wang Y. Toxicity of α-Fe2

10.1016/j.chemosphere.2014.01.054

DOI: 10.1016/j.scitotenv.2017.04.183

2017. DOI: 10.3389/fphys.2017.01039

10.3389/fphys.2016.0013

2014/256919

2 Experimental Zooprophylactic Institute of Sicily "A. Mirri", Catania, Italy

### **References**

[10] Šiller L, Lemloh ML, Piticharoenphun S, Mendis BG, Horrocks BR, Brümmer F, Medaković D. Silver nanoparticle toxicity in sea urchin *Paracentrotus lividus*. Environmental Pollution. 2013;**178**:498-502. DOI: doi.org/10.1016/j.envpol.2013.03.010

**Author details**

Catania, Italy

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**Chapter 8**

**Provisional chapter**

**The Monitoring and Assessment of Cd2+ Stress Using**

**The Monitoring and Assessment of Cd2+ Stress Using**

Pollution on the Earth is ubiquitous across ecosystems from the land to the ocean. Various sources contribute to pollution including industrial (e.g. chemicals), agricultural (e.g. pesticides) and domestic (e.g. transportation) pollutants' ecosystems and substrate environment (e.g. contamination in water). The extensive use of chemicals in agriculture, forests and wetlands may impair biological communities. Due to the lack of target specificity, these chemicals can cause severe and persistent toxic effects on nontarget aquatic species, including bacteria, invertebrates and vertebrates. Different degrees of biological response have been presented according to intensities of different chemicals. The cadmium (Cd2+) contamination in aquatic environment has attracted more and more attention due to its toxic characteristics, for example, accumulation in environment, nondegradability and the potential threat to the ecosystem. Knowledge and understanding of these conditions have led to the development of new monitoring and assessment technologies based on biological and chemical methods. This chapter covers new monitoring technologies and environment assessment of Cd2+ stress using zebrafish (*Danio rerio*), which include the

behaviour responses, metabolism and electrocardiogram (ECG).

**Keywords:** zebrafish, behaviour responses, metabolism, electrocardiogram (ECG),

With the development of industrialization, the efforts of decreasing pollutants from the natural environment cannot satisfy the fundamental requirement of health and safety due to the large and increasing amounts of waste materials [1]. Environmental pollution has become an

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.74454

**Zebrafish (***Danio rerio***)**

**Zebrafish (***Danio rerio***)**

Zongming Ren and Yuedan Liu

Zongming Ren and Yuedan Liu

http://dx.doi.org/10.5772/intechopen.74454

**Abstract**

cadmium stress

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **The Monitoring and Assessment of Cd2+ Stress Using Zebrafish (***Danio rerio***) The Monitoring and Assessment of Cd2+ Stress Using Zebrafish (***Danio rerio***)**

DOI: 10.5772/intechopen.74454

Zongming Ren and Yuedan Liu Zongming Ren and Yuedan Liu

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74454

#### **Abstract**

Pollution on the Earth is ubiquitous across ecosystems from the land to the ocean. Various sources contribute to pollution including industrial (e.g. chemicals), agricultural (e.g. pesticides) and domestic (e.g. transportation) pollutants' ecosystems and substrate environment (e.g. contamination in water). The extensive use of chemicals in agriculture, forests and wetlands may impair biological communities. Due to the lack of target specificity, these chemicals can cause severe and persistent toxic effects on nontarget aquatic species, including bacteria, invertebrates and vertebrates. Different degrees of biological response have been presented according to intensities of different chemicals. The cadmium (Cd2+) contamination in aquatic environment has attracted more and more attention due to its toxic characteristics, for example, accumulation in environment, nondegradability and the potential threat to the ecosystem. Knowledge and understanding of these conditions have led to the development of new monitoring and assessment technologies based on biological and chemical methods. This chapter covers new monitoring technologies and environment assessment of Cd2+ stress using zebrafish (*Danio rerio*), which include the behaviour responses, metabolism and electrocardiogram (ECG).

**Keywords:** zebrafish, behaviour responses, metabolism, electrocardiogram (ECG), cadmium stress

### **1. Introduction**

With the development of industrialization, the efforts of decreasing pollutants from the natural environment cannot satisfy the fundamental requirement of health and safety due to the large and increasing amounts of waste materials [1]. Environmental pollution has become an

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

increasingly serious problem in recent years [2, 3], and water pollution has caused more and more concern. The extensive use of chemicals in agriculture, forests and wetlands may impair biological communities [4, 5].

In the ecological assessment of environmental stress, many methods in different levels (molecular, cell, physiological and individual) are used [18]. Behaviour responses to toxic effects have been specifically observed [19, 20]. It has been reported that *Daphnia magna*, medaka (*Oryzias latipes*) and rare minnow (*Gobiocypris rarus*) are aquatic homeostatic organisms [21, 22] showing evident step-wise behaviour response, including "No effect", "Acclimation", "Adjustment (Readjustment)" and "Toxic effect" [23–25]. Wang et al. [26] reported a sequence of intoxication and recovery processes through data transformation (i.e. integration) at the time progressed after exposure to toxic chemicals and suggested that the behaviour response

The Monitoring and Assessment of Cd2+ Stress Using Zebrafish (*Danio rerio*)

http://dx.doi.org/10.5772/intechopen.74454

137

As an endpoint in the physiological level, organisms' metabolism plays an important role in the environmental stress assessment. Metabolism is the fundamental process of organisms, and it is related to energy assimilation, transformation and allocation that strongly influence the rate of individual growth and reproduction [27]. Standard metabolic rate (SMR), which is the minimum metabolic level of fish in the state of rest and starvation, means the metabolism level of organisms, which could be analysed directly by oxygen consumption (OC) monitoring system [28]. Respiration is an important physiological characteristic of the metabolic activities of fish, which can reflect the adaptation of fish to the external environment. Metabolism in the characteristic of SMR is ordinarily equal to OC [29] on the condition of no food supplied during the monitoring of fish respiration. Usually, there are many factors that can affect OC of fish including water temperature, body mass, body size, dioxide oxygen, sanity, atmospheric

Heart ECG provides a chart that represents the electrical activity of the heart, and it also provides a time voltage of the heartbeat [30]. The notation of ECG waveform suggested by Einthoven [31] is still in use today and has been used to detect and monitor disease in different animals [32–34]. ECG waves (P, Q, R, S and T) and the time intervals (PR, QRS, ST and QT) have been used to differentiate healthy and diseased fish [35]. Therefore, fish ECG can be

Zebrafish (*Danio rerio*), whose ether-a-go-go-related gene (zERG) which has high similarities in the protein sequence with the human gene (hERG) [35] has been frequently used as a representative in the toxicologic assessment of chemicals [36], providing sensitive, economical, practical and biological monitors for aquatic pollutants [37, 38]. First, it is economic to use zebrafish to assess water quality. Second, zebrafish can be a tool as good as higher vertebrates on toxic testing. Third, the use of zebrafish with other fish can realize the potential toxicity analysis [39]. Therefore, new monitoring technologies based on the behaviour responses, metabolism and electrocardiogram (ECG) of zebrafish can be applied to realize the monitor-

In the recording of the behaviour responses, metabolism and ECG, room temperature is controlled at 26 ± 2°C with a photoperiod of 16 h light and 8 h dark. Nonchlorinated water

is a good indicator for aquatic organisms to assess the water quality.

pressure, injury and disease [28].

a good tool to assess water quality.

ing and assessment of Cd2+ stress.

**2. Methods**

Due to the lack of target specificity, most contaminants can cause severe and persistent toxic effects on non-target aquatic species, including invertebrates and vertebrates [6, 7]. Knowledge and understanding of these conditions have led to the development of new monitoring, analysis and assessment technologies based on biological, physical and chemical methods [8–10]. Campanella et al. [11] achieved the monitoring of the evolution of photosynthetic oxygen (O2 ) and the detection of alterations due to toxic effects caused by environmental pollutants using a sensor by coupling a suited algal bioreceptor to an amperometric gas diffusion electrode. Glasgow et al. [12] illustrated that real-time remote monitoring and sensing technology which is a more important tool for evaluation of water quality. Zhang et al. [13] developed an online behaviour monitoring system to assess the toxic effects of carbamate pesticides on medaka (*Oryzias latipes*), a small fish native to East Asia. Alexakis [14] assessed the water samples based on chemical indexes such as sodium adsorption ratio, sodium percentage and residual sodium carbonate. These methods greatly contribute to environmental stress assessment and water resource management.

Heavy metal pollution caused worldwide attention due to their bioaccumulation. Cadmium (Cd) is one of the most easily accumulated toxic substances in humans and organisms [15]. Cd pollution commonly occurs in food and environment (e.g. water, air), and also Cd has a long half-life period in human and animal bodies. Hansen has investigated that rats accumulated Cd2+ at a greater rate firstly and the kidney toxicity due to Cd2+needed 6–8 weeks [16]. The accumulation of cadmium could be observed in different organs in fish [17] (**Figure 1**).

**Figure 1.** Step-wise behaviour responses according to previous reports: The model showed a series of different behaviours including "No effect," "Acclimation," "Adjustment (Readjustment)" and "Toxic effect". Threshold, the point at which a physiological effect begins to be specifically manifested on the individuals after exposure to stressors, decided the behaviour responses.

In the ecological assessment of environmental stress, many methods in different levels (molecular, cell, physiological and individual) are used [18]. Behaviour responses to toxic effects have been specifically observed [19, 20]. It has been reported that *Daphnia magna*, medaka (*Oryzias latipes*) and rare minnow (*Gobiocypris rarus*) are aquatic homeostatic organisms [21, 22] showing evident step-wise behaviour response, including "No effect", "Acclimation", "Adjustment (Readjustment)" and "Toxic effect" [23–25]. Wang et al. [26] reported a sequence of intoxication and recovery processes through data transformation (i.e. integration) at the time progressed after exposure to toxic chemicals and suggested that the behaviour response is a good indicator for aquatic organisms to assess the water quality.

As an endpoint in the physiological level, organisms' metabolism plays an important role in the environmental stress assessment. Metabolism is the fundamental process of organisms, and it is related to energy assimilation, transformation and allocation that strongly influence the rate of individual growth and reproduction [27]. Standard metabolic rate (SMR), which is the minimum metabolic level of fish in the state of rest and starvation, means the metabolism level of organisms, which could be analysed directly by oxygen consumption (OC) monitoring system [28]. Respiration is an important physiological characteristic of the metabolic activities of fish, which can reflect the adaptation of fish to the external environment. Metabolism in the characteristic of SMR is ordinarily equal to OC [29] on the condition of no food supplied during the monitoring of fish respiration. Usually, there are many factors that can affect OC of fish including water temperature, body mass, body size, dioxide oxygen, sanity, atmospheric pressure, injury and disease [28].

Heart ECG provides a chart that represents the electrical activity of the heart, and it also provides a time voltage of the heartbeat [30]. The notation of ECG waveform suggested by Einthoven [31] is still in use today and has been used to detect and monitor disease in different animals [32–34]. ECG waves (P, Q, R, S and T) and the time intervals (PR, QRS, ST and QT) have been used to differentiate healthy and diseased fish [35]. Therefore, fish ECG can be a good tool to assess water quality.

Zebrafish (*Danio rerio*), whose ether-a-go-go-related gene (zERG) which has high similarities in the protein sequence with the human gene (hERG) [35] has been frequently used as a representative in the toxicologic assessment of chemicals [36], providing sensitive, economical, practical and biological monitors for aquatic pollutants [37, 38]. First, it is economic to use zebrafish to assess water quality. Second, zebrafish can be a tool as good as higher vertebrates on toxic testing. Third, the use of zebrafish with other fish can realize the potential toxicity analysis [39]. Therefore, new monitoring technologies based on the behaviour responses, metabolism and electrocardiogram (ECG) of zebrafish can be applied to realize the monitoring and assessment of Cd2+ stress.

### **2. Methods**

increasingly serious problem in recent years [2, 3], and water pollution has caused more and more concern. The extensive use of chemicals in agriculture, forests and wetlands may impair

Due to the lack of target specificity, most contaminants can cause severe and persistent toxic effects on non-target aquatic species, including invertebrates and vertebrates [6, 7]. Knowledge and understanding of these conditions have led to the development of new monitoring, analysis and assessment technologies based on biological, physical and chemical methods [8–10]. Campanella et al. [11] achieved the monitoring of the evolution of photosynthetic oxygen (O2

and the detection of alterations due to toxic effects caused by environmental pollutants using a sensor by coupling a suited algal bioreceptor to an amperometric gas diffusion electrode. Glasgow et al. [12] illustrated that real-time remote monitoring and sensing technology which is a more important tool for evaluation of water quality. Zhang et al. [13] developed an online behaviour monitoring system to assess the toxic effects of carbamate pesticides on medaka (*Oryzias latipes*), a small fish native to East Asia. Alexakis [14] assessed the water samples based on chemical indexes such as sodium adsorption ratio, sodium percentage and residual sodium carbonate. These methods greatly contribute to environmental stress assessment and

Heavy metal pollution caused worldwide attention due to their bioaccumulation. Cadmium (Cd) is one of the most easily accumulated toxic substances in humans and organisms [15]. Cd pollution commonly occurs in food and environment (e.g. water, air), and also Cd has a long half-life period in human and animal bodies. Hansen has investigated that rats accumulated Cd2+ at a greater rate firstly and the kidney toxicity due to Cd2+needed 6–8 weeks [16]. The accumulation of cadmium could be observed in different organs in fish [17] (**Figure 1**).

**Figure 1.** Step-wise behaviour responses according to previous reports: The model showed a series of different behaviours including "No effect," "Acclimation," "Adjustment (Readjustment)" and "Toxic effect". Threshold, the point at which a physiological effect begins to be specifically manifested on the individuals after exposure to stressors, decided

)

biological communities [4, 5].

136 Recent Advances in Zebrafish Researches

water resource management.

the behaviour responses.

In the recording of the behaviour responses, metabolism and ECG, room temperature is controlled at 26 ± 2°C with a photoperiod of 16 h light and 8 h dark. Nonchlorinated water (hardness based on CaCO3 : 250 ± 25 mg/L, pH 7.8 and temperature 26 ± 2°C) is used. No food is provided to test organisms during the assessment.

**2.2. The recording of metabolism**

(DO) sensor, thermometer, pressure meter and water tubes.

water are detected by DO sensor, and then these data are recorded as *DOi*

Water temperature is recorded to remain constant through the experiments.

**Figure 3.** Online monitoring systems of metabolism based on oxygen consumption (OC).

room pressure is provided to ensure the stability of DO in the system. Prior to the start of the experiment, all sensors are warmed up for 5 min, and then the OC data begin to be collected.

and *DOo*

. Absolute

An online monitoring system based on OC is used to record the metabolism of zebrafish (**Figure 3**). The metabolism monitoring system is made up of water tank, flow-through sensor, peristaltic pump, three-way valve, digital control unit, data acquisition unit, dissolved oxygen

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Before the assessments, test zebrafish volumes are measured by 5 ml graduated glass cylinder (Va), and the body mass of test zebrafish is weighted as follows: A beaker (50 ml) with 10 ml water is weighted by a precision electronic balance (FA2204N, Shanghai Jinghai Instrument), which is regarded as a baseline weight. Then, test individuals are put into the beaker to get the wet weight (m). Test zebrafish are placed into the flow-through sensors (200 ml, Vr) with nylon nets (250 μm) at both sides to prevent test zebrafish running into water tubes. The water flow rate of the sensor is controlled by a peristaltic pump at approximately 2 L/h. The whole cycle of the three-way valve is designed as 150 s flushing phase (to ensure DO in test chamber is enough for test individuals) after 300 s circulation phase (to make measurement, regarded as t). During the circulation mode, the three-way valve is energized to close the loop. In this mode, the water flows from the flow-through sensor to the DO sensor. When the organisms breathe, the DO sensor can realize the measurement. In this phase, water tubes are installed in the correct direction, and all connections are sealed completely to ensure no external oxygen enters the system. During the flushing mode (dotted lines), the three-way valve will pump water (black dot) from water tank (brown box) to test chamber, and the water body will flow through chamber into water tank again (red dot). The flushing and circulation cycle are repeated until the end of the experiment. Oxygen concentrations of both inflow and outflow

### **2.1. The recording of behaviour responses**

An online monitoring system, built in the Research Center for Eco-Environmental Science, Chinese Academy of Sciences [40], is used to analyse the continuous swimming behaviour (**Figure 2**). Behaviour data are collected by behaviour sensors, which are made up of two pairs of electrodes that sent a high-frequency signal of alternating current by one pair and then received another. The behaviour strength is sampled automatically every second, and the average behaviour strength data are taken twice an hour in the first 2 h. Behaviour strength that changes from 0 (losing the ability of movement) to 1 (full behaviour expression) is applied to represent the differences of behavioural responses [13].

Test zebrafish were placed in behaviour sensors (10 cm long, 7 cm in diameter). These sensors were closed off on both sides with 250 μm nylon nets, and three replicates per concentration were used. Flow rate of each test channel was controlled about 2 L/h. Temperature and light conditions were the same as stock rearing. The flow rate in each test chamber was controlled to about 1 L/h in the online mixing system, and then the flow rate in the online monitoring system of behaviour strength was about 2 L/h.

**Figure 2.** Signal acquisition and transmission of the behaviour responses: (a) the signal acquisition, (b) the normal signal analysis, and (c) the signal analysis after Fast Fourier Transform.

### **2.2. The recording of metabolism**

(hardness based on CaCO3

138 Recent Advances in Zebrafish Researches

is provided to test organisms during the assessment.

to represent the differences of behavioural responses [13].

system of behaviour strength was about 2 L/h.

analysis, and (c) the signal analysis after Fast Fourier Transform.

**2.1. The recording of behaviour responses**

: 250 ± 25 mg/L, pH 7.8 and temperature 26 ± 2°C) is used. No food

An online monitoring system, built in the Research Center for Eco-Environmental Science, Chinese Academy of Sciences [40], is used to analyse the continuous swimming behaviour (**Figure 2**). Behaviour data are collected by behaviour sensors, which are made up of two pairs of electrodes that sent a high-frequency signal of alternating current by one pair and then received another. The behaviour strength is sampled automatically every second, and the average behaviour strength data are taken twice an hour in the first 2 h. Behaviour strength that changes from 0 (losing the ability of movement) to 1 (full behaviour expression) is applied

Test zebrafish were placed in behaviour sensors (10 cm long, 7 cm in diameter). These sensors were closed off on both sides with 250 μm nylon nets, and three replicates per concentration were used. Flow rate of each test channel was controlled about 2 L/h. Temperature and light conditions were the same as stock rearing. The flow rate in each test chamber was controlled to about 1 L/h in the online mixing system, and then the flow rate in the online monitoring

**Figure 2.** Signal acquisition and transmission of the behaviour responses: (a) the signal acquisition, (b) the normal signal

An online monitoring system based on OC is used to record the metabolism of zebrafish (**Figure 3**). The metabolism monitoring system is made up of water tank, flow-through sensor, peristaltic pump, three-way valve, digital control unit, data acquisition unit, dissolved oxygen (DO) sensor, thermometer, pressure meter and water tubes.

Before the assessments, test zebrafish volumes are measured by 5 ml graduated glass cylinder (Va), and the body mass of test zebrafish is weighted as follows: A beaker (50 ml) with 10 ml water is weighted by a precision electronic balance (FA2204N, Shanghai Jinghai Instrument), which is regarded as a baseline weight. Then, test individuals are put into the beaker to get the wet weight (m). Test zebrafish are placed into the flow-through sensors (200 ml, Vr) with nylon nets (250 μm) at both sides to prevent test zebrafish running into water tubes. The water flow rate of the sensor is controlled by a peristaltic pump at approximately 2 L/h. The whole cycle of the three-way valve is designed as 150 s flushing phase (to ensure DO in test chamber is enough for test individuals) after 300 s circulation phase (to make measurement, regarded as t). During the circulation mode, the three-way valve is energized to close the loop. In this mode, the water flows from the flow-through sensor to the DO sensor. When the organisms breathe, the DO sensor can realize the measurement. In this phase, water tubes are installed in the correct direction, and all connections are sealed completely to ensure no external oxygen enters the system. During the flushing mode (dotted lines), the three-way valve will pump water (black dot) from water tank (brown box) to test chamber, and the water body will flow through chamber into water tank again (red dot). The flushing and circulation cycle are repeated until the end of the experiment. Oxygen concentrations of both inflow and outflow water are detected by DO sensor, and then these data are recorded as *DOi* and *DOo* . Absolute room pressure is provided to ensure the stability of DO in the system. Prior to the start of the experiment, all sensors are warmed up for 5 min, and then the OC data begin to be collected. Water temperature is recorded to remain constant through the experiments.

**Figure 3.** Online monitoring systems of metabolism based on oxygen consumption (OC).

DO (mg/L) data in the metabolism monitoring system are converted to OC using Eq. (1):

DO (mg/L) data in the metastable monitoring system are converted to OC using Eq. (1):

$$\text{VO}\_2 = \frac{(\text{DO}\_i - \text{DO}\_i) \times (\text{V} - \text{V})}{m \times t} \tag{1}$$

are fixed in a damp sponge with the ventral side exposed for electrode placement (**Figure 4a**). Two micro-electrodes are positioned at 90° to the animal's epidermis near the heart and inside the medial pectoral fin; the positive electrode is on the right side and the negative electrode is at a relatively lower position on the left side. The grounding electrode is closer to the top of the pelvic fins as presented in **Figure 4a**. The relative distance between grounding electrode and other two electrodes is 8–10 mm. All electrodes are inserted into the skin to approximately 1 mm in depth. The exact position of the positive and negative electrodes was adjusted slightly to obtain the maximum voltage signals. ECG signals are characterized by the following parameters: waves P, Q, R, S, T and intervals PR, QRS, ST, QT (**Figure 4b**). ECG signals of each zebrafish are obtained over a 3 min period. Baseline ECG signals are recorded 24 h before assessment. The positive and negative electrodes are attached to the atrioventricular junction of the zebrafish, and the ECG signals are recorded at a sampling frequency of 4 kHz with the time constant 0.02 s [**Figure 4c(I)**]. The recorded signals were digitally processed by MATLAB (MATLAB R2009a, The MathWorks Inc., Natick, MA, USA). First, a zero-phase digital filter is used [**Figure 4c(II)**], and then the wavelet decomposition is acquired by performing a 10-level 1D wavelet analysis using coif5 wavelet. With the wavelet decomposition structure and the coif5 wavelet, the coefficients of the filtered signals are further reconstructed. The de-noised signals [**Figure 4c(III)**] are finally obtained from the above-reconstructed wavelet decomposition structure using the principle of Stein's unbiased risk; soft threshold, level-dependent estimation of level noise and coif5 wavelet at level 10 are used to analyse the ECG changes.

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According to the acute toxicity experiment following the guidelines for fish acute toxicity from OECD 203 [41], 48 h median lethal concentration (LC50–48 h) of cadmium chloride (CdCl2

zebrafish after probit analysis in MATLAB with 95% confidence interval is 42.6 mg/L. The LC50–48 h value (42.6 mg/L) is regarded as 1.0 TU, and then the exposure concentrations are

The behaviour responses based on behaviour strength (BS) of zebrafish are shown in **Figure 5**. The average BS in control kept about 0.8, and the higher the chemical concentrations, the

of zebrafish are closely related to the concentrations, which was defined as dose-effect relationship [42]. It is noteworthy that lower BS values could be observed in the dark period at 13–21 and 37 and 45 h in all treatments. Considering the ups and downs in BS curves matched to photo and scoot phase, this may suggest a possibility of circadian rhythm in test organisms

The behaviour activity (BA) as integrated BS was obtained by linear regression (**Figure 6**). If BA is in the positive range, the BS would be higher than the accumulated average of BS and could be considered as in active state [44]. The BA values were mostly negative at initial stage. The crossing times of BA between positive and negative values were commonly observed which show the circadian rhythms. The control group showed the high value between 21 and 37 h,

lower is the behaviour strength. These results suggest that the toxic effects of CdCl2

treatments, respectively.

) to

on the BS

**3. Results and discussion**

**3.1. The behaviour responses**

4.26, 42.6 and 85.2 mg/L in 0.1, 1.0 and 2.0 TU CdCl2

after being exposed to the chemical [43].

in which VO2 : OC of test zebrafish (mg/kg/h); *DOi* : DO concentration of water body flowing into SMR sensors (mg/L); *DOo* : DO concentration of water body flowing out SMR sensors (mg/L); *Vr* : the volume of flow-through sensor (L) and it is 0.2 L for zebrafish; *Va* : test zebrafish volume (L); *m*: test zebrafish mass (kg); and *t*: time of circulation cycle (h).

#### **2.3. The recording of ECG**

The ECGs are detected by RM-6240C Multichannel Physiological Signal Acquisition and Processing System (ChengDu Instrument Factory, China). During ECG acquisition, zebrafish

**Figure 4.** The acquisition and the analysis of zebrafish ECG.

are fixed in a damp sponge with the ventral side exposed for electrode placement (**Figure 4a**). Two micro-electrodes are positioned at 90° to the animal's epidermis near the heart and inside the medial pectoral fin; the positive electrode is on the right side and the negative electrode is at a relatively lower position on the left side. The grounding electrode is closer to the top of the pelvic fins as presented in **Figure 4a**. The relative distance between grounding electrode and other two electrodes is 8–10 mm. All electrodes are inserted into the skin to approximately 1 mm in depth. The exact position of the positive and negative electrodes was adjusted slightly to obtain the maximum voltage signals. ECG signals are characterized by the following parameters: waves P, Q, R, S, T and intervals PR, QRS, ST, QT (**Figure 4b**). ECG signals of each zebrafish are obtained over a 3 min period. Baseline ECG signals are recorded 24 h before assessment. The positive and negative electrodes are attached to the atrioventricular junction of the zebrafish, and the ECG signals are recorded at a sampling frequency of 4 kHz with the time constant 0.02 s [**Figure 4c(I)**]. The recorded signals were digitally processed by MATLAB (MATLAB R2009a, The MathWorks Inc., Natick, MA, USA). First, a zero-phase digital filter is used [**Figure 4c(II)**], and then the wavelet decomposition is acquired by performing a 10-level 1D wavelet analysis using coif5 wavelet. With the wavelet decomposition structure and the coif5 wavelet, the coefficients of the filtered signals are further reconstructed. The de-noised signals [**Figure 4c(III)**] are finally obtained from the above-reconstructed wavelet decomposition structure using the principle of Stein's unbiased risk; soft threshold, level-dependent estimation of level noise and coif5 wavelet at level 10 are used to analyse the ECG changes.

### **3. Results and discussion**

DO (mg/L) data in the metabolism monitoring system are converted to OC using Eq. (1):

: the volume of flow-through sensor (L) and it is 0.2 L for zebrafish; *Va*

The ECGs are detected by RM-6240C Multichannel Physiological Signal Acquisition and Processing System (ChengDu Instrument Factory, China). During ECG acquisition, zebrafish

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *m* × *t* (1)

: DO concentration of water body flowing out SMR sensors

: DO concentration of water body flowing

: test zebrafish

*VO*2 = (*DOi* − *DOo*) × (*Vr* − *Va*)

: OC of test zebrafish (mg/kg/h); *DOi*

volume (L); *m*: test zebrafish mass (kg); and *t*: time of circulation cycle (h).

in which VO2

(mg/L); *Vr*

into SMR sensors (mg/L); *DOo*

140 Recent Advances in Zebrafish Researches

**2.3. The recording of ECG**

**Figure 4.** The acquisition and the analysis of zebrafish ECG.

### **3.1. The behaviour responses**

According to the acute toxicity experiment following the guidelines for fish acute toxicity from OECD 203 [41], 48 h median lethal concentration (LC50–48 h) of cadmium chloride (CdCl2 ) to zebrafish after probit analysis in MATLAB with 95% confidence interval is 42.6 mg/L. The LC50–48 h value (42.6 mg/L) is regarded as 1.0 TU, and then the exposure concentrations are 4.26, 42.6 and 85.2 mg/L in 0.1, 1.0 and 2.0 TU CdCl2 treatments, respectively.

The behaviour responses based on behaviour strength (BS) of zebrafish are shown in **Figure 5**. The average BS in control kept about 0.8, and the higher the chemical concentrations, the lower is the behaviour strength. These results suggest that the toxic effects of CdCl2 on the BS of zebrafish are closely related to the concentrations, which was defined as dose-effect relationship [42]. It is noteworthy that lower BS values could be observed in the dark period at 13–21 and 37 and 45 h in all treatments. Considering the ups and downs in BS curves matched to photo and scoot phase, this may suggest a possibility of circadian rhythm in test organisms after being exposed to the chemical [43].

The behaviour activity (BA) as integrated BS was obtained by linear regression (**Figure 6**). If BA is in the positive range, the BS would be higher than the accumulated average of BS and could be considered as in active state [44]. The BA values were mostly negative at initial stage. The crossing times of BA between positive and negative values were commonly observed which show the circadian rhythms. The control group showed the high value between 21 and 37 h,

**Figure 5.** Behaviour responses of zebrafish in the treatments of CdCl2 . Grey shadow means the dark periods as the experiment started from 9 o'clock in the morning.

supported by a previous report on the effects of CdCl2

**Figure 7.** Changes in average oxygen consumption (mgO2/kg/h) after exposure to CdCl2

energy processes were changed correspondingly [48].

300 mg/kg/h in 1.0 TU and 200 mg/kg/h in 2.0 TU.

**Figure 8.** Continuous changes of zebrafish OC during 48 h exposure in differentCdCl2

environment of CdCl2

crab [46], which is a fundamental physiological function in organisms that can affect other metabolic processes, for example, feeding, food absorption and excretion [47]. In the toxic

concentrations, 0TU, 0.1TU, 1TU and 2TU. The bars in the histogram represent the mean ± SD of oxygen consumption in different TUs. Different alphabets on the bar represent statistical significance based on multiple comparison (*p* < 0.05).

suggested that the total tendency of the continuous results had the same order as presented in **Figure 7**. In control, a high respiration rate was maintained at about 617.39 mg/kg/h during the observation period. In different treatments, the observed levels of OC decreased with the increase in concentrations, for example, OC was approximately 500 mg/kg/h in 0.1 TU,

**Figure 8** shows the continuous changes of zebrafish OC during 48 h exposure of CdCl2

, the internal environment of the cell changed, and all the related

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on the respiration of fresh water

treatments.

across different toxic

, which

**Figure 6.** BA values for CdCl2 across different treatments. Control (blue), 0.1TU (green), 1.0 TU (yellow) and 2.0 TU (red). Shadows mean dark periods.

which matched photophase. However, BS values were not high at either previous (0–13 h) or next (45–48 h) photoperiod. Remarkably, the circadian rhythms appeared in different treatments, being substantially different with the control group. Except the last phase with 1.0 TU, rhythmic activity was clearly observed at dark phases, 13–21 and 37–45 h, which indicated that the pollutant is a stimulating agent to resume the circadian system in test organisms. BS was higher at scoot phase. It was noteworthy that the rhythm was disrupted with 1.0 TU at the end of the experiment, whereas the lower (0.1 TU) and higher (2 TU) levels showed clear rhythms. The circadian rhythms of the test group were quite different from the control group, indicating that the environmental stress stimulated the biological clock of zebrafish and made it more clear in pollutants than in control [45], which is consistent with the result of **Figure 5**.

#### **3.2. The changes of metabolism**

As presented in **Figure 7**, the exposure of CdCl2 significantly affected the overall oxygen consumption (OC) of zebrafish (*p* < 0.05). OC at 0.1 TU (463.11 ± 44.21 mg/kg/h) was significantly (p < 0.05) lower than the control (617.39 ± 30.48 mg/kg/h) and higher than 1.0 TU (314.40 ± 40.04 mg/kg/h) and 2.0 TU (229.07 ± 28.66 mg/kg/h). These results could be

**Figure 7.** Changes in average oxygen consumption (mgO2/kg/h) after exposure to CdCl2 across different toxic concentrations, 0TU, 0.1TU, 1TU and 2TU. The bars in the histogram represent the mean ± SD of oxygen consumption in different TUs. Different alphabets on the bar represent statistical significance based on multiple comparison (*p* < 0.05).

supported by a previous report on the effects of CdCl2 on the respiration of fresh water crab [46], which is a fundamental physiological function in organisms that can affect other metabolic processes, for example, feeding, food absorption and excretion [47]. In the toxic environment of CdCl2 , the internal environment of the cell changed, and all the related energy processes were changed correspondingly [48].

**Figure 8** shows the continuous changes of zebrafish OC during 48 h exposure of CdCl2 , which suggested that the total tendency of the continuous results had the same order as presented in **Figure 7**. In control, a high respiration rate was maintained at about 617.39 mg/kg/h during the observation period. In different treatments, the observed levels of OC decreased with the increase in concentrations, for example, OC was approximately 500 mg/kg/h in 0.1 TU, 300 mg/kg/h in 1.0 TU and 200 mg/kg/h in 2.0 TU.

which matched photophase. However, BS values were not high at either previous (0–13 h) or next (45–48 h) photoperiod. Remarkably, the circadian rhythms appeared in different treatments, being substantially different with the control group. Except the last phase with 1.0 TU, rhythmic activity was clearly observed at dark phases, 13–21 and 37–45 h, which indicated that the pollutant is a stimulating agent to resume the circadian system in test organisms. BS was higher at scoot phase. It was noteworthy that the rhythm was disrupted with 1.0 TU at the end of the experiment, whereas the lower (0.1 TU) and higher (2 TU) levels showed clear rhythms. The circadian rhythms of the test group were quite different from the control group, indicating that the environmental stress stimulated the biological clock of zebrafish and made it more clear in pollutants than in control [45], which is consistent with the result of **Figure 5**.

across different treatments. Control (blue), 0.1TU (green), 1.0 TU (yellow) and 2.0 TU (red).

consumption (OC) of zebrafish (*p* < 0.05). OC at 0.1 TU (463.11 ± 44.21 mg/kg/h) was significantly (p < 0.05) lower than the control (617.39 ± 30.48 mg/kg/h) and higher than 1.0 TU (314.40 ± 40.04 mg/kg/h) and 2.0 TU (229.07 ± 28.66 mg/kg/h). These results could be

significantly affected the overall oxygen

. Grey shadow means the dark periods as the

**3.2. The changes of metabolism**

**Figure 6.** BA values for CdCl2

Shadows mean dark periods.

As presented in **Figure 7**, the exposure of CdCl2

**Figure 5.** Behaviour responses of zebrafish in the treatments of CdCl2

experiment started from 9 o'clock in the morning.

142 Recent Advances in Zebrafish Researches

**Figure 8.** Continuous changes of zebrafish OC during 48 h exposure in differentCdCl2 treatments.

During 48 h exposure, OC in most treatments had some circadian rhythms according to the average values in different periods (**Table 1**) because OCs were significantly (p < 0.05) higher during photo phase (D1 and D2) than scoot period (N1 and N2).

When zebrafish were exposed to 0.1 TU and 1.0 TU CdCl2 , OCs were significantly (*p* < 0.05) different between sooct phase and photo phase. This indicated that the response of zebrafish to pollutants was stronger and had great effects on diurnal variation during 48 h exposure. There was a greater fluctuation of OC in CdCl2 exposures than in the control. In most treatments, higher values were observed during photo phases (0.1, 1 and 2 TU). At the beginning, OC decreased until the 11th h and then recovered during 21–36 h. In the end, OC decreased at all concentrations during the exposure of CdCl2 in 36–48 h.

### **3.3. The changes of ECG**

**Figure 9** shows the average values of zebrafish ECG characteristics in different treatments. The values of all exposure time points during the 48-h exposure are assigned to the corresponding concentration groups. After exposure to CdCl2 , the amplitudes of all waves (P, Q, R, S and T) and interval durations (PR, QRS, ST and QT) showed some difference with significance (*p* < 0.05 or *p* < 0.01). In the control group, the amplitudes of all waves were the largest, and they were almost the smallest in different intervals. Overall, the amplitudes of waves tended to show negative relationships, and intervals showed positive relationship with CdCl2 concentrations. The changes of waves that Q, T, QRS and QT intervals showed clearly observed dose-effect relationship. There were some exceptions: S and ST did not show an obvious dose-effect relationship, and R showed a reverse effect to other waves. These results suggested that it was not sufficient to analyse the toxic effects of environmental stress on zebrafish ECG alone, depending on the average values of these characteristics.

The observed continuous changes of ECG suggested that signals of P, Q, T and PR were disordered, in which the data of amplitudes and durations showed no clear regularity depending on either exposure time or concentrations. According to the analysis of the tendency (**Table 2** and **Figure 10**), R, QRS, ST and QT in different treatments showed dose dependency; however, due to some fluctuation changes at 8, 16 and 32 h, ST and QT did not show clear time

and T) is μν, the unit of duration (PR interval, ST segment, QRS complex and QT interval) is ms. Vertical bars indicate mean values and the solid lines are standard deviation. The data of every ECG parameter are representative of at least four independent experiments. **—** represents two experimental groups indicating the ends of a line segment. \**p* < 0.05,

a decrease in amplitude. A prolonged ST reflects delayed repolarization of ventricular function. As it is reported, ST depression hysteresis could offer a substantially better diagnostic accuracy for coronary artery disease [49], and ST elevation could be induced by CdCl2

A wide QRS reflecting left-sided intra-ventricular conduction delay and prolonged QRS reflects delayed depolarization of ventricular function too. Prolonged QT reflects delayed

terized by lengthening the repolarization phase of the cardiac action potential, manifested as prolongation of the QT on the surface ECG and predisposition to special arrhythmia [35].

in zebrafish. The R wave voltage changes were observed with doxorubicin (DXR) treatment. On the cellular level, DXR treatment led to a decrease in Vmax, with little increase or no change in resting potential and a marked prolongation in action potential duration at

relation coefficient *r*and significance *p*. We first checked the correlation coefficient *r* to see how much they correlated (*r* < 0.3, poor correlation, 0.3 < *r* < 0.5 moderate, *r* > 0.5 high correlation).

exposure and wave R showed

. The unit of wave amplitude (P, Q, R, S

The Monitoring and Assessment of Cd2+ Stress Using Zebrafish (*Danio rerio*)

http://dx.doi.org/10.5772/intechopen.74454

145

is highly toxic to the cardiomyocytes, charac-

induced bradycardia and arrhythmia

stress was performed based on the cor-

stress, the Pearson corre-

[50].

Overall, QRS, ST and QT showed prolonged effects after CdCl2

**Figure 9.** Comparison of zebrafish ECG characteristics after treated with CdCl2

repolarization of action potential [51]. CdCl2

50–75% repolarization levels [52].

Therefore, the QT prolonging suggested that CdCl2

lation analysis between ECG characteristics and CdCl2

To specify the role of ECG characteristics in the assessment of CdCl2

dependence.

\*\**p* < 0.01.

To assess water quality using zebrafish ECG characteristics, the continuous changes based on the de-noised ECG signals were applied (**Figure 10**). The difference of zebrafish ECG characteristics after two-way ANOVA is shown in **Table 2**. Some characteristics (Q, R, S and T) showed some significant difference (*p* < 0.05 or *p* < 0.01) at different exposure times in the control group (**Figure 10b**–**e**), but the differences of P, PR, QRS, ST and QT showed no significant changes, which suggests that P, PR, QRS, ST and QT can serve as normal control in the analysis of CdCl2 toxic effects on zebrafish ECG (**Figure 10a**, f, g, h, i).

D1, the first 0 day results, from 0 to 11 h and from 21 to 24 h; D2, the second-day results, from 24 to 36 h and from 46 to 48 h; N1, the first-night results, from 11 to 21 h; N2, the second-night results, from 36 to 46 h exposure. Data are shown as M ± S.D.\* *p* < 0.05, A, B, a and b mean the significant difference with D1, D2, N1 and N2, respectively.

**Table 1.** OC of zebrafish in different treatments during both photo phase (D) and scoot period (N).

The Monitoring and Assessment of Cd2+ Stress Using Zebrafish (*Danio rerio*) http://dx.doi.org/10.5772/intechopen.74454 145

During 48 h exposure, OC in most treatments had some circadian rhythms according to the average values in different periods (**Table 1**) because OCs were significantly (p < 0.05) higher

different between sooct phase and photo phase. This indicated that the response of zebrafish to pollutants was stronger and had great effects on diurnal variation during 48 h exposure.

ments, higher values were observed during photo phases (0.1, 1 and 2 TU). At the beginning, OC decreased until the 11th h and then recovered during 21–36 h. In the end, OC decreased

**Figure 9** shows the average values of zebrafish ECG characteristics in different treatments. The values of all exposure time points during the 48-h exposure are assigned to the corre-

R, S and T) and interval durations (PR, QRS, ST and QT) showed some difference with significance (*p* < 0.05 or *p* < 0.01). In the control group, the amplitudes of all waves were the largest, and they were almost the smallest in different intervals. Overall, the amplitudes of waves tended to show negative relationships, and intervals showed positive relationship with

To assess water quality using zebrafish ECG characteristics, the continuous changes based on the de-noised ECG signals were applied (**Figure 10**). The difference of zebrafish ECG characteristics after two-way ANOVA is shown in **Table 2**. Some characteristics (Q, R, S and T) showed some significant difference (*p* < 0.05 or *p* < 0.01) at different exposure times in the control group (**Figure 10b**–**e**), but the differences of P, PR, QRS, ST and QT showed no significant changes, which suggests that P, PR, QRS, ST and QT can serve as normal control in the

toxic effects on zebrafish ECG (**Figure 10a**, f, g, h, i).

D1, the first 0 day results, from 0 to 11 h and from 21 to 24 h; D2, the second-day results, from 24 to 36 h and from 46 to 48 h; N1, the first-night results, from 11 to 21 h; N2, the second-night results, from 36 to 46 h exposure. Data are shown

**Time periods Control 0.1 TU 1.0 TU 2.0 TU** D1 610.47 ± 25.69\*aBb 460.38 ± 52.88 310.53 ± 52.92 236.69 ± 32.28\*b N1 632.18 ± 23.31\*Ab 436.54 ± 36.44\*B 285.47 ± 21.01\*Bb 214.81 ± 22.22\*B D2 633.20 ± 33.03\*Ab 488.10 ± 37.48\*a 342.35 ± 27.01\*ab 246.67 ± 23.02\*ab N2 590.88 ± 17.78\*AaB 458.77 ± 30.42 309.99 ± 22.47\*aB 207.22 ± 12.83\*AB

as M ± S.D.\* *p* < 0.05, A, B, a and b mean the significant difference with D1, D2, N1 and N2, respectively.

**Table 1.** OC of zebrafish in different treatments during both photo phase (D) and scoot period (N).

zebrafish ECG alone, depending on the average values of these characteristics.

 concentrations. The changes of waves that Q, T, QRS and QT intervals showed clearly observed dose-effect relationship. There were some exceptions: S and ST did not show an obvious dose-effect relationship, and R showed a reverse effect to other waves. These results suggested that it was not sufficient to analyse the toxic effects of environmental stress on

in 36–48 h.

, OCs were significantly (*p* < 0.05)

, the amplitudes of all waves (P, Q,

exposures than in the control. In most treat-

during photo phase (D1 and D2) than scoot period (N1 and N2).

When zebrafish were exposed to 0.1 TU and 1.0 TU CdCl2

There was a greater fluctuation of OC in CdCl2

at all concentrations during the exposure of CdCl2

sponding concentration groups. After exposure to CdCl2

**3.3. The changes of ECG**

144 Recent Advances in Zebrafish Researches

CdCl2

analysis of CdCl2

**Figure 9.** Comparison of zebrafish ECG characteristics after treated with CdCl2 . The unit of wave amplitude (P, Q, R, S and T) is μν, the unit of duration (PR interval, ST segment, QRS complex and QT interval) is ms. Vertical bars indicate mean values and the solid lines are standard deviation. The data of every ECG parameter are representative of at least four independent experiments. **—** represents two experimental groups indicating the ends of a line segment. \**p* < 0.05, \*\**p* < 0.01.

The observed continuous changes of ECG suggested that signals of P, Q, T and PR were disordered, in which the data of amplitudes and durations showed no clear regularity depending on either exposure time or concentrations. According to the analysis of the tendency (**Table 2** and **Figure 10**), R, QRS, ST and QT in different treatments showed dose dependency; however, due to some fluctuation changes at 8, 16 and 32 h, ST and QT did not show clear time dependence.

Overall, QRS, ST and QT showed prolonged effects after CdCl2 exposure and wave R showed a decrease in amplitude. A prolonged ST reflects delayed repolarization of ventricular function. As it is reported, ST depression hysteresis could offer a substantially better diagnostic accuracy for coronary artery disease [49], and ST elevation could be induced by CdCl2 [50]. A wide QRS reflecting left-sided intra-ventricular conduction delay and prolonged QRS reflects delayed depolarization of ventricular function too. Prolonged QT reflects delayed repolarization of action potential [51]. CdCl2 is highly toxic to the cardiomyocytes, characterized by lengthening the repolarization phase of the cardiac action potential, manifested as prolongation of the QT on the surface ECG and predisposition to special arrhythmia [35]. Therefore, the QT prolonging suggested that CdCl2 induced bradycardia and arrhythmia in zebrafish. The R wave voltage changes were observed with doxorubicin (DXR) treatment. On the cellular level, DXR treatment led to a decrease in Vmax, with little increase or no change in resting potential and a marked prolongation in action potential duration at 50–75% repolarization levels [52].

To specify the role of ECG characteristics in the assessment of CdCl2 stress, the Pearson correlation analysis between ECG characteristics and CdCl2 stress was performed based on the correlation coefficient *r*and significance *p*. We first checked the correlation coefficient *r* to see how much they correlated (*r* < 0.3, poor correlation, 0.3 < *r* < 0.5 moderate, *r* > 0.5 high correlation).

**Figure 10.** The continuous changes of ECG characteristics in different treatments. a-e represent the characteristics of waves P, Q, R, S and T and f-i represent the characteristics PR interval, QRS complex, of ST segment and QT interval. Log scale was used in the x-axis.

Then, we checked *p* value to see whether these two variables are correlated significantly (*p* < 0.05). When r is high (absolute *r* > 0.5) with significance (*p* < 0.05), it indicates that the data correlation is significant [53, 54].

The environmental stress *E* is primarily decided by both chemical concentrations (*C*) and exposure time (*t*) with an exponential function as shown in Eq.  When r is high (absolute *r* > 0.5) with significance (*p* < 0.05), it indicates that the data

**Figure 10.** The continuous changes of ECG characteristics in different treatments. a-e represent the characteristics of waves P, Q, R, S and T and f-i represent the characteristics PR interval, QRS complex, of ST segment and QT interval.

The environmental stress *E* is primarily decided by both chemical concentrations (*C*) and

+ *Ef* (2)

exposure time (*t*) with an exponential function as shown in Eq. (2) [55]:

correlation is significant [53, 54].

Log scale was used in the x-axis.

146 Recent Advances in Zebrafish Researches

*E* = *e*(*C*×*t*)

of the heart [61], which may prolong QRS. On a cellular level, depolarizing inward current

upstroke of the ventricular action potential that initiates the conduction of the excitatory wave front throughout the ventricular wall [58]. Visentin et al. investigated the dependence of Na+

ventricular myocytes [62]. In other words, exposure to Cd2+ could have an effect on Na + cur-

As a by-product of industry, wastewater with Cd2+ should not be discharged into aquatic environment only after basic treatment according to the Environmental Quality Standards for Surface Water, GB3838–2002 [63], in which the limitation of Cd is 0.01 mg/L. However, some industrial wastewater may be discharged without any treatment, in which Cd2+concentration might be higher than 26 mg/L [64] and the concentration of Cd2+ in some sediments could

The online behaviour responses of zebrafish showed that BS has obvious dose-effect relationship with Cd2+, and the online behaviour responses could illustrate the toxicity of Cd2+ directly. The circadian rhythms could be observed even in higher concentrations (1.0 and 2.0 TU). Meanwhile, Cd2+ has an inhibitory effect on the standard metabolic rate of zebrafish, and respiratory parameter of zebrafish with ECG can be regarded as sensitive biological monitoring indexes to realize the online assessment of Cd2+ pollution. It is noteworthy that there is an extreme significant correlation between QRS complex and Cd2+ stress with the highest *r* and the smallest *p* among all ECG characteristics, and it may be a good way to monitor Cd2+ pollu-

These results provide an objective ground for analysing complex stress response that could be applied to test the changes of organisms (exposed in different treatments) quantitatively in

This study was financially supported by the Natural Science Foundation of China (21107135, 41501538), the High-level Talent Project of Shandong Normal University, the Taishan Leader Talent Project of Shandong (tscy20150707), the Oversea High-level Talent Project of Ji'nan (2013041), Jinan Livelihood Major Projects (201704139), Guangdong Provincial Department of Science and Technology (2016A020221016, 2015A020216011), Guangzhou Science Technology and Innovation Commission<PHONE_NUMBER>94), and Central Research Institutes of Basic Research

tion in aquatic environment by observing and analysing QRS complex.

and Public Service Special Operations (PM-zx703-201701-007).

currents, resulting in delayed ventricular conduction and prolonged QRS in the ECG.

current by Cd2+ was essentially voltage independent, in agreement with it being

) channel is responsible for the rapid

http://dx.doi.org/10.5772/intechopen.74454

149

current in Purkinje fibres and in

channel and decrease inward Na+

concentration in ventricular myocytes. Depression of

The Monitoring and Assessment of Cd2+ Stress Using Zebrafish (*Danio rerio*)

passing through the voltage-gated cardiac sodium (Na+

caused by channel block. The data show that Cd2+ reduces Na+

rent in zebrafish cardiac myocytes. Cd2+ could block the Na+

current block by Cd2+ on external Na+

inward Na+

**4. Conclusions**

reach 359.8 g/kg [65].

toxic physiology and ecology.

**Acknowledgements**

The significance shows the difference between exposure times and start time (0 h) in each treatment.\* *p* < 0.05, \*\* *p* < 0.01 and \ represents no significant differences.

**Table 2.** The difference of zebrafish ECG characteristics after the two-way ANOVA.

in which *C* is based on TU values and *t* presents the time with 6 min per unit (0–480) according to our previous results [13]. *Ef* is the environmental stress due to the effects of all other physico-chemical factors, including water temperature, turbidity, pH, dioxide oxygen and conductivity. As physico-chemical factors are controlled under experimental conditions as shown in our previous research results [56], the effects of *Ef* are not considered (*Ef* = 0) for simplicity of model execution in this study. Then, if *C* × *t* = 0, *E* = 1, it means that the minimum value of *E* is 1 in a pollution-free environment [57].

The correlation between ECG parameters and CdCl2 stress (*E*) after Pearson correlation analysis is shown in **Table 3**. The results suggest that the relationship between E and wave S, intervals PR, QRS, ST and QT, showed a high correlation with absolute *r* > 0.5 and *p* < 0.05. With the correlation coefficient *r* = 0.729 (the highest) and correlation significance *p* = 0.002 (the smallest), the relationship between *E* and QRS was extremely significant, which suggested that QRS could be significantly affected by CdCl2 stress.

On the surface ECG, QRS reflects ventricular depolarization and propagation of the excitatory cardiac impulse throughout the ventricles [58]. Cardiac conduction system excitability was depressed preferentially in Cd2+ [59]. Cd2+ could lead to changes in ECG, which may be attributed to its effects on ventricular conduction. Yin et al. found that workers exposed to Cd2+ had a significantly longer QRS [60]. However, Cd2+ administration caused a reduction in myocardiac contractile performance, slowing of heart rate and disturbances in metabolism

**Table 3.** The correlation between ECG parameters and environmental stress (*E*) after Pearson correlation analysis.

of the heart [61], which may prolong QRS. On a cellular level, depolarizing inward current passing through the voltage-gated cardiac sodium (Na+ ) channel is responsible for the rapid upstroke of the ventricular action potential that initiates the conduction of the excitatory wave front throughout the ventricular wall [58]. Visentin et al. investigated the dependence of Na+ current block by Cd2+ on external Na+ concentration in ventricular myocytes. Depression of inward Na+ current by Cd2+ was essentially voltage independent, in agreement with it being caused by channel block. The data show that Cd2+ reduces Na+ current in Purkinje fibres and in ventricular myocytes [62]. In other words, exposure to Cd2+ could have an effect on Na + current in zebrafish cardiac myocytes. Cd2+ could block the Na+ channel and decrease inward Na+ currents, resulting in delayed ventricular conduction and prolonged QRS in the ECG.

### **4. Conclusions**

in which *C* is based on TU values and *t* presents the time with 6 min per unit (0–480) accord-

The significance shows the difference between exposure times and start time (0 h) in each treatment.\* *p* < 0.05, \*\* *p* < 0.01

QT (ms) Control — \ \ \ \ \ \

physico-chemical factors, including water temperature, turbidity, pH, dioxide oxygen and conductivity. As physico-chemical factors are controlled under experimental conditions as

simplicity of model execution in this study. Then, if *C* × *t* = 0, *E* = 1, it means that the minimum

sis is shown in **Table 3**. The results suggest that the relationship between E and wave S, intervals PR, QRS, ST and QT, showed a high correlation with absolute *r* > 0.5 and *p* < 0.05. With the correlation coefficient *r* = 0.729 (the highest) and correlation significance *p* = 0.002 (the smallest), the relationship between *E* and QRS was extremely significant, which suggested

On the surface ECG, QRS reflects ventricular depolarization and propagation of the excitatory cardiac impulse throughout the ventricles [58]. Cardiac conduction system excitability was depressed preferentially in Cd2+ [59]. Cd2+ could lead to changes in ECG, which may be attributed to its effects on ventricular conduction. Yin et al. found that workers exposed to Cd2+ had a significantly longer QRS [60]. However, Cd2+ administration caused a reduction in myocardiac contractile performance, slowing of heart rate and disturbances in metabolism

*E* Correlation (*r*) −0.094 0.317 −0.487 0.517 −0.443 0.685 **0.729** 0.667 0.635 Significance (*p*) 0.739 0.25 0.066 0.048\* 0.098 0.005\*\* **0.002**\*\* 0.007\*\* 0.011\*

**Table 3.** The correlation between ECG parameters and environmental stress (*E*) after Pearson correlation analysis.

stress.

**P Q R S T PR QRS ST QT**

is the environmental stress due to the effects of all other

**0 h 2 h 4 h 8 h 16 h 32 h 48 h**

0.1 TU — \ \ \* \ \ \*\* 0.5 TU — \ \ \ \*\* \ \*\* 1.0 TU — \ \ \ \*\* \ \*\*

are not considered (*Ef* = 0) for

stress (*E*) after Pearson correlation analy-

ing to our previous results [13]. *Ef*

and \ represents no significant differences.

148 Recent Advances in Zebrafish Researches

**Pearson correlation analysis**

\**p* < 0.05 \*\**p* < 0.01.

shown in our previous research results [56], the effects of *Ef*

**Table 2.** The difference of zebrafish ECG characteristics after the two-way ANOVA.

**ECG characteristics Treatments Start time Exposure time**

value of *E* is 1 in a pollution-free environment [57].

The correlation between ECG parameters and CdCl2

that QRS could be significantly affected by CdCl2

**ECG parameters**

As a by-product of industry, wastewater with Cd2+ should not be discharged into aquatic environment only after basic treatment according to the Environmental Quality Standards for Surface Water, GB3838–2002 [63], in which the limitation of Cd is 0.01 mg/L. However, some industrial wastewater may be discharged without any treatment, in which Cd2+concentration might be higher than 26 mg/L [64] and the concentration of Cd2+ in some sediments could reach 359.8 g/kg [65].

The online behaviour responses of zebrafish showed that BS has obvious dose-effect relationship with Cd2+, and the online behaviour responses could illustrate the toxicity of Cd2+ directly. The circadian rhythms could be observed even in higher concentrations (1.0 and 2.0 TU). Meanwhile, Cd2+ has an inhibitory effect on the standard metabolic rate of zebrafish, and respiratory parameter of zebrafish with ECG can be regarded as sensitive biological monitoring indexes to realize the online assessment of Cd2+ pollution. It is noteworthy that there is an extreme significant correlation between QRS complex and Cd2+ stress with the highest *r* and the smallest *p* among all ECG characteristics, and it may be a good way to monitor Cd2+ pollution in aquatic environment by observing and analysing QRS complex.

These results provide an objective ground for analysing complex stress response that could be applied to test the changes of organisms (exposed in different treatments) quantitatively in toxic physiology and ecology.

### **Acknowledgements**

This study was financially supported by the Natural Science Foundation of China (21107135, 41501538), the High-level Talent Project of Shandong Normal University, the Taishan Leader Talent Project of Shandong (tscy20150707), the Oversea High-level Talent Project of Ji'nan (2013041), Jinan Livelihood Major Projects (201704139), Guangdong Provincial Department of Science and Technology (2016A020221016, 2015A020216011), Guangzhou Science Technology and Innovation Commission<PHONE_NUMBER>94), and Central Research Institutes of Basic Research and Public Service Special Operations (PM-zx703-201701-007).

### **Author details**

Zongming Ren1 and Yuedan Liu2 \*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Institute of Environment and Ecology, Shandong Normal University, Jinan, PR China

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2 The Key Laboratory of Water and Air Pollution Control of Guangdong Province, South China Institute of Environmental Sciences, MEP, Guangzhou, PR China

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**Author details**

150 Recent Advances in Zebrafish Researches