Nanoscience and Nanoengineering

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

[31] Borzooeian Z, Taslim M, Borzooeian G, Ghasemi O, Aminlari M. Activity and stability analysis of covalent conjugated lysozyme-single walled carbon nanotubes: Potential biomedical and industrial applications. RSC Advances. 2017;**7**:48692-48701. DOI: 10.1039/

c7ra07189b

activity of a nanoformulation of

DOI: 10.1016/j.jgar.2018.09.004

[26] Manoukianab O, Arulb M, Rudraiahbc S, Kalajzicd I,

jconrel.2019.01.013

10.3390/ma7127770

cej.2016.02.001

ejic.201600359

[27] Li H, Nie B, Zhang S,

coating on enoxacin loaded titania nanotubes for improved osteogenesis and osseointegration in ovariectomized rats. Colloids and Surfaces B. 2019;**175**:409-420. DOI: 10.1016/j.colsurfb.2018.12.033

cisplatin with carbon nanotubes against *Leishmania major*. Journal of Global Antimicrobial Resistance. 2019;**16**:11-16.

Kumbar S. Aligned microchannel polymernanotube composites for peripheral nerve regeneration: Small molecule drug delivery. Journal of Controlled Release. 2019;**296**:54-67. DOI: 10.1016/j.

Long T, Yue B. Immobilization of type I collagen/hyaluronic acid multilayer

[28] Sánchez A, Peña L, Vidaltamayo R, Cué R, Mendoza A, Zomosa V, et al. Synthesization, characterization, and in vitro evaluation of cytotoxicity of biomaterial son halloysite nanotubes. Materials. 2014;**7**:7770-7780. DOI:

[29] Paseta L, Simón-Gaudó E, Gracia F, Coronas J. Encapsulation of essential oils in porous silica and MOFs for trichloroisocyanuric acid tablets used for water treatment in swimming pools. Chemical Engineering Journal. 2016;**292**:28-34. DOI: 10.1016/j.

[30] Roch-Marchal C, Hidalgo T, Banh H, Fischer R, Horcajada P. A promising catalytic and theranostic agent

obtained through the in-situ synthesis of Au nanoparticles with a reduced polyoxometalate incorporated within mesoporous MIL-101. European Journal of Inorganic Chemistry. 2016;**2016**:4387-4394. DOI: 10.1002/

**136**

**139**

**Chapter 8**

**Abstract**

Biochemical Toxicology: Heavy

The synthesis and application of nanoparticles have been actively studied in the modern era as it holds promises for effective and targeted strategies to deliver drugs inside the human body. Nanoparticles (NPs) play a big role in cancer diagnosis and have various advantages over other conventional chemotherapeutic drug delivery systems. But, the application of emerging engineered NPs to heavy toxic metals such as zinc, cobalt, and iron has resulted in a major source of toxicity. The toxicity of nanomaterials is majorly determined by their physical and chemical properties such as size, charge, and surface area. Also, the mechanism of nanotoxicity is majorly via the production of reactive oxygen species that create oxidative stress, thereby activating inflammatory cytokines and the mechanism of DNA damage that ultimately results in the cell death. So, mechanistic study needs to be done on nanomaterials to elucidate the mechanism involved in nanotoxicity and to generate

**Keywords:** nanoparticle, heavy metals, nanotoxicity, ROS, inflammation

Nanotechnology is one of the rapidly emerging fields in the twenty-first century

with extensive increase of nanoparticle application for the treatment of a wide variety of chronic diseases such as cancer. P. Ehrlich's visionary concept of "magic bullet" based on the use of targeted medicines to effectively attack cancer cells has provided a promising field for cancer therapy [1]. Targeted delivery to solid cancers provides more bioavailability and effective approach for cancer treatment. The characteristics of nanocarriers such as their nanoscale, high surface-to-volume ratio, favorable drug release profiles, and targeting modifications allow them to target tumor tissue in an effective manner and release drugs in a stable and controlled manner [2]. NPs can accumulate in the leaky vasculatures of tumor tissue in an enhanced permeability and retention effect (EPR). The potential of nanomedicine can be explored in the field of early detection of cancer as well as in combination therapies for treating tumor earlier and effectively. NPs effectively solve the physiological barriers such as renal, hepatic, and immune related for effective drug delivery of conventional chemotherapeutic drugs [3]. NPs may be modified to utilize passive and active targeting mechanism to reach the tumor tissue. The nanodelivery-based carriers range from natural polymeric materials to nonbiodegradable gold NP, and magnetic mesoporous silica-based, metal-based NP. The surface of the NP can be suitably modified with ligands or drugs to offer multimodular treatment options [4]. The nanoparticle shape also plays an important role

Metals and Nanomaterials

*Sibi Raj and Dhruv Kumar*

less toxic and efficient nanomaterials.

**1. Introduction**

#### **Chapter 8**

## Biochemical Toxicology: Heavy Metals and Nanomaterials

*Sibi Raj and Dhruv Kumar*

#### **Abstract**

The synthesis and application of nanoparticles have been actively studied in the modern era as it holds promises for effective and targeted strategies to deliver drugs inside the human body. Nanoparticles (NPs) play a big role in cancer diagnosis and have various advantages over other conventional chemotherapeutic drug delivery systems. But, the application of emerging engineered NPs to heavy toxic metals such as zinc, cobalt, and iron has resulted in a major source of toxicity. The toxicity of nanomaterials is majorly determined by their physical and chemical properties such as size, charge, and surface area. Also, the mechanism of nanotoxicity is majorly via the production of reactive oxygen species that create oxidative stress, thereby activating inflammatory cytokines and the mechanism of DNA damage that ultimately results in the cell death. So, mechanistic study needs to be done on nanomaterials to elucidate the mechanism involved in nanotoxicity and to generate less toxic and efficient nanomaterials.

**Keywords:** nanoparticle, heavy metals, nanotoxicity, ROS, inflammation

#### **1. Introduction**

Nanotechnology is one of the rapidly emerging fields in the twenty-first century with extensive increase of nanoparticle application for the treatment of a wide variety of chronic diseases such as cancer. P. Ehrlich's visionary concept of "magic bullet" based on the use of targeted medicines to effectively attack cancer cells has provided a promising field for cancer therapy [1]. Targeted delivery to solid cancers provides more bioavailability and effective approach for cancer treatment. The characteristics of nanocarriers such as their nanoscale, high surface-to-volume ratio, favorable drug release profiles, and targeting modifications allow them to target tumor tissue in an effective manner and release drugs in a stable and controlled manner [2]. NPs can accumulate in the leaky vasculatures of tumor tissue in an enhanced permeability and retention effect (EPR). The potential of nanomedicine can be explored in the field of early detection of cancer as well as in combination therapies for treating tumor earlier and effectively. NPs effectively solve the physiological barriers such as renal, hepatic, and immune related for effective drug delivery of conventional chemotherapeutic drugs [3]. NPs may be modified to utilize passive and active targeting mechanism to reach the tumor tissue. The nanodelivery-based carriers range from natural polymeric materials to nonbiodegradable gold NP, and magnetic mesoporous silica-based, metal-based NP. The surface of the NP can be suitably modified with ligands or drugs to offer multimodular treatment options [4]. The nanoparticle shape also plays an important role

in specific and effective nanodrug delivery. Nano-based drug delivery system has enhanced pharmacokinetic parameters, such as clearance value, volume distribution, and bioavailability to cancer cells through EPR. Unfortunately, these novel drug delivery systems still face barriers when delivered into the body, which can reduce the targeting efficiency as well as have increased toxic side effects. NPs have shown distinct toxicity patterns as compared with their larger counterparts [5]. As the size of NPs gets reduced for effective targeting, the number of surface molecules and surface area increase exponentially, which leads to complex bio-physiochemical interactions at the bio-nano interfaces when exposed to physiological environments. The potential paradigms of nanotoxicity can be understood possibly by understanding these bio-nano interactions. Since nanomaterials and therapeutic drug in combination work against cancer, the unfavorable toxicity of nanomaterials causes side effects and dysfunctions. Since the nanomedicines and therapeutic drugs share the same fate in the body, understanding the interconnections between nanotoxicity and drug delivery can widen our knowledge to improve the possibilities for cancer therapy. The effect of NPs can be divided into two categories, that is, primary and secondary depending upon the exposure time period [6]. The direct contact of NPs with cells results in primary effect, which involves toxicity, oxidative stress, DNA damage, and inflammation. Due to their nano-based size, the nanoparticles can translocate into the blood through tissue barriers where they can circulate and eventually accumulate in other organs, thereby, generating a secondary response of the NP. The secondary toxic effect of NPs might occur at the site of nanoparticle accumulation in organs such as the liver, spleen, or kidneys, and can stimulate systemic inflammation or can alter their systemic function [7]. The toxicity of NPs has been studied in different biological systems involving the cell lines as well as different organisms, which involve humans, rodents, zebra fish, catfish, algae, and macrophages. Carbon and metallic NPs are the most widely studied and used engineered nanomaterials. Nanometals, such as nanogold (nano-Au), nanosilver (nano-Ag), nanocopper, nanoaluminum, nanonickel, nanocobalt, and other NPs, have also been extensively studied. Toxic effect of metal oxide NPs such as nano-TiO2, nano-ZnO, nano-CuO, nano-CuZn, nano-Fe3O4, and nano-Fe2O3, with nano-TiO2 and nano-ZnO in particular, has been reported [8]. As expected, different nanomaterials exhibit different toxic potency. For example, Zhu et al. compared the toxicity of three nanometal oxides, nano-CuO, nano-CdO, and nano-TiO2. Nano-CuO was determined to be the most potent in cytotoxicity and DNA damage, leading to 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation, while nano-TiO2 was the least, without inducing a significant level of 8-OHdG [9]. The production of carbon nanotubes (CNTs) and graphene oxide is becoming commercially important. Under some experimental conditions, investigators have found that CNTs and graphene oxide are toxic. So, understanding the matter of safety and toxicity of nanomaterials has become an issue of interest to the public. Therefore, understanding the interactions of nanomaterials with biological systems is a particularly important scientific issue.

#### **2. Physical and chemical properties of NPs in nanotoxicity**

Toxic effect of NPs can proceed through a variety of mechanisms. Toxicity from a nanoparticle depends on its physical and chemical properties as well as the testing systems such as different cell types. The fundamental physical and chemical properties, which include molecular shape, size, oxidation status, surface area, bonded surface species, surface coating, solubility, and degree of aggregation and agglomeration of nanomaterials, majorly lead to the generation of reactive oxygen

**141**

*Biochemical Toxicology: Heavy Metals and Nanomaterials*

biological factors and lead to the mechanism of toxicity.

species and toxicity [10]. These intrinsic properties of nanomaterials can stimulate and generate toxic effects inside the biological system. Also, interaction with environmental factors such as light also determines how nanomaterials interact with the

Their nanosize and large surface area are the unique physiochemical properties of nanomaterials that determine their toxicity. Due to their very small size, they have the ability to penetrate into cell membrane and other biological barriers into living organisms and can inhibit cellular functions [11]. The increased nanoparticle size decreases its ability for cellular uptake. Majorly due to their nanosize, nanomaterials can even target the lungs and give rise to several toxic effects. Yoshida et al. had reported that particle size plays a major role in intracellular disruption of amorphous silica and its induced reactive oxygen species (ROS) formation, leading to DNA damage in human skin HaCaT cells [12]. Moreover, as the size of nanoparticle decreases, the toxic effects increase. Alpha-MnO2 nanowire, which is a wire-shaped nanomaterial, induces cytotoxicity, DNA oxidative damage, and apoptosis in HeLa cells [13]. In support of this statement, it was shown that long nanowires in cultured fibroblasts inhibited cell division, DNA damage, and increased ROS. Similarly, WISH cells when exposed to TiO2 induced cytotoxicity alterations in morphology, production of ROS, and DNA damage. Sohaebuddin et al. determined the effects of the chemical composition of nano-TiO2, nano-SiO2, and multiwall CNTs on their toxicity in 3T3 fibroblasts, RAW 264.7 macrophages, and telomerase-immortalized bronchiolar epithelial cells [14]. The results indicated that the composition, molecular size, and target cell type are all critical determinants of intracellular responses, degree of cytotoxicity, and potential mechanisms of toxicity. Moreover, these nanomaterials induced cell-specific responses, resulting in variable toxicity and subsequent cell damage. A study by Yin et al. showed that the smaller the particle size, the greater the cellular damage induced. He studied the photocytotoxicity of four different sized (<25, 31, <100, and 325 nm) nano-TiO2 and two different crystal forms antase and rutile in human skin keratinocytes. Upon exposure to UVA radiation, all nano-TiO2 particles induced cytotoxicity and cell membrane damage in a light- and dose-dependent manner. Similarly, in a study with different sizes of silica-titania hollow particle with uniform diameters of 25, 50, 75, 100, and 125 nm, the 50-nm silica-titania hollow NP showed the largest

The shape of the nanoparticle is one of the major determinants of nanomaterial-

induced cytotoxicity. This was supported by the study done by Ray and his coworkers where they determined that a set of gold NPs with different shapes had similar cytotoxicity [16]. The shape of the nanoparticle is considered as a major determinant in the process of engineering and application. The characteristic shapes of NP are mainly spherical, ellipsoidal, sheet-like, cubic, and rod-like. Spherical NPs have shown to be more prone to endocytosis than nanotubes and nanofibers. Similarly, a study with different shapes (needle-like, plate-like, rodlike, and spherical) of hydroxyapatite NPs on cultured BEAS-2B cells showed that plant-like and needle-like NPs showed higher cell death than spherical and rod-like NPs [17]. This might be due to the fact that needle-like NPs have the capacity of damaging cells upon direct contact to the cell surface. An interesting study with graphene oxide nanosheets showed that the toxicity of these NPs was determined by their shape allowing them to physically damage the cell membrane. However, the toxicity of these NPs was reduced with increasing concentration of the fetal calf serum in the cell culture media. This phenomenon was explained on the basis

*DOI: http://dx.doi.org/10.5772/intechopen.90928*

toxicity effect in macrophages [15].

**2.1 Size and shape**

species and toxicity [10]. These intrinsic properties of nanomaterials can stimulate and generate toxic effects inside the biological system. Also, interaction with environmental factors such as light also determines how nanomaterials interact with the biological factors and lead to the mechanism of toxicity.

#### **2.1 Size and shape**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

in specific and effective nanodrug delivery. Nano-based drug delivery system has enhanced pharmacokinetic parameters, such as clearance value, volume distribution, and bioavailability to cancer cells through EPR. Unfortunately, these novel drug delivery systems still face barriers when delivered into the body, which can reduce the targeting efficiency as well as have increased toxic side effects. NPs have shown distinct toxicity patterns as compared with their larger counterparts [5]. As the size of NPs gets reduced for effective targeting, the number of surface molecules and surface area increase exponentially, which leads to complex bio-physiochemical interactions at the bio-nano interfaces when exposed to physiological environments. The potential paradigms of nanotoxicity can be understood possibly by understanding these bio-nano interactions. Since nanomaterials and therapeutic drug in combination work against cancer, the unfavorable toxicity of nanomaterials causes side effects and dysfunctions. Since the nanomedicines and therapeutic drugs share the same fate in the body, understanding the interconnections between nanotoxicity and drug delivery can widen our knowledge to improve the possibilities for cancer therapy. The effect of NPs can be divided into two categories, that is, primary and secondary depending upon the exposure time period [6]. The direct contact of NPs with cells results in primary effect, which involves toxicity, oxidative stress, DNA damage, and inflammation. Due to their nano-based size, the nanoparticles can translocate into the blood through tissue barriers where they can circulate and eventually accumulate in other organs, thereby, generating a secondary response of the NP. The secondary toxic effect of NPs might occur at the site of nanoparticle accumulation in organs such as the liver, spleen, or kidneys, and can stimulate systemic inflammation or can alter their systemic function [7]. The toxicity of NPs has been studied in different biological systems involving the cell lines as well as different organisms, which involve humans, rodents, zebra fish, catfish, algae, and macrophages. Carbon and metallic NPs are the most widely studied and used engineered nanomaterials. Nanometals, such as nanogold (nano-Au), nanosilver (nano-Ag), nanocopper, nanoaluminum, nanonickel, nanocobalt, and other NPs, have also been extensively studied. Toxic effect of metal oxide NPs such as nano-TiO2, nano-ZnO, nano-CuO, nano-CuZn, nano-Fe3O4, and nano-Fe2O3, with nano-TiO2 and nano-ZnO in particular, has been reported [8]. As expected, different nanomaterials exhibit different toxic potency. For example, Zhu et al. compared the toxicity of three nanometal oxides, nano-CuO, nano-CdO, and nano-TiO2. Nano-CuO was determined to be the most potent in cytotoxicity and DNA damage, leading to 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation, while nano-TiO2 was the least, without inducing a significant level of 8-OHdG [9]. The production of carbon nanotubes (CNTs) and graphene oxide is becoming commercially important. Under some experimental conditions, investigators have found that CNTs and graphene oxide are toxic. So, understanding the matter of safety and toxicity of nanomaterials has become an issue of interest to the public. Therefore, understanding the interactions of nanomaterials with biological systems is a particularly

**140**

important scientific issue.

**2. Physical and chemical properties of NPs in nanotoxicity**

Toxic effect of NPs can proceed through a variety of mechanisms. Toxicity from a nanoparticle depends on its physical and chemical properties as well as the testing systems such as different cell types. The fundamental physical and chemical properties, which include molecular shape, size, oxidation status, surface area, bonded surface species, surface coating, solubility, and degree of aggregation and agglomeration of nanomaterials, majorly lead to the generation of reactive oxygen

Their nanosize and large surface area are the unique physiochemical properties of nanomaterials that determine their toxicity. Due to their very small size, they have the ability to penetrate into cell membrane and other biological barriers into living organisms and can inhibit cellular functions [11]. The increased nanoparticle size decreases its ability for cellular uptake. Majorly due to their nanosize, nanomaterials can even target the lungs and give rise to several toxic effects. Yoshida et al. had reported that particle size plays a major role in intracellular disruption of amorphous silica and its induced reactive oxygen species (ROS) formation, leading to DNA damage in human skin HaCaT cells [12]. Moreover, as the size of nanoparticle decreases, the toxic effects increase. Alpha-MnO2 nanowire, which is a wire-shaped nanomaterial, induces cytotoxicity, DNA oxidative damage, and apoptosis in HeLa cells [13]. In support of this statement, it was shown that long nanowires in cultured fibroblasts inhibited cell division, DNA damage, and increased ROS. Similarly, WISH cells when exposed to TiO2 induced cytotoxicity alterations in morphology, production of ROS, and DNA damage. Sohaebuddin et al. determined the effects of the chemical composition of nano-TiO2, nano-SiO2, and multiwall CNTs on their toxicity in 3T3 fibroblasts, RAW 264.7 macrophages, and telomerase-immortalized bronchiolar epithelial cells [14]. The results indicated that the composition, molecular size, and target cell type are all critical determinants of intracellular responses, degree of cytotoxicity, and potential mechanisms of toxicity. Moreover, these nanomaterials induced cell-specific responses, resulting in variable toxicity and subsequent cell damage. A study by Yin et al. showed that the smaller the particle size, the greater the cellular damage induced. He studied the photocytotoxicity of four different sized (<25, 31, <100, and 325 nm) nano-TiO2 and two different crystal forms antase and rutile in human skin keratinocytes. Upon exposure to UVA radiation, all nano-TiO2 particles induced cytotoxicity and cell membrane damage in a light- and dose-dependent manner. Similarly, in a study with different sizes of silica-titania hollow particle with uniform diameters of 25, 50, 75, 100, and 125 nm, the 50-nm silica-titania hollow NP showed the largest toxicity effect in macrophages [15].

The shape of the nanoparticle is one of the major determinants of nanomaterialinduced cytotoxicity. This was supported by the study done by Ray and his coworkers where they determined that a set of gold NPs with different shapes had similar cytotoxicity [16]. The shape of the nanoparticle is considered as a major determinant in the process of engineering and application. The characteristic shapes of NP are mainly spherical, ellipsoidal, sheet-like, cubic, and rod-like. Spherical NPs have shown to be more prone to endocytosis than nanotubes and nanofibers. Similarly, a study with different shapes (needle-like, plate-like, rodlike, and spherical) of hydroxyapatite NPs on cultured BEAS-2B cells showed that plant-like and needle-like NPs showed higher cell death than spherical and rod-like NPs [17]. This might be due to the fact that needle-like NPs have the capacity of damaging cells upon direct contact to the cell surface. An interesting study with graphene oxide nanosheets showed that the toxicity of these NPs was determined by their shape allowing them to physically damage the cell membrane. However, the toxicity of these NPs was reduced with increasing concentration of the fetal calf serum in the cell culture media. This phenomenon was explained on the basis

that graphene oxide NPs had the capacity to adsorb the protein molecules, which covered the nanoparticle surface which changed the shape of the nanoparticle and partly prevented cell damage.

#### **2.2 Surface charge**

The surface charge of NPs plays an important role in determining the nanotoxicity as it largely determines the interactions of the NP with biological systems. Positively charged NPs have been reported to have high toxicity due to their easy penetration into cells rather than the negatively charged nanoparticles [18]. This is due to the electrostatic attraction between the negatively charged cell membrane and positively charged NP. A comparative study of the toxic effects of negatively and positively charged polystyrene NPs on HeLa and HIH/3T3 cells has shown that the positively charged NPs were relatively more toxic. This is due to the ability of positively charged cells to easily penetrate through the cell membrane; also, they strongly bind to the negatively charged DNA, causing its damage, and prolong the G0/G1 phase of the cells. Negatively charged NPs have not been reported to have any effect on cell cycle. Similar observations have been reported with gold NPs where positively charged NPs were highly adsorbed and showed toxic effects rather than the negatively charged gold nanoparticle. Positively charged NPs have increased capacity of opsonization, which involves the process of adsorption of proteins facilitating phagocytosis, including antibodies and complement components from blood and biological fluids [19]. The adsorbed protein to the surface of nanoparticle which is normally referred to as protein crown may affect the surface properties of the NP. The protein crown contains serum proteins such as albumin, fibrinogens, and immunoglobulin G and several other functional molecules. *In vitro* experiments with quantum dots coated with a hydrophilic polymer enhance the fibril formation of human β2 microglobulin, which is arranged into multilayered structures on the surface of nanoparticle resulting in local increase in the protein concentration on the nanoparticle surface, precipitation, and formation of oligomers [20]. The charge of the nanoparticle can be modified from negative to positive via various modifications of the surface. So, Xu et al. had developed a method of changing the charge in polymer NP with the help of a pH-sensitive polymer that helps the negatively charged particles in a neutral medium acquire a positive charge in an acidic medium of pH 5–6 [21]. The cytotoxic effect estimated from surface-modified cerium oxide NP in H9C2, HEK293, A549, and MCF-7 cells showed that different polymers enable the nanoparticle charge modification, thereby showing different biological and toxic effects. Specifically, positive and neutral charged NPs are absorbed by all cell types at the same rate, whereas negatively charged NPs have the tendency to accumulate inside the biological tissues. So, modifying the charge of NPs allows to control their localization and toxicity, which can help in improving effective systems for targeted chemotherapeutic drug delivery to the tumor site.

#### **3. Nanoparticle shell**

Improving the optical, magnetic, and electrical properties of nanomaterials application of a shell onto the surface of NP is quite important as it also improves the biocompatibility and solubility of NPs in water and other biological fluids by decreasing their capacity to aggregate and increasing their stability. Therefore, the shell reduces the toxic effect of NPs and provides them the capacity to selectively

**143**

(**Figure 1**).

*Biochemical Toxicology: Heavy Metals and Nanomaterials*

interact with different types of cells and biological molecules [22]. In addition, the shell influences the pharmacokinetics of NP, which considerably changes the pattern of nanoparticle distribution and accumulation inside the body. Most of the nanoparticle toxicity has been reported due to the formation of free radicals inside the cells [23]. However, the shell has the capability to: reduce or eliminate these negative side effects as well as stabilize the NP, increase the resistance of NPs toward environmental factors, and enable them to acquire the capacity to selectively interact with the biological molecules. In regard to this point, Cho et al. demonstrated that polymer NPs could be modified with lectins and were able to selectively bind to the tumor cells presenting sialic acid on their surface, which made the nanoparticle suitable for specifically labeling cancer cells [24]. The surface of the NP can be modified using both organic and inorganic compounds such as polyethylene glycol, polyglycolic acid, lipids, proteins, low-molecular weight compounds and silicon. These modifiers make complex nanoparticle surface and change the nanoparticle properties for their specific transport and accumulation. The toxicity of quantum dots is significantly reduced using shells as the core of quantum dots is mostly hydrophobic and mainly consists of toxic heavy metals such as cadmium, tellurium, and mercury [25]. The shell enhances the stability of the core of quantum dots, thereby preventing its desalinization and oxidative or photolytic degradation. This ultimately prevents the leakage of heavy metal ions from the quantum core,

Nanotechnology has been an emerging field to determine the set standards or to formulate a set of designed rules for designing safe nanomaterials. The ability of nanomaterials to accumulate in different organs has resulted in some severe side effects and has hindered their use in the field of nanomedicine. So, understanding the mechanism that underlies the toxicity of nanomaterials may provide clues for overcoming the toxic effects of NPs. A major mechanism of nanotoxicity is by the generation of reactive oxygen species (ROS), which results in the subsequent formation of oxidative stress in tissues [27]. The induction of oxidative stress simultaneously activates the pro-inflammatory mediators via the principle cascades such as the nuclear factor-κB (NF-κb), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K) pathways [28]. The most widely used nanomaterials are mostly the carbon nanotubes and metallic nanomaterials. Radomski et al. reported that engineered carbon NPs and nanotubes induced the aggregation of platelets in vitro, and enhanced vascular thrombosis in rat carotid artery [29]. Similarly, the single-walled carbon nanotubes showed enhanced cell apoptosis and decreased cell adhesion by upregulating genes involved in cell death or downregulating genes involved in cell proliferation and survival in cellular models of human kidney and bronchi. With the application of skin lotion and creams that majorly contain nano-TiO2 and nano-ZnO, the skin is in continuous exposure to the toxic nanometals that can accumulate in the brain and can cause auxiliary toxicity resulting in the disruption of normal metabolism of neurotransmitters and ultimately leading to the cause of brain damage. While comparing the toxicity of three nanometal oxides, nano-CuO, nano-CdO, and nano-TiO2, nano-CuO was determined to be the most potent in regard to cytotoxicity and DNA damage, leading to 8-hydroxy-20-deoxyguanosine (8-OHdG) formation, while nano-TiO2 was the least potent, without inducing a significant level of 8-OHdG [9]

*DOI: http://dx.doi.org/10.5772/intechopen.90928*

thereby preventing nanotoxicity [26].

**4. Mechanism of nanotoxicity**

*Biochemical Toxicology: Heavy Metals and Nanomaterials DOI: http://dx.doi.org/10.5772/intechopen.90928*

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

partly prevented cell damage.

**2.2 Surface charge**

that graphene oxide NPs had the capacity to adsorb the protein molecules, which covered the nanoparticle surface which changed the shape of the nanoparticle and

The surface charge of NPs plays an important role in determining the nanotoxicity as it largely determines the interactions of the NP with biological systems. Positively charged NPs have been reported to have high toxicity due to their easy penetration into cells rather than the negatively charged nanoparticles [18]. This is due to the electrostatic attraction between the negatively charged cell membrane and positively charged NP. A comparative study of the toxic effects of negatively and positively charged polystyrene NPs on HeLa and HIH/3T3 cells has shown that the positively charged NPs were relatively more toxic. This is due to the ability of positively charged cells to easily penetrate through the cell membrane; also, they strongly bind to the negatively charged DNA, causing its damage, and prolong the G0/G1 phase of the cells. Negatively charged NPs have not been reported to have any effect on cell cycle. Similar observations have been reported with gold NPs where positively charged NPs were highly adsorbed and showed toxic effects rather than the negatively charged gold nanoparticle. Positively charged NPs have increased capacity of opsonization, which involves the process of adsorption of proteins facilitating phagocytosis, including antibodies and complement components from blood and biological fluids [19]. The adsorbed protein to the surface of nanoparticle which is normally referred to as protein crown may affect the surface properties of the NP. The protein crown contains serum proteins such as albumin, fibrinogens, and immunoglobulin G and several other functional molecules. *In vitro* experiments with quantum dots coated with a hydrophilic polymer enhance the fibril formation of human β2 microglobulin, which is arranged into multilayered structures on the surface of nanoparticle resulting in local increase in the protein concentration on the nanoparticle surface, precipitation, and formation of oligomers [20]. The charge of the nanoparticle can be modified from negative to positive via various modifications of the surface. So, Xu et al. had developed a method of changing the charge in polymer NP with the help of a pH-sensitive polymer that helps the negatively charged particles in a neutral medium acquire a positive charge in an acidic medium of pH 5–6 [21]. The cytotoxic effect estimated from surface-modified cerium oxide NP in H9C2, HEK293, A549, and MCF-7 cells showed that different polymers enable the nanoparticle charge modification, thereby showing different biological and toxic effects. Specifically, positive and neutral charged NPs are absorbed by all cell types at the same rate, whereas negatively charged NPs have the tendency to accumulate inside the biological tissues. So, modifying the charge of NPs allows to control their localization and toxicity, which can help in improving effective systems for targeted chemotherapeutic drug

Improving the optical, magnetic, and electrical properties of nanomaterials application of a shell onto the surface of NP is quite important as it also improves the biocompatibility and solubility of NPs in water and other biological fluids by decreasing their capacity to aggregate and increasing their stability. Therefore, the shell reduces the toxic effect of NPs and provides them the capacity to selectively

**142**

delivery to the tumor site.

**3. Nanoparticle shell**

interact with different types of cells and biological molecules [22]. In addition, the shell influences the pharmacokinetics of NP, which considerably changes the pattern of nanoparticle distribution and accumulation inside the body. Most of the nanoparticle toxicity has been reported due to the formation of free radicals inside the cells [23]. However, the shell has the capability to: reduce or eliminate these negative side effects as well as stabilize the NP, increase the resistance of NPs toward environmental factors, and enable them to acquire the capacity to selectively interact with the biological molecules. In regard to this point, Cho et al. demonstrated that polymer NPs could be modified with lectins and were able to selectively bind to the tumor cells presenting sialic acid on their surface, which made the nanoparticle suitable for specifically labeling cancer cells [24]. The surface of the NP can be modified using both organic and inorganic compounds such as polyethylene glycol, polyglycolic acid, lipids, proteins, low-molecular weight compounds and silicon. These modifiers make complex nanoparticle surface and change the nanoparticle properties for their specific transport and accumulation. The toxicity of quantum dots is significantly reduced using shells as the core of quantum dots is mostly hydrophobic and mainly consists of toxic heavy metals such as cadmium, tellurium, and mercury [25]. The shell enhances the stability of the core of quantum dots, thereby preventing its desalinization and oxidative or photolytic degradation. This ultimately prevents the leakage of heavy metal ions from the quantum core, thereby preventing nanotoxicity [26].

#### **4. Mechanism of nanotoxicity**

Nanotechnology has been an emerging field to determine the set standards or to formulate a set of designed rules for designing safe nanomaterials. The ability of nanomaterials to accumulate in different organs has resulted in some severe side effects and has hindered their use in the field of nanomedicine. So, understanding the mechanism that underlies the toxicity of nanomaterials may provide clues for overcoming the toxic effects of NPs. A major mechanism of nanotoxicity is by the generation of reactive oxygen species (ROS), which results in the subsequent formation of oxidative stress in tissues [27]. The induction of oxidative stress simultaneously activates the pro-inflammatory mediators via the principle cascades such as the nuclear factor-κB (NF-κb), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K) pathways [28]. The most widely used nanomaterials are mostly the carbon nanotubes and metallic nanomaterials. Radomski et al. reported that engineered carbon NPs and nanotubes induced the aggregation of platelets in vitro, and enhanced vascular thrombosis in rat carotid artery [29]. Similarly, the single-walled carbon nanotubes showed enhanced cell apoptosis and decreased cell adhesion by upregulating genes involved in cell death or downregulating genes involved in cell proliferation and survival in cellular models of human kidney and bronchi. With the application of skin lotion and creams that majorly contain nano-TiO2 and nano-ZnO, the skin is in continuous exposure to the toxic nanometals that can accumulate in the brain and can cause auxiliary toxicity resulting in the disruption of normal metabolism of neurotransmitters and ultimately leading to the cause of brain damage. While comparing the toxicity of three nanometal oxides, nano-CuO, nano-CdO, and nano-TiO2, nano-CuO was determined to be the most potent in regard to cytotoxicity and DNA damage, leading to 8-hydroxy-20-deoxyguanosine (8-OHdG) formation, while nano-TiO2 was the least potent, without inducing a significant level of 8-OHdG [9] (**Figure 1**).

#### **Figure 1.**

*Mechanism of nanotoxicity. The major mechanism of nanotoxicity is by the generation of reactive oxygen species (ROS), which results in the subsequent formation of oxidative stress in tissues. The induction of oxidative stress simultaneously activates the pro-inflammatory mediators via the principle cascades such as the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K) pathways. The other major effects are protein oxidation and DNA damage, which leads to apoptosis or cell cycle inhibition.*

#### **4.1 Nanotoxicity via ROS production**

The ROS generation and the subsequent production of oxidative stress are major causes of nanotoxicity, which involves DNA damage, unregulated cell signaling, changes in cell motility, cytotoxicity, apoptosis, and cancer initiation and progression. The amount and effect of ROS generation are completely dependent on the chemical nature of the nanomaterials [30]. Engineered nanomaterials have relatively small size, high specific volume-to-area ratio, and high surface reactivity, which results in higher production of ROS, simultaneously resulting in cytotoxicity and genotoxicity [31]. A variety of nanomaterials has been reported to induce nanotoxicity, that is, mediated by ROS in many biological systems such as human erythrocytes and fibroblasts. Quantum dots have been reported to have toxic effects produced by ROS-mediated oxidative stress and cell death. Akhtar et al. reported that silica NPs induced cellular stress and cytotoxicity in a dose-dependent manner, which is mediated by the induction of ROS and lipid peroxidation in cell membranes [32]. Nano-CuO induced cytotoxicity in mouse embryonic fibroblasts (BALB 3T3) by releasing lactate dehydrogenase, causing oxidative stress in a dose-dependent manner mediated by the induction of ROS and lipid peroxidation. Nano-ZnO has been reported to induce cytotoxicity that is mostly mediated by the induction of ROS, causing oxidative injury simultaneously releasing inflammatory mediators resulting in cell death in phagocytic RAW 264.7 cells, and transformation in human bronchial epithelial BEAS-2B cells [17]. Nano-Ag has been reported to induce apoptosis in NIH3T3 cells, which is mainly mediated via ROS and C-Jun terminal kinase-dependent mechanism involving the mitochondrial pathway. Also, nano-Ag–induced mutation and oxidative stress in mouse lymphoma cells. Shvedova et al. reported that keratinocytes incubated with high doses of single-walled CNTs resulted in ROS production, thereby leading to cellular and mitochondrial

**145**

*Biochemical Toxicology: Heavy Metals and Nanomaterials*

dysfunction. Comparison of cytotoxicity of the four nanometal oxides nano-ZnO, nano-TiO2, nano-Co3O4, and nano-CuO in catfish hepatocytes and human HepG2 cells induced toxicity in the order of TiO2 < Co3O4 < ZnO < CuO and the major cause

DNA is one of the major targets of ROS. Toxicity of NPs is often specified for ROS production that ultimately damages the genetic material, thereby causing cell death. NPs are responsible for a wide variety of DNA damage such as chromosomal fragmentation, DNA strand breakages, and the induction of mutation in genes [34]. Gold NPs 20 nm in size at concentration of 1 nM have been reported to exhibit DNA damage in the form of 8-hydroxydeoxyguanosine (8OHdG), adduct formation in the embryonic lung fibroblasts, having a very low expression for DNA repair and cell cycle check point genes [35]. Several reports have also confirmed that metal oxide NPs induce DNA fragmentation and formation of oxidation-induced DNA adducts. The main functional molecule that comes into play in response to DNA damage is p53. Metal oxide NPs including TiO2, ZnO, Fe3O4, Al2O3, and CrO3 of particle sizes ranging from 30 to 45 nm have been reported to induce apoptosis [36]. Cadmium telluride quantum dots were found to significantly increase p53 levels and upregulate the p53-downstream effectors Bax, Puma, and Noxa in human breast

Oxidative stress induction is relatively linked to inflammation through the release of pro-inflammatory mediators through the cascade such as the NF-κB (nuclear factor-κB), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K) pathways [38]. Inflammation is majorly a type of defense mechanism of the body that involves several immune regulatory molecules followed by the infiltration of phagocytic cells. The induction of inflammation in several cell types such as the alveolar and bronchial epithelial cells, epidermal keratinocytes, and cultured monocyte-macrophage cells has been reported with single and multi-walled carbon nanotubes and fullerene derivatives. A recent study has been able to provide a mechanistic explanation for immune and inflammatory responses initiated upon exposure to carbon NPs [39]. This observation reported that the immune system receptors like toll-like receptors recognize carbon nanotubes and C60 fullerenes as pathogens and thereby trigger the inflammatory responses by secreting inflammatory protein mediators such as interleukins and chemokines. Similarly, exposure of liposomes and other lipid-based NPs trigger the activation of the complementary cascade leading to hypersensitivity reactions and anaphylaxis [40]. However, the exact mechanism through which these complement proteins mediate nanotoxicity has not been elucidated. In the absence of a stimulus, NF-κB is degraded in the cytoplasm by the Inhibitor of κB (IκB) family of inhibitors. The reactive oxygen species play a major role in the induction of the NF-κB, resulting in the inflammatory responses. Both *in vitro* and *in vivo* studies showed that nanoparticle-induced lung injury and pulmonary fibrosis lead to the ROS-mediated activation of NF-κB and production of pro-inflammatory mediators such as TNF-α, IL-8, IL-2, and IL-6 [41]. Metal oxide NPs such as zinc, cadmium, silica, and iron have also been reported to show toxic effects via the induction of inflammatoryrelated cytokine release induced by NF-κB. The single-walled and multiple-walled carbon nanotubes were also shown to promote inflammatory responses in mice by generating the TNF-α and monocyte chemoattractant protein-1 (MCP-1) [42].

was the ROS generation leading to cell and mitochondrial damage [15, 33].

*DOI: http://dx.doi.org/10.5772/intechopen.90928*

**4.2 DNA damage**

carcinoma cells [37].

**4.3 Inflammation**

dysfunction. Comparison of cytotoxicity of the four nanometal oxides nano-ZnO, nano-TiO2, nano-Co3O4, and nano-CuO in catfish hepatocytes and human HepG2 cells induced toxicity in the order of TiO2 < Co3O4 < ZnO < CuO and the major cause was the ROS generation leading to cell and mitochondrial damage [15, 33].

#### **4.2 DNA damage**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

**4.1 Nanotoxicity via ROS production**

**Figure 1.**

*inhibition.*

The ROS generation and the subsequent production of oxidative stress are major causes of nanotoxicity, which involves DNA damage, unregulated cell signaling, changes in cell motility, cytotoxicity, apoptosis, and cancer initiation and progression. The amount and effect of ROS generation are completely dependent on the chemical nature of the nanomaterials [30]. Engineered nanomaterials have relatively small size, high specific volume-to-area ratio, and high surface reactivity, which results in higher production of ROS, simultaneously resulting in cytotoxicity and genotoxicity [31]. A variety of nanomaterials has been reported to induce nanotoxicity, that is, mediated by ROS in many biological systems such as human erythrocytes and fibroblasts. Quantum dots have been reported to have toxic effects produced by ROS-mediated oxidative stress and cell death. Akhtar et al. reported that silica NPs induced cellular stress and cytotoxicity in a dose-dependent manner, which is mediated by the induction of ROS and lipid peroxidation in cell membranes [32]. Nano-CuO induced cytotoxicity in mouse embryonic fibroblasts (BALB 3T3) by releasing lactate dehydrogenase, causing oxidative stress in a dose-dependent manner mediated by the induction of ROS and lipid peroxidation. Nano-ZnO has been reported to induce cytotoxicity that is mostly mediated by the induction of ROS, causing oxidative injury simultaneously releasing inflammatory mediators resulting in cell death in phagocytic RAW 264.7 cells, and transformation in human bronchial epithelial BEAS-2B cells [17]. Nano-Ag has been reported to induce apoptosis in NIH3T3 cells, which is mainly mediated via ROS and C-Jun terminal kinase-dependent mechanism involving the mitochondrial pathway. Also, nano-Ag–induced mutation and oxidative stress in mouse lymphoma cells. Shvedova et al. reported that keratinocytes incubated with high doses of single-walled CNTs resulted in ROS production, thereby leading to cellular and mitochondrial

*Mechanism of nanotoxicity. The major mechanism of nanotoxicity is by the generation of reactive oxygen species (ROS), which results in the subsequent formation of oxidative stress in tissues. The induction of oxidative stress simultaneously activates the pro-inflammatory mediators via the principle cascades such as the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K) pathways. The other major effects are protein oxidation and DNA damage, which leads to apoptosis or cell cycle* 

**144**

DNA is one of the major targets of ROS. Toxicity of NPs is often specified for ROS production that ultimately damages the genetic material, thereby causing cell death. NPs are responsible for a wide variety of DNA damage such as chromosomal fragmentation, DNA strand breakages, and the induction of mutation in genes [34]. Gold NPs 20 nm in size at concentration of 1 nM have been reported to exhibit DNA damage in the form of 8-hydroxydeoxyguanosine (8OHdG), adduct formation in the embryonic lung fibroblasts, having a very low expression for DNA repair and cell cycle check point genes [35]. Several reports have also confirmed that metal oxide NPs induce DNA fragmentation and formation of oxidation-induced DNA adducts. The main functional molecule that comes into play in response to DNA damage is p53. Metal oxide NPs including TiO2, ZnO, Fe3O4, Al2O3, and CrO3 of particle sizes ranging from 30 to 45 nm have been reported to induce apoptosis [36]. Cadmium telluride quantum dots were found to significantly increase p53 levels and upregulate the p53-downstream effectors Bax, Puma, and Noxa in human breast carcinoma cells [37].

#### **4.3 Inflammation**

Oxidative stress induction is relatively linked to inflammation through the release of pro-inflammatory mediators through the cascade such as the NF-κB (nuclear factor-κB), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K) pathways [38]. Inflammation is majorly a type of defense mechanism of the body that involves several immune regulatory molecules followed by the infiltration of phagocytic cells. The induction of inflammation in several cell types such as the alveolar and bronchial epithelial cells, epidermal keratinocytes, and cultured monocyte-macrophage cells has been reported with single and multi-walled carbon nanotubes and fullerene derivatives. A recent study has been able to provide a mechanistic explanation for immune and inflammatory responses initiated upon exposure to carbon NPs [39]. This observation reported that the immune system receptors like toll-like receptors recognize carbon nanotubes and C60 fullerenes as pathogens and thereby trigger the inflammatory responses by secreting inflammatory protein mediators such as interleukins and chemokines. Similarly, exposure of liposomes and other lipid-based NPs trigger the activation of the complementary cascade leading to hypersensitivity reactions and anaphylaxis [40]. However, the exact mechanism through which these complement proteins mediate nanotoxicity has not been elucidated. In the absence of a stimulus, NF-κB is degraded in the cytoplasm by the Inhibitor of κB (IκB) family of inhibitors. The reactive oxygen species play a major role in the induction of the NF-κB, resulting in the inflammatory responses. Both *in vitro* and *in vivo* studies showed that nanoparticle-induced lung injury and pulmonary fibrosis lead to the ROS-mediated activation of NF-κB and production of pro-inflammatory mediators such as TNF-α, IL-8, IL-2, and IL-6 [41]. Metal oxide NPs such as zinc, cadmium, silica, and iron have also been reported to show toxic effects via the induction of inflammatoryrelated cytokine release induced by NF-κB. The single-walled and multiple-walled carbon nanotubes were also shown to promote inflammatory responses in mice by generating the TNF-α and monocyte chemoattractant protein-1 (MCP-1) [42].

The MAP-kinase pathway regulates critical cellular processes such as cell proliferation, differentiation, mitosis, cell survival, and apoptosis. Treatment of human bronchial epithelial cell lines with titanium dioxide NPs showed interleukin (IL)-8 production via p38 MAPK and/or ERK pathway and mediated toxicity in the cell lines [43]. The model organism *C. elegans* used for in vivo toxicity assay studies of silver NPs with a size range of 20–30 nm showed that the toxicity mediated was due to the production of ROS, which consequently increased the expression of PMK-1 p38 MAPK and hypoxia-inducible factor (HIF-1) [44]. The toxicity of silica NPs, which hinders their application as drug delivery systems, has been attributed to the activation of JNK, p53, and NF-κB pathways and an elevated expression of pro-inflammatory factors IL-6, IL-8, and MCP-1 [45]. Also, single-walled nanocarbon of size range 0.8–2 nm was reported to have potential adverse cytotoxic effects in mesothelial cells via the activation of signaling molecules, including PARP, AP-1, NF-κB, p38, and Akt, in a dosedependent manner [46].

#### **5. Organ-/tissue-specific nanotoxicity**

Nanoparticles can easily penetrate the tissue system and damage body organs because of their smaller size and high specificity to the tissue system. It has been observed that nanoparticles can move fast in the blood stream and easily cross the blood-brain barrier, this may induce toxicity, which can be harmful for the human organ system (e.g., pulmonary system, reticuloendothelial systems, cardiovascular systems, central nervous system, skin, and embryonic cells) (**Figure 2**).

#### **5.1 Toxicity in pulmonary system**

The small-sized NPs have the ability to penetrate easily through the lungs and can cause lung injuries and generate ROS [47]. The pulmonary toxicity studies in rats with ultrafine and fine NPs such as carbon black, nickel, and TiO2 particles have shown enhanced pulmonary inflammation by the ultrafine NPs [48]. It is being reported that the toxic effects of NPs on lungs show characteristics such as development of exaggerated lung responses, high rate of pulmonary inflammation, failed clearance, cellular proliferation, fibroproliferative effects, and inflammatory-derived mutagenesis, ultimately leading to chronic effects like tumor development in lungs. Factors that mainly influence nanotoxicity in lungs are the particulate characteristics of NPs, such as particle size, number, surface area, surface dose, surface modifications, degree of aggregation, and method of particle synthesis [49, 50].

#### **5.2 Toxicity in reticuloendothelial systems**

The reticuloendothelial system in the liver is the main source of biological system where all the NPs get absorbed from the gastrointestinal tract into the cardiovascular systems, as all blood exiting from the gastrointestinal tract transport from the hepatic portal vein that directly diffuses to the liver. Carbon black and polystyrene NPs being less toxic NPs stimulate macrophages by the generation of ROS and activation of calcium signaling to release pro-inflammatory cytokines such as tumor necrosis factor-alpha [51]. Pro-inflammatory cytokines are also associated with pathology of liver disease where the generation of ROS molecule inhibits the hepatocyte function and bile formation.

**147**

microvessels [54].

**Figure 2.**

*immune disorders.*

*Biochemical Toxicology: Heavy Metals and Nanomaterials*

*DOI: http://dx.doi.org/10.5772/intechopen.90928*

**5.3 Toxicity in cardiovascular systems**

**5.4 Toxicity in central nervous system**

The positively charged NPs such as gold and polystyrene have been reported to cause hemolysis and clotting of blood, while the negatively charged NPs are reported to be nontoxic in nature. Increased exposure to diesel-exposed particles (DEP) in hypertensive rats through the process of inhalation resulted in altered heart rate in rats as interpreted through the pacemaker that determines the activity of the heart [52]. Exposure to single-walled NPs also showed altered cardiovascular effects [53]. The injection of ultrafine carbon black NPs into the blood of normal rats caused platelet accumulation in the hepatic microvasculature of the rats and also caused prothrombotic changes on the endothelial surface of the hepatic

*Tissue- and organ-specific nanotoxicity. The toxic accumulation of nanoparticles can affect any of the tissue types in the body. The small-sized nanoparticles have the ability to penetrate easily through the lungs and can cause lung injuries and have an ability to generate ROS that lead to toxic effects in lungs. The reticuloendothelial system in the liver is the main source of biological system where all the nanoparticles get absorbed from the gastrointestinal tract into the cardiovascular systems, as all blood exiting from the gastrointestinal tract transport from the hepatic portal vein that directly diffuses to the liver. Increased exposure to nanoparticles majorly happens through the process of inhalation, which results in altered heart rate. The inhalation of nanoparticles acquires the ability to reach the brain system mainly through the route of olfactory epithelium by the mechanism of transsynaptic transport or through their uptake via the blood-brain barrier. Nanoparticles acquire the ability to penetrate inside the skin and cause toxic effects such as dermatitis and auto* 

NPs on inhalation of acquire the ability to reach the brain system mainly through

the route of olfactory epithelium by the mechanism of transsynaptic transport or through their uptake via the blood-brain barrier [55]. Enhanced permeability of NPs through the blood-brain barrier has been reported to have increased the number of pathologies including hypertension and allergic encephalomyelitis. The surface charge of the nanoparticle has also been shown to have toxic effects on the brain leading to brain toxicity altering the blood-brain integrity [56]. NPs have *Biochemical Toxicology: Heavy Metals and Nanomaterials DOI: http://dx.doi.org/10.5772/intechopen.90928*

#### **Figure 2.**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

dependent manner [46].

**5. Organ-/tissue-specific nanotoxicity**

**5.1 Toxicity in pulmonary system**

**5.2 Toxicity in reticuloendothelial systems**

hepatocyte function and bile formation.

synthesis [49, 50].

The MAP-kinase pathway regulates critical cellular processes such as cell proliferation, differentiation, mitosis, cell survival, and apoptosis. Treatment of human bronchial epithelial cell lines with titanium dioxide NPs showed interleukin (IL)-8 production via p38 MAPK and/or ERK pathway and mediated toxicity in the cell lines [43]. The model organism *C. elegans* used for in vivo toxicity assay studies of silver NPs with a size range of 20–30 nm showed that the toxicity mediated was due to the production of ROS, which consequently increased the expression of PMK-1 p38 MAPK and hypoxia-inducible factor (HIF-1) [44]. The toxicity of silica NPs, which hinders their application as drug delivery systems, has been attributed to the activation of JNK, p53, and NF-κB pathways and an elevated expression of pro-inflammatory factors IL-6, IL-8, and MCP-1 [45]. Also, single-walled nanocarbon of size range 0.8–2 nm was reported to have potential adverse cytotoxic effects in mesothelial cells via the activation of signaling molecules, including PARP, AP-1, NF-κB, p38, and Akt, in a dose-

Nanoparticles can easily penetrate the tissue system and damage body organs because of their smaller size and high specificity to the tissue system. It has been observed that nanoparticles can move fast in the blood stream and easily cross the blood-brain barrier, this may induce toxicity, which can be harmful for the human organ system (e.g., pulmonary system, reticuloendothelial systems, cardiovascular

The small-sized NPs have the ability to penetrate easily through the lungs and can cause lung injuries and generate ROS [47]. The pulmonary toxicity studies in rats with ultrafine and fine NPs such as carbon black, nickel, and TiO2 particles have shown enhanced pulmonary inflammation by the ultrafine NPs [48]. It is being reported that the toxic effects of NPs on lungs show characteristics such as development of exaggerated lung responses, high rate of pulmonary inflammation, failed clearance, cellular proliferation, fibroproliferative effects, and inflammatory-derived mutagenesis, ultimately leading to chronic effects like tumor development in lungs. Factors that mainly influence nanotoxicity in lungs are the particulate characteristics of NPs, such as particle size, number, surface area, surface dose, surface modifications, degree of aggregation, and method of particle

The reticuloendothelial system in the liver is the main source of biological system where all the NPs get absorbed from the gastrointestinal tract into the cardiovascular systems, as all blood exiting from the gastrointestinal tract transport from the hepatic portal vein that directly diffuses to the liver. Carbon black and polystyrene NPs being less toxic NPs stimulate macrophages by the generation of ROS and activation of calcium signaling to release pro-inflammatory cytokines such as tumor necrosis factor-alpha [51]. Pro-inflammatory cytokines are also associated with pathology of liver disease where the generation of ROS molecule inhibits the

systems, central nervous system, skin, and embryonic cells) (**Figure 2**).

**146**

*Tissue- and organ-specific nanotoxicity. The toxic accumulation of nanoparticles can affect any of the tissue types in the body. The small-sized nanoparticles have the ability to penetrate easily through the lungs and can cause lung injuries and have an ability to generate ROS that lead to toxic effects in lungs. The reticuloendothelial system in the liver is the main source of biological system where all the nanoparticles get absorbed from the gastrointestinal tract into the cardiovascular systems, as all blood exiting from the gastrointestinal tract transport from the hepatic portal vein that directly diffuses to the liver. Increased exposure to nanoparticles majorly happens through the process of inhalation, which results in altered heart rate. The inhalation of nanoparticles acquires the ability to reach the brain system mainly through the route of olfactory epithelium by the mechanism of transsynaptic transport or through their uptake via the blood-brain barrier. Nanoparticles acquire the ability to penetrate inside the skin and cause toxic effects such as dermatitis and auto immune disorders.*

#### **5.3 Toxicity in cardiovascular systems**

The positively charged NPs such as gold and polystyrene have been reported to cause hemolysis and clotting of blood, while the negatively charged NPs are reported to be nontoxic in nature. Increased exposure to diesel-exposed particles (DEP) in hypertensive rats through the process of inhalation resulted in altered heart rate in rats as interpreted through the pacemaker that determines the activity of the heart [52]. Exposure to single-walled NPs also showed altered cardiovascular effects [53]. The injection of ultrafine carbon black NPs into the blood of normal rats caused platelet accumulation in the hepatic microvasculature of the rats and also caused prothrombotic changes on the endothelial surface of the hepatic microvessels [54].

#### **5.4 Toxicity in central nervous system**

NPs on inhalation of acquire the ability to reach the brain system mainly through the route of olfactory epithelium by the mechanism of transsynaptic transport or through their uptake via the blood-brain barrier [55]. Enhanced permeability of NPs through the blood-brain barrier has been reported to have increased the number of pathologies including hypertension and allergic encephalomyelitis. The surface charge of the nanoparticle has also been shown to have toxic effects on the brain leading to brain toxicity altering the blood-brain integrity [56]. NPs have

also been associated with the production of reactive oxidative species and oxidative stress, which are also associated with brain diseases such as Parkinson's and Alzheimer's [57].

#### **5.5 Toxicity in skin**

The widely used cosmetic products for application in the skin contains mostly 3% NPs of size range approximately 50–500 nm [58]. These NPs behold the scattering properties that enhance the entering of UV photons from the optical source into the skin layer although the dermatological effects of NPs able to penetrate the skin are still under investigation. In vitro study with multi-walled carbon nanotubes reported that the carbon NPs have the ability to localize within and initiate an irritation response in human keratinocytes, which are the primary route of occupational exposure [59].

#### **5.6 Toxicity in embryonic cells**

Fluorescence correlation spectroscopy played a major role in identifying the toxicity of nanomaterials in embryonic cells. The observation through this microscopy revealed that the accumulation of NPs especially NPs with carboxylate group on their surface takes place more in smaller blood vessels rather than larger blood vessels [60]. These findings are majorly important for finding the aggregation state that can likely influence nanoparticle accumulation in angiogenic tissue. The fluorescence correlation spectroscopy helps to measure the loss of NPs from the blood streams of live embryo [61]. This kinetic loss of NPs can be correlated to surface characteristics of NPs such as surface charge and size. Also, it has been reported that in a mature organism, the renal clearance of nanoparticles occurs only for NPs with size less than 5 nm in lateral dimension. NPs are being reported to act as effective targeted delivery agents in angiogenic tissues of adults as well as embryonic tissues. Larson et al. reported that quantum dots could be used to image vasculature (using two-photon excitation) in the dermis of mice [61]. Semiconductor quantum dots are NPs with intense, stable fluorescence and are a very good source to detect ten to hundreds of cancer biomarkers in blood assays, on cancer tissue biopsies, or as contrast agents for medical imaging. Smith and coworkers have developed some functionalized quantum dots for tumor targeting in mice; however, no study has been made to measure directly the concentration of the quantum dots in the blood or whether or not they were aggregated; hence, the toxicity level of these quantum dots has not been checked [62].

#### **6. Conclusion**

The use of nanomaterials in biomedical sciences and health sciences has increased in recent years due to their size and surface characteristics appropriate for targeted and site-specific delivery of drugs to the affected areas. In cancer research, nanomedicine holds the massive potential for cancer therapy. The surface and tiny size and shape of NPs have been used as unique properties of NPs to play a key role for an efficient treatment and specific targeting. Nano-based therapeutic and diagnostic strategies pose as highly promising tools for easy and cost-effective diagnosis of cancer. But, the public interest's in accurate, relevant, and predictive nanotoxicological assessments also has been growing. Due to the complication of ROS formation and disruption to the normal biological events, the use of nanomaterials has created complicated situation. The usage of nanomaterials has been highly reported

**149**

**Author details**

Sibi Raj and Dhruv Kumar\*

University, Noida, Uttar Pradesh, India

provided the original work is properly cited.

Amity Institute of Molecular Medicine and Stem Cell Research (AIMMSCR), Amity

© 2020 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,

\*Address all correspondence to: dhruvbhu@gmail.com; dkumar13@amity.edu

*Biochemical Toxicology: Heavy Metals and Nanomaterials*

to cause toxic events such as DNA damage, oxidative stress damage, and inflammatory responses. Major organs such as heart, brain, skin, etc. have been reported to have toxic responses related to nanoparticle applications. So, the development of a set of rules is needed for developing safe engineered nanomaterials, which can be

*DOI: http://dx.doi.org/10.5772/intechopen.90928*

determined by *in vitro* toxicity studies.

*Biochemical Toxicology: Heavy Metals and Nanomaterials DOI: http://dx.doi.org/10.5772/intechopen.90928*

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

Alzheimer's [57].

exposure [59].

**5.6 Toxicity in embryonic cells**

dots has not been checked [62].

**6. Conclusion**

**5.5 Toxicity in skin**

also been associated with the production of reactive oxidative species and oxidative stress, which are also associated with brain diseases such as Parkinson's and

The widely used cosmetic products for application in the skin contains mostly 3% NPs of size range approximately 50–500 nm [58]. These NPs behold the scattering properties that enhance the entering of UV photons from the optical source into the skin layer although the dermatological effects of NPs able to penetrate the skin are still under investigation. In vitro study with multi-walled carbon nanotubes reported that the carbon NPs have the ability to localize within and initiate an irritation response in human keratinocytes, which are the primary route of occupational

Fluorescence correlation spectroscopy played a major role in identifying the toxicity of nanomaterials in embryonic cells. The observation through this microscopy revealed that the accumulation of NPs especially NPs with carboxylate group on their surface takes place more in smaller blood vessels rather than larger blood vessels [60]. These findings are majorly important for finding the aggregation state that can likely influence nanoparticle accumulation in angiogenic tissue. The fluorescence correlation spectroscopy helps to measure the loss of NPs from the blood streams of live embryo [61]. This kinetic loss of NPs can be correlated to surface characteristics of NPs such as surface charge and size. Also, it has been reported that in a mature organism, the renal clearance of nanoparticles occurs only for NPs with size less than 5 nm in lateral dimension. NPs are being reported to act as effective targeted delivery agents in angiogenic tissues of adults as well as embryonic tissues. Larson et al. reported that quantum dots could be used to image vasculature (using two-photon excitation) in the dermis of mice [61]. Semiconductor quantum dots are NPs with intense, stable fluorescence and are a very good source to detect ten to hundreds of cancer biomarkers in blood assays, on cancer tissue biopsies, or as contrast agents for medical imaging. Smith and coworkers have developed some functionalized quantum dots for tumor targeting in mice; however, no study has been made to measure directly the concentration of the quantum dots in the blood or whether or not they were aggregated; hence, the toxicity level of these quantum

The use of nanomaterials in biomedical sciences and health sciences has increased in recent years due to their size and surface characteristics appropriate for targeted and site-specific delivery of drugs to the affected areas. In cancer research, nanomedicine holds the massive potential for cancer therapy. The surface and tiny size and shape of NPs have been used as unique properties of NPs to play a key role for an efficient treatment and specific targeting. Nano-based therapeutic and diagnostic strategies pose as highly promising tools for easy and cost-effective diagnosis of cancer. But, the public interest's in accurate, relevant, and predictive nanotoxicological assessments also has been growing. Due to the complication of ROS formation and disruption to the normal biological events, the use of nanomaterials has created complicated situation. The usage of nanomaterials has been highly reported

**148**

to cause toxic events such as DNA damage, oxidative stress damage, and inflammatory responses. Major organs such as heart, brain, skin, etc. have been reported to have toxic responses related to nanoparticle applications. So, the development of a set of rules is needed for developing safe engineered nanomaterials, which can be determined by *in vitro* toxicity studies.

### **Author details**

Sibi Raj and Dhruv Kumar\* Amity Institute of Molecular Medicine and Stem Cell Research (AIMMSCR), Amity University, Noida, Uttar Pradesh, India

\*Address all correspondence to: dhruvbhu@gmail.com; dkumar13@amity.edu

© 2020 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.

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mesothelial cells. Environmental Health Perspectives. 2008

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mesothelial cells. Environmental Health

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Weydert JA, Grassian VH. Inflammatory response of mice following inhalation

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particle size, in vivo particle persistence, and lung injury. Environmental Health

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Thorne PS, O'Shaughnessy PT,

exposure to iron and copper NP. Nanotoxicology. 2008

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[50] Oberdorster G, Ferin J, Lehnert BE. Correlation between

Molecular Physiology. 2004

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[54] Simeonova PP, Erdely A.

[52] Hansen CS et al. Diesel exhaust particles induce endothelial dysfunction in apoE−/− mice. Toxicology and Applied Pharmacology. 2007

[53] Li Z et al. Cardiovascular effects of pulmonary exposure to single-wall carbon nanotubes. Environmental

Engineered nanoparticle respiratory exposure and potential risks for

Perspectives. 1994

Perspectives. 2008

2006

[38] Huang YW, Wu CH, Aronstam RS. Toxicity of transition metal oxide NP: Recent insights from in vitro studies.

[39] Yuan X, Zhang X, Sun L, Wei Y,

immunological effects of carbon-based nanomaterials. Particle and Fibre

[40] Szebeni J et al. Animal models of complement-mediated hypersensitivity reactions to liposomes and other lipid-based NP. Journal of Liposome

[41] Monteiller C et al. The proinflammatory effects of low-toxicity low-solubility particles, NP and fine particles, on epithelial cells in vitro: The role of surface area. Occupational and Environmental Medicine. 2007

[42] Nygaard UC, Hansen JS,

[43] Torres M, Forman HJ. Redox

BioFactors. 2003

signaling and the MAP kinase pathways.

[44] Lim D, Roh JY, Eom HJ, Choi JY, Hyun J, Choi J. Oxidative stress-related PMK-1 P38 MAPK activation as a mechanism for toxicity of silver NP to reproduction in the nematode *Caenorhabditis elegans*. Environmental Toxicology and Chemistry. 2012

[45] Liu X, Sun J. Endothelial cells dysfunction induced by silica NP through oxidative stress via JNK/P53 and NF-κB pathways. Biomaterials.

[46] Pacurari M et al. Raw single-wall carbon nanotubes induce oxidative stress and activate MAPKs, AP-1, NF-κB, and Akt in normal and malignant human

Samuelsen M, Alberg T, Marioara CD, Løvik M. Single-walled and multiwalled carbon nanotubes promote allergic immune responses in mice. Toxicological Sciences. 2009

Wei X. Cellular toxicity and

Materials. 2010

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[59] Zvyagin AV, Zhao X, Gierden A, Sanchez W, Ross JA, Roberts MS. Imaging of zinc oxide nanoparticle penetration in human skin in vitro and in vivo. Journal of Biomedical Optics. 2008

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**155**

**Chapter 9**

**Abstract**

Properties

Few-Layered Hexagonal Boron

Physicochemical and Biological

*Magdalena Jedrzejczak-Silicka, Martyna Trukawka,* 

Hexagonal boron nitride (h-BN) is an analogue of graphite called "white graphene." In the structure of h-BN, B and N atoms substitute C atoms. The boron and nitrogen atoms are linked via strong B-N covalent bonds and form interlocking hexagonal rings. h-BN is used in different areas due to its interesting physical and chemical properties, e.g., in electronics as an insulator and in ceramics, resins, plastics, and paints. Therefore, boron nitride (BN) is also a popular inorganic compound in cosmetic industry (the highest BN concentration up to 25% can be found in eye shadow formulation). It is also widely used in dental cement production (for dental and orthodontic applications). Boron nitride seems to be suitable for biomedical applications; therefore, the cytotoxicity in vitro and in vivo observations of h-BN nanoplates and novel few-layered h-BN-based nanocomposites are still needed. The short-time studies confirm their low cytotoxicity and suggest that BN can be used as a novel drug delivery system; however, medical application needs

**Keywords:** boron nitride, few-layered hexagonal boron nitride, exfoliation, functionalization, hydroxyl groups, gold nanoparticles, h-BN nanocomposites,

Nanotechnology became a crucial technology in many science fields, not only in organic and inorganic chemistry, materials and surface sciences, semiconductor physics, microfabrication, and molecular engineering but also has significant impact on biological and medical science. The results of nanotechnology activity create a new reality. On the one hand, it gives the possibility to develop novel methods of diagnosis, drug delivery, and cancer treatment. On the other hand, methods implemented in nanomaterial production and development can affect human health and the state of the environment. The knowledge of the effect on

Nitride: Functionalization,

Nanocomposites, and

*Katarzyna Piotrowska and Ewa Mijowska*

additional verification in long-term studies.

biocompatibility, cellular uptake

**1. Introduction**

#### **Chapter 9**

## Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites, and Physicochemical and Biological Properties

*Magdalena Jedrzejczak-Silicka, Martyna Trukawka, Katarzyna Piotrowska and Ewa Mijowska*

### **Abstract**

Hexagonal boron nitride (h-BN) is an analogue of graphite called "white graphene." In the structure of h-BN, B and N atoms substitute C atoms. The boron and nitrogen atoms are linked via strong B-N covalent bonds and form interlocking hexagonal rings. h-BN is used in different areas due to its interesting physical and chemical properties, e.g., in electronics as an insulator and in ceramics, resins, plastics, and paints. Therefore, boron nitride (BN) is also a popular inorganic compound in cosmetic industry (the highest BN concentration up to 25% can be found in eye shadow formulation). It is also widely used in dental cement production (for dental and orthodontic applications). Boron nitride seems to be suitable for biomedical applications; therefore, the cytotoxicity in vitro and in vivo observations of h-BN nanoplates and novel few-layered h-BN-based nanocomposites are still needed. The short-time studies confirm their low cytotoxicity and suggest that BN can be used as a novel drug delivery system; however, medical application needs additional verification in long-term studies.

**Keywords:** boron nitride, few-layered hexagonal boron nitride, exfoliation, functionalization, hydroxyl groups, gold nanoparticles, h-BN nanocomposites, biocompatibility, cellular uptake

#### **1. Introduction**

Nanotechnology became a crucial technology in many science fields, not only in organic and inorganic chemistry, materials and surface sciences, semiconductor physics, microfabrication, and molecular engineering but also has significant impact on biological and medical science. The results of nanotechnology activity create a new reality. On the one hand, it gives the possibility to develop novel methods of diagnosis, drug delivery, and cancer treatment. On the other hand, methods implemented in nanomaterial production and development can affect human health and the state of the environment. The knowledge of the effect on

living organisms is limited due to relatively short-time *in vitro* and especially *in vivo* experiments that highlight mechanism of nanomaterials—cell-tissue-organism interactions.

Application of nanomaterials in biology and medicine has a multidirectional character. The different nanomaterials with many unique physicochemical properties are tested to develop new nanomaterial-based approaches: fluorescent labels (e.g., quantum dots), detection of pathogens and other biological samples (e.g., nucleic acids, proteins), methods of separation and purification of single biomolecules or cells, pharmacokinetic analysis, biosensing, final drug or gene delivery, cancer treatment via hyperthermia method, tissue engineering, and contrast enhancement of medical imaging technique (e.g., magnetic resonance imaging) [1].

Hexagonal boron nitride (h-BN) is one of the most unique and promising layered nanomaterial widely used as in cosmetic production. As it was stated by Fiume and co-workers [2], although the *International Cosmetic Ingredient Dictionary and Handbook* does not specify which crystal form/forms is/are used in cosmetics, it is presumed that the hexagonal form of boron nitride is applied for that proposes. The form of h-BN presents the most appropriate functionality in cosmetic production/properties (e.g., as a slip modifier). The use of h-BN in cosmetic formulation suggests the lack of toxicity/cytotoxicity [2–4]; thus, the new approach of h-BN, or its exfoliated form, to study its modification and functionalization to obtain a potentially interesting nanomaterial in, e.g., the context of theranostic concept is expected.

#### **2. General information about hexagonal boron nitride (h-BN)**

In recent years, 2D materials have become very attractive due to their properties. The most popular among them are graphene, graphene oxide (GO), and reduced graphene oxide (rGO). The big advantage of these materials is their potential multifunctionality, so they can be applied, for example, in transistors, sensing, energy devices [5] and biomedical devices [6], or nanomedicine [7]. Even though these materials are studied widely, there is a plenty of room to explore their properties, e.g., very complexed bio-response on many levels. Another attractive layered material, which is not fully explored, is hexagonal boron nitride. Its exfoliated form is considered as a graphene analogue.

Boron nitride is a chemical compound with equal number of boron and nitrogen atoms. Just like carbon, it occurs in amorphous and crystalline forms. The major crystalline forms are hexagonal boron nitride (h-BN) compared to graphite, sphalerite boron nitride (β-BN) similar to cubic diamond, and rhombohedral (r-BN) and wurtzite boron nitride (γ-BN), which is in hexagonal diamond form [3, 8–10]. Boron nitride nanotubes (BNNT) are also known. All the forms are electrical insulators [11]. The most popular form of BN, due to its stability, is hexagonal boron nitride. In its structure the boron and nitrogen atoms are linked with each other via strong B-N covalent bonds and form interlocking hexagonal rings [12, 13]. Atoms are bound via strong covalent bonds in-plane, and each layer is held together via van der Waals forces [12] (**Figure 1**).

The multilayered form stabilizes the whole structure. Hexagonal boron nitride systems (e.g., nanotubes, flakes) are highly thermally and chemically stable, but at the same time, they are equally thermally conductive and mechanically robust. **Table 1** presents the basic properties of hexagonal boron nitride. Thus, h-BN systems are widely used for durable high-temperature crucibles, antioxidation lubricants, and protective coatings and as a substrate for semiconductors, lens coatings, etc. in industry [2, 15].

**157**

**Table 1.**

**Figure 1.**

*Hexagonal boron nitride structure [13].*

Std enthalpy of formation (Δf*H*<sup>o</sup>

*Properties of hexagonal boron nitride [2, 14].*

**Properties of hexagonal boron nitride (h-BN)**

*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites...*

Boron nitride nanosheets (BNNSs) were found to be used in polymeric film reinforcement, for example, the elastic modulus of polymethyl methacrylate (PMMA) film was increased when BN nanosheets were incorporated into the polymer [16]. It is also a popular inorganic compound in cosmetic industry used as a slip modifier [13, 17]. The data from the US Food and Drug Administration (FDA) report showed that boron nitride was used in 643 cosmetic formulations (data from 2013). The highest BN concentration (up to 25%) can be found in eye shadow formulation, up to 16% in powders and 2% in lipstick formulation [2, 13]. The successful use of BNNTs in dental adhesive and sealants has been also reported. Moreover, h-BN nanoplatelets modified by the presence of quaternary ammonium compounds (QACs) loaded on h-BN's surface to form fillers for linear

298) −254.4 kJ/mol

Appearance White powder, photostable, odorless (hexagonal, cosmetic grade)

/g (varies by grade)

Bond length 1.466 Å (with interlayer spacing of 3.331 Å)

Molar mass 24.82 g mol<sup>−</sup><sup>1</sup> Density ~2.1 g/cm3 Structure Crystal; hexagonal Melting point 2.973°C; sublimes

Surface area 0.82–30 m2

Refractive index (*n*D) 1.74

Gibbs free energy (Δf*G*°) −22 kJ/mol Coefficient of friction <0.3

Stability Chemical inert and stable Hardness 1–2 on the Mohs scale Specific heat capacity (*C*) 19.7 J/(K·mol)

*DOI: http://dx.doi.org/10.5772/intechopen.90528*

*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites... DOI: http://dx.doi.org/10.5772/intechopen.90528*

#### **Figure 1.** *Hexagonal boron nitride structure [13].*

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

interactions.

expected.

is considered as a graphene analogue.

van der Waals forces [12] (**Figure 1**).

ings, etc. in industry [2, 15].

living organisms is limited due to relatively short-time *in vitro* and especially *in vivo* experiments that highlight mechanism of nanomaterials—cell-tissue-organism

Application of nanomaterials in biology and medicine has a multidirectional character. The different nanomaterials with many unique physicochemical properties are tested to develop new nanomaterial-based approaches: fluorescent labels (e.g., quantum dots), detection of pathogens and other biological samples (e.g., nucleic acids, proteins), methods of separation and purification of single biomolecules or cells, pharmacokinetic analysis, biosensing, final drug or gene delivery, cancer treatment via hyperthermia method, tissue engineering, and contrast enhancement of medical imaging technique (e.g., magnetic resonance imaging) [1]. Hexagonal boron nitride (h-BN) is one of the most unique and promising layered nanomaterial widely used as in cosmetic production. As it was stated by Fiume and co-workers [2], although the *International Cosmetic Ingredient Dictionary and Handbook* does not specify which crystal form/forms is/are used in cosmetics, it is presumed that the hexagonal form of boron nitride is applied for that proposes. The form of h-BN presents the most appropriate functionality in cosmetic production/properties (e.g., as a slip modifier). The use of h-BN in cosmetic formulation suggests the lack of toxicity/cytotoxicity [2–4]; thus, the new approach of h-BN, or its exfoliated form, to study its modification and functionalization to obtain a potentially interesting nanomaterial in, e.g., the context of theranostic concept is

**2. General information about hexagonal boron nitride (h-BN)**

In recent years, 2D materials have become very attractive due to their properties. The most popular among them are graphene, graphene oxide (GO), and reduced graphene oxide (rGO). The big advantage of these materials is their potential multifunctionality, so they can be applied, for example, in transistors, sensing, energy devices [5] and biomedical devices [6], or nanomedicine [7]. Even though these materials are studied widely, there is a plenty of room to explore their properties, e.g., very complexed bio-response on many levels. Another attractive layered material, which is not fully explored, is hexagonal boron nitride. Its exfoliated form

Boron nitride is a chemical compound with equal number of boron and nitrogen atoms. Just like carbon, it occurs in amorphous and crystalline forms. The major crystalline forms are hexagonal boron nitride (h-BN) compared to graphite, sphalerite boron nitride (β-BN) similar to cubic diamond, and rhombohedral (r-BN) and wurtzite boron nitride (γ-BN), which is in hexagonal diamond form [3, 8–10]. Boron nitride nanotubes (BNNT) are also known. All the forms are electrical insulators [11]. The most popular form of BN, due to its stability, is hexagonal boron nitride. In its structure the boron and nitrogen atoms are linked with each other via strong B-N covalent bonds and form interlocking hexagonal rings [12, 13]. Atoms are bound via strong covalent bonds in-plane, and each layer is held together via

The multilayered form stabilizes the whole structure. Hexagonal boron nitride systems (e.g., nanotubes, flakes) are highly thermally and chemically stable, but at the same time, they are equally thermally conductive and mechanically robust. **Table 1** presents the basic properties of hexagonal boron nitride. Thus, h-BN systems are widely used for durable high-temperature crucibles, antioxidation lubricants, and protective coatings and as a substrate for semiconductors, lens coat-

**156**


#### **Table 1.**

*Properties of hexagonal boron nitride [2, 14].*

Boron nitride nanosheets (BNNSs) were found to be used in polymeric film reinforcement, for example, the elastic modulus of polymethyl methacrylate (PMMA) film was increased when BN nanosheets were incorporated into the polymer [16]. It is also a popular inorganic compound in cosmetic industry used as a slip modifier [13, 17]. The data from the US Food and Drug Administration (FDA) report showed that boron nitride was used in 643 cosmetic formulations (data from 2013). The highest BN concentration (up to 25%) can be found in eye shadow formulation, up to 16% in powders and 2% in lipstick formulation [2, 13]. The successful use of BNNTs in dental adhesive and sealants has been also reported. Moreover, h-BN nanoplatelets modified by the presence of quaternary ammonium compounds (QACs) loaded on h-BN's surface to form fillers for linear low-density polyethylene (LLDPE) were tested for inhibition of growth of both *E. coli* and *S. aureus* bacteria [18–20].

Therefore, boron nitride seems to be suitable for biomedical applications as well. Several cytotoxicity studies based on boron nitride nanotubes confirmed its low cytotoxicity and suggested that BN can be used as a novel drug delivery system. In contrast, other studies showed that BNNT was cytotoxic and affected relative cell viability even at low concentrations [14, 21–24].

For the sake of discrepancies occurring in the literature, a deeper understanding of the toxicity of h-BN-based samples is crucial. Here, we will present the synthesis and cytotoxicity study on exfoliated and functionalized hexagonal boron nitride called few-layered BN.

#### **2.1 Synthesis methods of few-layered h-BN (fBN)**

There are two typical approaches to obtain exfoliated hexagonal boron nitrogen nanosheets: top-down (exfoliation methods) or bottom-up (chemical vapor deposition (CVD) or other deposition techniques).

An example of top-down had been described by Liu et al. [25]. They used one-pot solvothermal synthesis involving mixing bulk h-BN, ethanol, and sodium hydroxide in Teflon autoclave. In this method they obtained boron nitride nanosheets and quantum dots at the same time. Marsh with co-workers found even a simpler method [26]. They produced boron nitride nanosheets (BNNSs) from bulk h-BN powders using a simple cosolvent approach. Authors used common organic solvents and water to create a mixture. It was more efficient than using the individual components to get h-BN exfoliated and suspended. They maintain that cosolvent system is inexpensive, safe to work with, and completely scalable. Han et al. [27] used 1,2-dichloroethane solution of poly[(m-phenylenevinylene) co-(2,5-dioctoxy-p-phenylenevinylene)] to disperse and break up van der Waals forces between h-BN layers, while Zhi et al. [28] reported the large-scale fabrication of 2D h-BN nanosheets by vigorous sonication of h-BN in dimethylformamide (DMF). The choice of solvent should be optimized to overcome van der Waals forces.

The optimization to use two-step exfoliation technique combining chemical and mechanical exfoliation was also reported [29]. Chemical exfoliation of h-BN was carried out by a modified Hummer's method. h-BN was additionally delaminated mechanically. Mechanical exfoliation was performed using a tip sonicator. Chemically exfoliated h-BN was added into 1-methyl-2-pyrrolidinone (NMP) in a volume ratio of 0.5%. After the sonication, the mixture was left to evaporate the solvent. This method is simple and fast.

The most important representative of bottom-up method is chemical vapor deposition. In general, it can be divided into two types: one that requires a substrate and another which does not need it. A lot of optional substrates have been used in the process. CVD can be carried out on metals (Cu [30], Ni [31], Co [32], etc.) as well as on metal oxides (Al2O3) [33] or graphite [34]. The precursors can also be in different forms. Most popular are borazine [35], ammonia borane [36], and diborane [37].

Each method has its advantages and disadvantages. Exfoliation techniques ensure higher crystallinity of the material, but high-scale synthesis is very difficult. In contrast, the materials obtained from CVD give the possibility to control over the thickness or size of the sheets, but their crystallinity is lower. Therefore, the technique of material synthesis should be adapted to the requirements of a specific application.

**159**

CVD (LPCVD)

CVD (LPCVD)

CVD (LPCVD)

CVD (AP-CVD)

CVD (AP-CVD)

**Table 2.**

*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites...*

Depending on the synthesis method, a material with different properties can be expected. CVD is more suitable for large-scale synthesis in industry, but exfoliation methods have been also used for various applications, especially on a laboratory scale. Therefore, these materials cannot be clearly compared. **Table 2** presents a summary of properties of fBN such as flake size and material thickness in relation

Following data from state of the art, it can be concluded that chemical vapor deposition provides thinner and larger layers of h-BN than in the case of exfoliation. It should be noted that in the case of exfoliation, the starting bulk material is used, while in the case of CVD, a completely new material is obtained from various molecular precursors. Mostly, the number of the obtained h-BN layers is strongly related with the used substrates. It can be also concluded that the combination of

**Method Substrates Thickness [nm] Flake size** 

Exfoliation Dimethylformamide 3–7 Smaller

Exfoliation Octadecylamine 1–2 0.3–0.5 [38]

2–6 1–3

Temperature depending 40–228

Sulfuric acid 1-Methyl-2-pyrrolidinone

Sodium hydroxide

1-Propanol 2-Propanol acetone Tert-butanol (cosolvents)

phenylenevinylene)-co-(2,5-dictoxyp-phenylenevinylene)]

Potassium chloride

Cu foil Borazane

Co film Ammonia borane

Al2O3 substrate Borazane

Borazine

Ag foil Borazine

Pt foil Borazane

*Properties of fBN (flake size, material thickness) in relation to the synthesis method.*

**[μm]**

5 0.3–0.6 This

~1 ~1.2 [25]

6–10 — [26]

~1.2 Several [27]

than pristine material

> 0.5 0.2

0.42 0.05–0.1 [30]

~ 1 > 5 [32]

5–15 10 × 10 [35]

0.7–1.3 0.1 [27]

0.32–0.809 1–2 [40]

— [33]

**Ref.**

work

[28]

[39]

*DOI: http://dx.doi.org/10.5772/intechopen.90528*

Exfoliation Potassium permanganate

Exfoliation Ethanol

Exfoliation Methanol ethanol

Exfoliation 1,2-Dichloroethane poly[(m-

Exfoliation Zinc chloride

CVD Fe foil

to the method of synthesis.

**2.2 Characterization of few-layered h-BN (fBN)**

#### **2.2 Characterization of few-layered h-BN (fBN)**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

viability even at low concentrations [14, 21–24].

**2.1 Synthesis methods of few-layered h-BN (fBN)**

tion (CVD) or other deposition techniques).

solvent. This method is simple and fast.

*E. coli* and *S. aureus* bacteria [18–20].

called few-layered BN.

low-density polyethylene (LLDPE) were tested for inhibition of growth of both

Therefore, boron nitride seems to be suitable for biomedical applications as well. Several cytotoxicity studies based on boron nitride nanotubes confirmed its low cytotoxicity and suggested that BN can be used as a novel drug delivery system. In contrast, other studies showed that BNNT was cytotoxic and affected relative cell

For the sake of discrepancies occurring in the literature, a deeper understanding of the toxicity of h-BN-based samples is crucial. Here, we will present the synthesis and cytotoxicity study on exfoliated and functionalized hexagonal boron nitride

There are two typical approaches to obtain exfoliated hexagonal boron nitrogen nanosheets: top-down (exfoliation methods) or bottom-up (chemical vapor deposi-

An example of top-down had been described by Liu et al. [25]. They used one-pot solvothermal synthesis involving mixing bulk h-BN, ethanol, and

sodium hydroxide in Teflon autoclave. In this method they obtained boron nitride nanosheets and quantum dots at the same time. Marsh with co-workers found even a simpler method [26]. They produced boron nitride nanosheets (BNNSs) from bulk h-BN powders using a simple cosolvent approach. Authors used common organic solvents and water to create a mixture. It was more efficient than using the individual components to get h-BN exfoliated and suspended. They maintain that cosolvent system is inexpensive, safe to work with, and completely scalable. Han et al. [27] used 1,2-dichloroethane solution of poly[(m-phenylenevinylene) co-(2,5-dioctoxy-p-phenylenevinylene)] to disperse and break up van der Waals forces between h-BN layers, while Zhi et al. [28] reported the large-scale fabrication of 2D h-BN nanosheets by vigorous sonication of h-BN in dimethylformamide (DMF). The choice of solvent should be optimized to overcome van der Waals

The optimization to use two-step exfoliation technique combining chemical and mechanical exfoliation was also reported [29]. Chemical exfoliation of h-BN was carried out by a modified Hummer's method. h-BN was additionally delaminated mechanically. Mechanical exfoliation was performed using a tip sonicator. Chemically exfoliated h-BN was added into 1-methyl-2-pyrrolidinone (NMP) in a volume ratio of 0.5%. After the sonication, the mixture was left to evaporate the

The most important representative of bottom-up method is chemical vapor deposition. In general, it can be divided into two types: one that requires a substrate and another which does not need it. A lot of optional substrates have been used in the process. CVD can be carried out on metals (Cu [30], Ni [31], Co [32], etc.) as well as on metal oxides (Al2O3) [33] or graphite [34]. The precursors can also be in different forms. Most popular are borazine [35], ammonia borane [36], and

Each method has its advantages and disadvantages. Exfoliation techniques ensure higher crystallinity of the material, but high-scale synthesis is very difficult. In contrast, the materials obtained from CVD give the possibility to control over the thickness or size of the sheets, but their crystallinity is lower. Therefore, the technique of material synthesis should be adapted to the requirements of a specific

**158**

diborane [37].

application.

forces.

Depending on the synthesis method, a material with different properties can be expected. CVD is more suitable for large-scale synthesis in industry, but exfoliation methods have been also used for various applications, especially on a laboratory scale. Therefore, these materials cannot be clearly compared. **Table 2** presents a summary of properties of fBN such as flake size and material thickness in relation to the method of synthesis.

Following data from state of the art, it can be concluded that chemical vapor deposition provides thinner and larger layers of h-BN than in the case of exfoliation. It should be noted that in the case of exfoliation, the starting bulk material is used, while in the case of CVD, a completely new material is obtained from various molecular precursors. Mostly, the number of the obtained h-BN layers is strongly related with the used substrates. It can be also concluded that the combination of


**Table 2.**

*Properties of fBN (flake size, material thickness) in relation to the synthesis method.*

chemical and mechanical exfoliation is a very effective and repeatable method. **Figure 2A** presents the bulk hexagonal boron nitride. The multilayered material is clearly visible. Many flakes are aggregated and connected to each other. After the exfoliation process, even individual flakes of the material are detected (**Figure 2B**).

The exfoliation efficiency was also confirmed by atomic force microscopy (AFM). The thickness of bulk nanomaterial was estimated to be ~40 nm (**Figure 3A**). After chemical and mechanical exfoliation, the number of layers had been greatly reduced. The obtained thickness was ~5 nm, which corresponded to several layers of h-BN (**Figure 3B**).

Scanning electron microscope (SEM) was also used to analyze the flake size of fBN in greater details (**Figure 4**). The observation revealed that most of the flakes were in a size range of 0.3–1.2 μm.

Concluding this observation, using various methods the materials with similar parameters can be obtained. The issue of choosing the method of preparation should be adapted to the properties that the material should exhibit to perform the best in required applications.

#### **2.3 Functionalization of few-layered h-BN (fBN)**

Functionalization process is undertaken to provide additional/new properties. In the case of hexagonal boron nitride, the biggest challenge is to raise the materials' water solubility/dispersibility. This can be achieved by introducing the functional groups on fBN surface. One of the simplest routes is functionalization with hydroxyl groups. At the same time, as it was proved, the presence of -OH groups determines the stability of dispersion in water-based solution [41].

The procedure was easy and repeatable. Chemically and mechanically exfoliated hexagonal boron nitrides were refluxed in hydrogen peroxide for a longer time. To confirm the functionalization, FT-IR spectra were analyzed (**Figure 5**). Except for the peaks characteristic for hexagonal boron nitride (810 and 1370 cm<sup>−</sup><sup>1</sup> ), clearly visible new bonds corresponding to hydroxyl groups at 2525 and 3400 cm<sup>−</sup><sup>1</sup> are detected [42].

Pristine BN nanomaterials exhibit notable hydrophobicity when interacting with water or aqueous solutions. Therefore, this functionalization allows stable dispersion in phosphate buffer solution, which is crucial for biological application [41].

**161**

**Figure 5.**

*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites...*

Other methods for obtaining hydroxylated hexagonal boron nitride are also known. Sainsbury et al. [43] used boron nitride nanosheets with tert-butoxy groups on the surface. To induce hydroxyl groups, they mixed and sonicated piranha solution

*DOI: http://dx.doi.org/10.5772/intechopen.90528*

*Height profile of bulk h-BN (A) and exfoliated fBN (B).*

*Flake size distribution and SEM image of exfoliated fBN.*

*FT-IR spectra of pristine and hydroxylated fBN.*

**Figure 3.**

**Figure 4.**

**Figure 2.** *Transmission electron microscope (TEM) images of bulk h-BN (A) and exfoliated fBN (B).*

*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites... DOI: http://dx.doi.org/10.5772/intechopen.90528*

**Figure 3.** *Height profile of bulk h-BN (A) and exfoliated fBN (B).*

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

several layers of h-BN (**Figure 3B**).

were in a size range of 0.3–1.2 μm.

**2.3 Functionalization of few-layered h-BN (fBN)**

determines the stability of dispersion in water-based solution [41].

the peaks characteristic for hexagonal boron nitride (810 and 1370 cm<sup>−</sup><sup>1</sup>

*Transmission electron microscope (TEM) images of bulk h-BN (A) and exfoliated fBN (B).*

visible new bonds corresponding to hydroxyl groups at 2525 and 3400 cm<sup>−</sup><sup>1</sup>

best in required applications.

detected [42].

chemical and mechanical exfoliation is a very effective and repeatable method. **Figure 2A** presents the bulk hexagonal boron nitride. The multilayered material is clearly visible. Many flakes are aggregated and connected to each other. After the exfoliation process, even individual flakes of the material are detected (**Figure 2B**). The exfoliation efficiency was also confirmed by atomic force microscopy

(**Figure 3A**). After chemical and mechanical exfoliation, the number of layers had been greatly reduced. The obtained thickness was ~5 nm, which corresponded to

Scanning electron microscope (SEM) was also used to analyze the flake size of fBN in greater details (**Figure 4**). The observation revealed that most of the flakes

Concluding this observation, using various methods the materials with similar

Functionalization process is undertaken to provide additional/new properties. In the case of hexagonal boron nitride, the biggest challenge is to raise the materials' water solubility/dispersibility. This can be achieved by introducing the functional groups on fBN surface. One of the simplest routes is functionalization with hydroxyl groups. At the same time, as it was proved, the presence of -OH groups

The procedure was easy and repeatable. Chemically and mechanically exfoliated hexagonal boron nitrides were refluxed in hydrogen peroxide for a longer time. To confirm the functionalization, FT-IR spectra were analyzed (**Figure 5**). Except for

Pristine BN nanomaterials exhibit notable hydrophobicity when interacting with water or aqueous solutions. Therefore, this functionalization allows stable dispersion in phosphate buffer solution, which is crucial for biological application [41].

), clearly

are

parameters can be obtained. The issue of choosing the method of preparation should be adapted to the properties that the material should exhibit to perform the

(AFM). The thickness of bulk nanomaterial was estimated to be ~40 nm

**160**

**Figure 2.**

**Figure 4.** *Flake size distribution and SEM image of exfoliated fBN.*

**Figure 5.** *FT-IR spectra of pristine and hydroxylated fBN.*

Other methods for obtaining hydroxylated hexagonal boron nitride are also known. Sainsbury et al. [43] used boron nitride nanosheets with tert-butoxy groups on the surface. To induce hydroxyl groups, they mixed and sonicated piranha solution

(H2SO4:H2O2, 3:1) [43]. Moreover, the authors carried out further functionalization. They used hydroxylated material to obtain isocyanate-functionalized BNNSs. A completely different approach to obtain hydroxylated BN has been shown by Pakdel et al. [44]. Boron nitride nanostructure films were subjected to direct ion/electron bombardment in a plasma generator device. In this way they aimed to control the wetting properties of nanomaterial.

It turned out that hydroxylated h-BN can also be obtained by ball milling. Lee with co-workers [45] presented a simple ball milling of BN powders in the presence of sodium hydroxide. They connected the synergetic effect of chemical peeling and mechanical shear forces. There are many other functionalities that allow h-BN to be used in even more applications. For example, it is possible to decorate hexagonal boron nitride with metal nanoparticles. There are reports, for example, of platinum, silver, and gold decoration. Anna Harley-Trochimczyk et al. [46] described crystalline boron nitride aerogel loaded with crystalline platinum nanoparticles. They found out that this material can be used in catalytic gas sensing. Dai et al. [47] produced Ag nanoparticle coverage on porous BN microfibers and examined it for a novel pollutant-capturing surface-enhanced Raman scattering (SERS) substrate. There are already several reports on BN materials functionalized with gold; however, such a composite had not been previously tested for biological response as described below. Until now, such nanocomposite has found application in electrocatalysis [48] or hydrogen peroxide detection [49].

In research [29] simple and repeatable Au-fBN nanocomposite synthesis method was demonstrated. Briefly, exfoliated h-BN was sonicated in distilled water. The mixture was heated with gold(III) chloride trihydrate under the reflux. After a few minutes, trisodium citrate was added to the boiling mixture. The whole system was heated. **Figure 6** shows transmission electron microscope (TEM) images of the obtained nanocomposite (Au-fBN).

Based on TEM micrographs, the size of gold nanoparticles was determined (**Figure 7**). The nanoparticle size distribution was in the range from 6 to 25 nm with a majority of ~16 nm.

The presence of gold nanoparticles was confirmed by Raman spectroscopy (**Figure 8**). The peak at 1366 cm<sup>−</sup><sup>1</sup> is the most characteristic for hexagonal boron nitride. It is resulting from the E2g phonon mode and is an analogue of the G peak in graphene [50]. It is very clearly visible in the spectrum of pure material and much

**163**

the chapter.

**Figure 8.**

**Figure 7.**

*Gold nanoparticle size distribution.*

*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites...*

less intense in the nanocomposite. This is due to the presence of gold nanoparticles

another intense peak in the spectrum of gold nanoparticles. It occurs at 2130 cm<sup>−</sup><sup>1</sup>

and it can also be clearly seen in the nanocomposite spectrum, which additionally

Nanomaterials prepared in the method described above (fBN-OH and Au-fBN) have been subjected to biological tests, which will be discussed in further parts of

The biocompatibility of h-BN-based nanocomposites synthesized in studies [29, 41]

[51]. There is

,

with characteristic peaks in the region from 1300 to 1600 cm<sup>−</sup><sup>1</sup>

*Raman spectra of h-BN bulk, nanocomposite (Au-fBN), and gold nanoparticles (Au NP).*

**3. The effect of few-layered BN-OH (fBN-OH) and Au-fBN** 

ensures efficient functionalization.

was determined in three main steps.

**nanocomposites on cellular viability**

*DOI: http://dx.doi.org/10.5772/intechopen.90528*

**Figure 6.** *TEM images of Au-fBN nanocomposite.*

*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites... DOI: http://dx.doi.org/10.5772/intechopen.90528*

**Figure 7.** *Gold nanoparticle size distribution.*

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

talysis [48] or hydrogen peroxide detection [49].

obtained nanocomposite (Au-fBN).

(**Figure 8**). The peak at 1366 cm<sup>−</sup><sup>1</sup>

a majority of ~16 nm.

wetting properties of nanomaterial.

(H2SO4:H2O2, 3:1) [43]. Moreover, the authors carried out further functionalization. They used hydroxylated material to obtain isocyanate-functionalized BNNSs. A completely different approach to obtain hydroxylated BN has been shown by Pakdel et al. [44]. Boron nitride nanostructure films were subjected to direct ion/electron bombardment in a plasma generator device. In this way they aimed to control the

It turned out that hydroxylated h-BN can also be obtained by ball milling. Lee with co-workers [45] presented a simple ball milling of BN powders in the presence of sodium hydroxide. They connected the synergetic effect of chemical peeling and mechanical shear forces. There are many other functionalities that allow h-BN to be used in even more applications. For example, it is possible to decorate hexagonal boron nitride with metal nanoparticles. There are reports, for example, of platinum, silver, and gold decoration. Anna Harley-Trochimczyk et al. [46] described crystalline boron nitride aerogel loaded with crystalline platinum nanoparticles. They found out that this material can be used in catalytic gas sensing. Dai et al. [47] produced Ag nanoparticle coverage on porous BN microfibers and examined it for a novel pollutant-capturing surface-enhanced Raman scattering (SERS) substrate. There are already several reports on BN materials functionalized with gold; however, such a composite had not been previously tested for biological response as described below. Until now, such nanocomposite has found application in electroca-

In research [29] simple and repeatable Au-fBN nanocomposite synthesis method was demonstrated. Briefly, exfoliated h-BN was sonicated in distilled water. The mixture was heated with gold(III) chloride trihydrate under the reflux. After a few minutes, trisodium citrate was added to the boiling mixture. The whole system was heated. **Figure 6** shows transmission electron microscope (TEM) images of the

Based on TEM micrographs, the size of gold nanoparticles was determined (**Figure 7**). The nanoparticle size distribution was in the range from 6 to 25 nm with

The presence of gold nanoparticles was confirmed by Raman spectroscopy

nitride. It is resulting from the E2g phonon mode and is an analogue of the G peak in graphene [50]. It is very clearly visible in the spectrum of pure material and much

is the most characteristic for hexagonal boron

**162**

**Figure 6.**

*TEM images of Au-fBN nanocomposite.*

**Figure 8.** *Raman spectra of h-BN bulk, nanocomposite (Au-fBN), and gold nanoparticles (Au NP).*

less intense in the nanocomposite. This is due to the presence of gold nanoparticles with characteristic peaks in the region from 1300 to 1600 cm<sup>−</sup><sup>1</sup> [51]. There is another intense peak in the spectrum of gold nanoparticles. It occurs at 2130 cm<sup>−</sup><sup>1</sup> , and it can also be clearly seen in the nanocomposite spectrum, which additionally ensures efficient functionalization.

Nanomaterials prepared in the method described above (fBN-OH and Au-fBN) have been subjected to biological tests, which will be discussed in further parts of the chapter.

#### **3. The effect of few-layered BN-OH (fBN-OH) and Au-fBN nanocomposites on cellular viability**

The biocompatibility of h-BN-based nanocomposites synthesized in studies [29, 41] was determined in three main steps.

The first step of the *in vitro* study was based on morphological cell analysis. This step often is omitted by the researchers, although the morphology observation is the simplest and direct method that gives possibility to identify the changes of cellular shape and adhesion ability. Both of them may be changed upon specific environmental stress [52]. **Figure 9** presents the cellular morphology and distribution of L929 and MCF-7 cells after 48-hour incubation with fBN-OH and Au-fBN nanocomposites (analysis was conducted using phase contrast microscopy, ×100, Nikon TS-100 microscope). Both cell lines grown in monolayers display typical morphology—the cells did not change shape. The cells did not show a tendency to form clusters, and the adhesion process was not impaired after 48 hours.

Moreover, in the second step, the cellular uptake and distribution of fBN-OH and Au-fBN nanocomposites were analyzed using confocal microscopy (**Figure 10**). The nanomaterials were labeled with FITC, and the presence of nanocomposites in cells was confirmed by green fluorescent signal. The internalized h-BN-based nanocomposites were accumulated in the peripheral cytoplasm and the perinuclear region, but the presence of fBN-OH and Au-fBN was not confirmed in the nucleus. The labeled nanocomposites formed small intercellular aggregates. The uptake

**Figure 9.**

*Cellular morphology of cultures incubated with fBN-OH and Au-fBN nanoplates for 48 hours. Magnification ×100.*

**165**

**Figure 10.**

*Uptake of tested nanomaterials after 48-hour incubation.*

*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites...*

efficiency process of hexagonal boron nitride nanocomposites by normal and

In the third step of the study, a comparison of biocompatibility results of the fBN-OH and Au-fBN nanocomposites at 3.125, 6.25, 10.0, 12.5, 25.0, 50.0, 100.0,

(CCK-8), neutral red uptake (NRU), and lactate dehydrogenase leaking (LDH) assays (**Figure 10**) [29]. The highest reduction of the cell viability was recorded

Au-fBN in L929 cell cultures incubated for 48 hours (**Figure 10**). The MCF-7 cell cultures exhibited higher reduction of cell viability for fBN-OH at the concentra-

cells incubated with nanocomposites loaded with gold NPs was reduced at the

In contrast to CCK-8 assay results, NRU assay showed higher reduction of the viability of both cell lines (**Figure 10**). Both cell lines showed higher sensitivity of

concentrations was determined using Cell Counting Kit-8

for fBN-OH and 200.0 μg mL<sup>−</sup><sup>1</sup>

(**Figure 10**). The cell viability of MCF-7

, in *comparison to free-grown MCF-7 control* 

for

*DOI: http://dx.doi.org/10.5772/intechopen.90528*

and 200.0 μg mL<sup>−</sup><sup>1</sup>

*culture* (**Figure 10**).

cancer cells was established at a similar level.

at the concentrations of 100.0–200.0 μg mL<sup>−</sup><sup>1</sup>

tion range between 12.5 and 200.0 μg mL<sup>−</sup><sup>1</sup>

concentration of 10.0 and 200.0 μg mL<sup>−</sup><sup>1</sup>

*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites... DOI: http://dx.doi.org/10.5772/intechopen.90528*

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

The first step of the *in vitro* study was based on morphological cell analysis. This step often is omitted by the researchers, although the morphology observation is the simplest and direct method that gives possibility to identify the changes of cellular shape and adhesion ability. Both of them may be changed upon specific environmental stress [52]. **Figure 9** presents the cellular morphology and distribution of L929 and MCF-7 cells after 48-hour incubation with fBN-OH and Au-fBN nanocomposites (analysis was conducted using phase contrast microscopy, ×100, Nikon TS-100 microscope). Both cell lines grown in monolayers display typical morphology—the cells did not change shape. The cells did not show a tendency to

form clusters, and the adhesion process was not impaired after 48 hours.

*Cellular morphology of cultures incubated with fBN-OH and Au-fBN nanoplates for 48 hours.* 

Moreover, in the second step, the cellular uptake and distribution of fBN-OH and Au-fBN nanocomposites were analyzed using confocal microscopy (**Figure 10**). The nanomaterials were labeled with FITC, and the presence of nanocomposites in cells was confirmed by green fluorescent signal. The internalized h-BN-based nanocomposites were accumulated in the peripheral cytoplasm and the perinuclear region, but the presence of fBN-OH and Au-fBN was not confirmed in the nucleus. The labeled nanocomposites formed small intercellular aggregates. The uptake

**164**

**Figure 9.**

*Magnification ×100.*

efficiency process of hexagonal boron nitride nanocomposites by normal and cancer cells was established at a similar level.

In the third step of the study, a comparison of biocompatibility results of the fBN-OH and Au-fBN nanocomposites at 3.125, 6.25, 10.0, 12.5, 25.0, 50.0, 100.0, and 200.0 μg mL<sup>−</sup><sup>1</sup> concentrations was determined using Cell Counting Kit-8 (CCK-8), neutral red uptake (NRU), and lactate dehydrogenase leaking (LDH) assays (**Figure 10**) [29]. The highest reduction of the cell viability was recorded at the concentrations of 100.0–200.0 μg mL<sup>−</sup><sup>1</sup> for fBN-OH and 200.0 μg mL<sup>−</sup><sup>1</sup> for Au-fBN in L929 cell cultures incubated for 48 hours (**Figure 10**). The MCF-7 cell cultures exhibited higher reduction of cell viability for fBN-OH at the concentration range between 12.5 and 200.0 μg mL<sup>−</sup><sup>1</sup> (**Figure 10**). The cell viability of MCF-7 cells incubated with nanocomposites loaded with gold NPs was reduced at the concentration of 10.0 and 200.0 μg mL<sup>−</sup><sup>1</sup> , in *comparison to free-grown MCF-7 control culture* (**Figure 10**).

In contrast to CCK-8 assay results, NRU assay showed higher reduction of the viability of both cell lines (**Figure 10**). Both cell lines showed higher sensitivity of

**Figure 10.** *Uptake of tested nanomaterials after 48-hour incubation.*

neutral red dye uptake based on functional lysosomes in the presence of few-layered h-BN-based nanocomposites (**Figure 10**). In the case of fBN-OH, the L929 and MCF-7 responded to the presence of NPs in a dose-dependent manner, but MCF-7 seems to be more sensitive than L929 cell cultures. The presence of Au-fBN affected the cell viability less than the fBN-OH.

The integrity of cellular plasma membranes (analyzed via LDH leakage assay) was impaired less than the other cell's features. L929 cells as well as MCF-7 showed minimal changes in lactate dehydrogenase leakage even at the highest concentrations of fBN-OH and Au-fBN (**Figure 10**). The reduction of cell membrane integrity was the highest (to 80% vs. control cultures) for L929 and MCF-7 at concentrations of 200.0 μg mL<sup>−</sup><sup>1</sup> fBN-OH (**Figure 11**).

Pristine bulk h-BN is known to be poorly soluble in water-based solutions. Thus, the h-BN preparation (e.g., exfoliation, functionalization) should be optimized to obtain a nanomaterial that exhibits suitable properties in required applications (e.g., higher hydrophobicity that allows stable dispersion in aqueous solutions) [41]. The functionalization of the h-BN by hydroxyl groups improve h-BN hydrophobicity and allows to obtain stable dispersion in phosphate buffer solution [41] or in phosphate buffer solution supplemented with dispersant Pluronic F-127 [29]. This is crucial for cytotoxicity experimentations and biological/medical applications.

Another crucial factor in biocompatibility analysis is experiment in short- and long-term studies. In studies it was demonstrated that the effect of the fBN-OH on cells may vary depending on the species, type of cells tested, their function, and time of exposure of cells to these nanoparticles. The short-term in vitro study on L929 cell cultures and human erythrocytes as well as in vivo study on insect (*T. molitor*) hemocytes demonstrated a low cytotoxicity of this fBN-OH (dispersed in PBS), whereas a long-term study in *T. molitor* has shown a significant effect of fBN-OH on the behavior of immunocompetent cells and their function during the immune response [41].

In the preliminary study based on hexagonal boron nitride (exfoliated and functionalized with Au nanoparticles), it was found that Au-fBN nanoflakes

#### **Figure 11.**

*Biocompatibility of fBN-OH and Au-fBN nanoplates incubated for 48 hours—L929 cell cultures (A, B) and MCF-7 cell cultures (C, D). Bars represent standard deviation.*

**167**

*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites...*

did not affect the cellular metabolism (CCK-8) and membrane integrity (LDH assays). However, the function of lysosomes in both normal and cancer cell lines during 24-hour exposition was modified. Longer incubation, for 48 and 72 hours, affected the cell relative viability and proliferation activity of the MCF-7 cancer cell line in comparison to normal L929 cell line after 72-hour incubation period [29]. Additionally, fBN-based nanocomposites have been tested as a platform for drug delivery. Both the hexagonal boron nitride nanocomposites (fBN-OH and Au-fBN) were loaded chemically with an anticancer drug—10-hydroxycamptothecin (HCPT)—and were tested against human breast adenocarcinoma cells [53]. It was found that both nanocomposites conjugated with HCPT were effectively internalized and cumulated inside the cell cytoplasm, but not in the nuclear region of cells. Both the tested boron nitride nanocomposites loaded with 10-hydroxycomptothecin significantly reduced relative cell viability of MCF-7 cells. The slightly higher reduction was observed for Au-fBN-HCPT against human breast adenocarcinoma cells [53].

The hexagonal boron nitride (h-BN) is an attractive layered material that can be used in different industry sectors (due to its interesting physical and chemical properties). Its exfoliated form is considered as a graphene analogue. The pristine bulk h-BN is poorly soluble and exhibits a hydrophobic character in water/aqueous solutions. That is why the h-BN preparation (e.g., exfoliation, functionalization) is crucial to obtain a nanomaterial that exhibits the best properties that are required especially in in vitro and/or in vivo applications. The effect of the presented fewlayered h-BN-based nanocomposites on biological environment may vary depending on the type of cells tested, their function, and time of exposure of cells to these nanoparticles. Boron nitride seems to be suitable for biomedical applications; therefore, the cytotoxicity in vitro and in vivo observations of novel few-layered h-BN-based nanocomposites are needed. The short-time studies confirmed their low cytotoxicity and suggest that h-BN can be used as a novel drug delivery system. However, medical applications need additional verification in long-term studies.

The authors are grateful for the financial support of National Science Centre within MINIATURA 2 program (no. 2018/02/X/NZ3/00161) and PRELUDIUM 11

*DOI: http://dx.doi.org/10.5772/intechopen.90528*

**4. Conclusions**

**Acknowledgements**

**Conflict of interest**

program (no. 2016/21/N/ST8/02397).

The authors declare no conflict of interest.

#### *Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites... DOI: http://dx.doi.org/10.5772/intechopen.90528*

did not affect the cellular metabolism (CCK-8) and membrane integrity (LDH assays). However, the function of lysosomes in both normal and cancer cell lines during 24-hour exposition was modified. Longer incubation, for 48 and 72 hours, affected the cell relative viability and proliferation activity of the MCF-7 cancer cell line in comparison to normal L929 cell line after 72-hour incubation period [29]. Additionally, fBN-based nanocomposites have been tested as a platform for drug delivery. Both the hexagonal boron nitride nanocomposites (fBN-OH and Au-fBN) were loaded chemically with an anticancer drug—10-hydroxycamptothecin (HCPT)—and were tested against human breast adenocarcinoma cells [53]. It was found that both nanocomposites conjugated with HCPT were effectively internalized and cumulated inside the cell cytoplasm, but not in the nuclear region of cells. Both the tested boron nitride nanocomposites loaded with 10-hydroxycomptothecin significantly reduced relative cell viability of MCF-7 cells. The slightly higher reduction was observed for Au-fBN-HCPT against human breast adenocarcinoma cells [53].

### **4. Conclusions**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

the cell viability less than the fBN-OH.

concentrations of 200.0 μg mL<sup>−</sup><sup>1</sup>

immune response [41].

neutral red dye uptake based on functional lysosomes in the presence of few-layered h-BN-based nanocomposites (**Figure 10**). In the case of fBN-OH, the L929 and MCF-7 responded to the presence of NPs in a dose-dependent manner, but MCF-7 seems to be more sensitive than L929 cell cultures. The presence of Au-fBN affected

The integrity of cellular plasma membranes (analyzed via LDH leakage assay) was impaired less than the other cell's features. L929 cells as well as MCF-7 showed minimal changes in lactate dehydrogenase leakage even at the highest concentrations of fBN-OH and Au-fBN (**Figure 10**). The reduction of cell membrane integrity was the highest (to 80% vs. control cultures) for L929 and MCF-7 at

 fBN-OH (**Figure 11**). Pristine bulk h-BN is known to be poorly soluble in water-based solutions. Thus,

the h-BN preparation (e.g., exfoliation, functionalization) should be optimized to obtain a nanomaterial that exhibits suitable properties in required applications (e.g., higher hydrophobicity that allows stable dispersion in aqueous solutions) [41]. The functionalization of the h-BN by hydroxyl groups improve h-BN hydrophobicity and allows to obtain stable dispersion in phosphate buffer solution [41] or in phosphate buffer solution supplemented with dispersant Pluronic F-127 [29]. This is crucial for cytotoxicity experimentations and biological/medical applications. Another crucial factor in biocompatibility analysis is experiment in short- and long-term studies. In studies it was demonstrated that the effect of the fBN-OH on cells may vary depending on the species, type of cells tested, their function, and time of exposure of cells to these nanoparticles. The short-term in vitro study on L929 cell cultures and human erythrocytes as well as in vivo study on insect (*T. molitor*) hemocytes demonstrated a low cytotoxicity of this fBN-OH (dispersed in PBS), whereas a long-term study in *T. molitor* has shown a significant effect of fBN-OH on the behavior of immunocompetent cells and their function during the

In the preliminary study based on hexagonal boron nitride (exfoliated and functionalized with Au nanoparticles), it was found that Au-fBN nanoflakes

*Biocompatibility of fBN-OH and Au-fBN nanoplates incubated for 48 hours—L929 cell cultures (A, B) and* 

*MCF-7 cell cultures (C, D). Bars represent standard deviation.*

**166**

**Figure 11.**

The hexagonal boron nitride (h-BN) is an attractive layered material that can be used in different industry sectors (due to its interesting physical and chemical properties). Its exfoliated form is considered as a graphene analogue. The pristine bulk h-BN is poorly soluble and exhibits a hydrophobic character in water/aqueous solutions. That is why the h-BN preparation (e.g., exfoliation, functionalization) is crucial to obtain a nanomaterial that exhibits the best properties that are required especially in in vitro and/or in vivo applications. The effect of the presented fewlayered h-BN-based nanocomposites on biological environment may vary depending on the type of cells tested, their function, and time of exposure of cells to these nanoparticles. Boron nitride seems to be suitable for biomedical applications; therefore, the cytotoxicity in vitro and in vivo observations of novel few-layered h-BN-based nanocomposites are needed. The short-time studies confirmed their low cytotoxicity and suggest that h-BN can be used as a novel drug delivery system. However, medical applications need additional verification in long-term studies.

#### **Acknowledgements**

The authors are grateful for the financial support of National Science Centre within MINIATURA 2 program (no. 2018/02/X/NZ3/00161) and PRELUDIUM 11 program (no. 2016/21/N/ST8/02397).

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Magdalena Jedrzejczak-Silicka1 \*, Martyna Trukawka<sup>2</sup> , Katarzyna Piotrowska<sup>3</sup> \* and Ewa Mijowska<sup>2</sup>

1 Laboratory of Cytogenetics, West Pomeranian University of Technology, Szczecin, Poland

2 Department of Physicochemistry of Nanomaterials, West Pomeranian University of Technology, Szczecin, Poland

3 Department of Physiology, Pomeranian Medical University, Szczecin, Poland

\*Address all correspondence to: mjedrzejczak@zut.edu.pl and piot.kata@gmail.com

© 2020 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.

**169**

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[10] Ribeiro H, Luciano MA, Randow P, Vilela DN, Andrade LM. Functionalized

boron nitride applications in biotechnology. In: Recent Advances in Boron Containing Materials. IntechOpen; 2019. pp. 1-15. DOI: 10.5772/intechopen.80849

[11] Kim JH, Pham TV, Hwang JH, Kim CS, Kim MJ. Boron nitride

10.1186/s40580-018-0149-y

DOI: 10.1039/C5CS00869G

[Accessed: 25 March 2019]

cr00099a004

nanotubes: Synthesis and applications. Nano Convergence. 2018;**5**:17. DOI:

[12] Weng Q, Wang X, Wang X, Bando Y, Golberg D. Functionalized hexagonal boron nitride nanomaterials: Emerging properties and applications. Chemical Society Reviews. 2016;**45**:3989-4012.

[13] Safety assessment of boron nitride as used in cosmetics. Scientific literature review for public comment. Cosmetics ingredient review. Washington, DC, USA [Internet]. 2012. Available from: https://www.cir-safety.org/sites/default/ files/Boron%20Nitride\_faa-final.pdf

[14] Haynes WM, editor. CRC Handbook of Chemistry and Physics. 92nd ed. Boca Raton, FL: CRC Press; 2011. p. 5.6

[15] Paine RT, Narula CK. Synthetic routes to boron nitride. Chemical Reviews. 1990;**90**:73-91. DOI: 10.1021/

[16] Golberg D, Bando Y, Huang Y, Terano T, Mitome M, Tang C, et al. Boron nitride nanotubes and

nanosheets. ACS Nano. 2010;**4**:2979.

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*DOI: http://dx.doi.org/10.5772/intechopen.90528*

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[3] Ansaloni LMS, Barros de Sousa EM. Boron nitride nanostructured: Synthesis, characterization and potential use in cosmetics. Materials Sciences and Applications. 2013;**4**:22-28. DOI:

[4] Su CY, Wang JC, Chen CY, Chu K, Lin CK. Spherical composite powder by coupling polymethyl methacrylate and boron nitride via spray drying for cosmetic application. Materials. 2019;**12**:706. DOI: 10.3390/ma12050706

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*Few-Layered Hexagonal Boron Nitride: Functionalization, Nanocomposites... DOI: http://dx.doi.org/10.5772/intechopen.90528*

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**168**

**Author details**

and Ewa Mijowska<sup>2</sup>

Szczecin, Poland

Magdalena Jedrzejczak-Silicka1

of Technology, Szczecin, Poland

provided the original work is properly cited.

and piot.kata@gmail.com

\*, Martyna Trukawka<sup>2</sup>

2 Department of Physicochemistry of Nanomaterials, West Pomeranian University

© 2020 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,

3 Department of Physiology, Pomeranian Medical University, Szczecin, Poland

\*Address all correspondence to: mjedrzejczak@zut.edu.pl

1 Laboratory of Cytogenetics, West Pomeranian University of Technology,

, Katarzyna Piotrowska<sup>3</sup>

\*

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10.1039/C4CC07324J

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10.1002/adma.200900323

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[35] Kim SM, Hsu A, Park MH, Chae SH, Yun SJ, Lee JS, et al. Synthesis of largearea multilayer hexagonal boron nitride for high material performance. Nature Communications. 2015;**6**:8662. DOI: 10.1038/ncomms9662

[36] Han R, Khan MH, Angeloski A, Casillas G, Yoon CW, Sun X, et al. Hexagonal boron nitride nanosheets grown via chemical vapor deposition for silver protection. ACS Applied Nano Materials. 2019;**2**:2830-2835. DOI: 10.1021/acsanm.9b00298

[37] Auwärter W. Hexagonal boron nitride monolayers on metal supports: Versatile templates for atoms, molecules and nanostructures. Surface Science Reports. 2019;**74**:1-95. DOI: 10.1016/j. surfrep.2018.10.001

[38] Lin Y, Williams TV, Xu TB, Cao W, Elsayed-Ali HE, Connell JW. Aqueous dispersions of few-layered and monolayered hexagonal boron nitride nanosheets from sonication-assisted hydrolysis: Critical role of water. Journal of Physical Chemistry C. 2011;**115**:2679- 2685. DOI: 10.1021/jp110985w

[39] Gonzalez Ortiz D, Pochat-Bohatier C, Cambedouzo J, Bechelany M, Miele P. Exfoliation of hexagonal boron nitride (h-BN) in liquide phase by ion intercalation. Nanomaterials. 2018;**8**:716. DOI: 10.3390/nano8090716

[40] Gao T, Song X, Du H, Nie Y, Chen Y, Ji Q, et al. Temperaturetriggered chemical switching growth of in-plane and vertically stacked graphene-boron nitride heterostructures. Nature Communications. 2015;**6**:6835. DOI: 10.1038/ncomms7835

[41] Czarniewska E, Mrówczyńska L, Jędrzejczak-Silicka M, Nowicki P, Trukawka M, Mijowska E. Non-cytotoxic hydroxyl-functionalized exfoliated boron nitride nanoflakes impair the immunological function of insect haemocytes in vivo. Scientific Reports. 2019;**9**:14027. DOI: 10.1038/ s41598-019-50097-0

[42] Sudeep PM, Vinod S, Ozden S, Sruthi R, Kukovecz A, Kónya Z, et al. Functionalized boron nitride porous solids. RSC Advances. 2015;**5**:93964- 93968. DOI: 10.1039/C5RA19091F

[43] Sainsbury T, Satti A, May P, Wang Z, McGovern I, Gun'ko YK, et al. Oxygen radical functionalization of boron nitride nanosheets. Journal of the American Chemical Society. 2012;**134**:18758-18771. DOI: 10.1021/ ja3080665

[44] Pakdel A, Bando Y, Golberg D. Plasma-assisted interface engineering of boron nitride nanostructure films. ACS Nano. 2014;**8**:10631-10639. DOI: 10.1021/nn5041729

[45] Lee D, Lee B, Park HK, Ryu HJ, Jeon S, Hong SH. Scalable exfoliation process for highly soluble boron nitride nanoplatelets by hydroxide-assisted ball milling. Nano Letters. 2015;**15**:1238- 1244. DOI: 10.1021/nl504397h

[46] Harley-Trochimczyk A, Pham T, Chang J, Chen E, Worsley MA, Zettl A, et al. Platinum nanoparticle loading of boron nitride aerogel and its use as a novel material for low-power catalytic gas sensing. Advanced Functional Materials. 2016;**26**:433-439. DOI: 10.1002/adfm.201503605

[47] Dai PC, Xue YM, Wang XB, Weng QH, Zhang C, Jiang XF, et al. Pollutant capturing SERS substrate: Porous boron nitride microfibers with uniform silver nanoparticle decoration. Nanoscale. 2014;**118**:21110-21118. DOI: 10.1039/C5NR05625J

[48] Elumalai G, Noguchi H, Lyalin A, Taketsugu T, Uosaki K. Gold nanoparticle decoration of insulating boron nitride nanosheet on inert gold electrode toward an efficient electrocatalyst for the reduction of oxygen to water. Electrochemistry Communications. 2016;**66**:53-57. DOI: 10.1016/j.elecom.2016.02.021

[49] Yang GH, Abulizi A, Zhu JJ. Sonochemical fabrication of gold nanoparticles-boron nitride sheets nanocomposites for enzymeless hydrogen peroxide detection. Ultrasonics Sonochemistry. 2014;**21**:1958-1963. DOI: 10.1016/j. elecom.2016.02.021

[50] Griffin A, Harvey A, Cunningham B, Scullion D, Tian T, Shih CJ, et al. Spectroscopic size and thickness metrics for liquid-exfoliated h-BN. Chemistry of Materials. 2018;**30**:1998-2005. DOI: 10.1021/acs. chemmater.7b05188

[51] Draz MS, Lu X. Development of a loop mediated isothermal amplification (LAMP)—Surface Enhanced Raman spectroscopy (SERS) assay for the detection of *Salmonella enterica* serotype enteritidis. Theranostics. 2016;**6**:522- 532. DOI: 10.7150/thno.14391

[52] Chen S, Zhao M, Wu G, Yao C, Zhang J. Recent advances in morphological cell image analysis. Computational and Mathematical Methods in Medicine. 2012;**2012**:101536. DOI: 10.1155/2012/101536

[53] Trukawka M, Piotrowska K, Jedrzejczak-Silicka M, Mijowska E. A preliminary in vitro study of boron nitride nanocomposites loaded with 10-hydroxycamptothecin on mammalian cells. In: 5th Polish Conference Graphene and 2D Materials; 19-21 September 2019; Szczecin. Szczecin: ZUT; 2019. pp. 49-50

**173**

Section 5

Environmental

Management and Risk

Assessment

Section 5

## Environmental Management and Risk Assessment

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

h-BN. Chemistry of Materials. 2018;**30**:1998-2005. DOI: 10.1021/acs.

532. DOI: 10.7150/thno.14391

DOI: 10.1155/2012/101536

[53] Trukawka M, Piotrowska K, Jedrzejczak-Silicka M, Mijowska E. A preliminary in vitro study of boron nitride nanocomposites loaded with 10-hydroxycamptothecin on mammalian cells. In: 5th Polish

19-21 September 2019; Szczecin. Szczecin: ZUT; 2019. pp. 49-50

Conference Graphene and 2D Materials;

[52] Chen S, Zhao M, Wu G, Yao C, Zhang J. Recent advances in morphological cell image analysis. Computational and Mathematical Methods in Medicine. 2012;**2012**:101536.

[51] Draz MS, Lu X. Development of a loop mediated isothermal amplification (LAMP)—Surface Enhanced Raman spectroscopy (SERS) assay for the detection of *Salmonella enterica* serotype enteritidis. Theranostics. 2016;**6**:522-

chemmater.7b05188

[44] Pakdel A, Bando Y, Golberg D. Plasma-assisted interface engineering of boron nitride nanostructure films. ACS Nano. 2014;**8**:10631-10639. DOI:

[45] Lee D, Lee B, Park HK, Ryu HJ, Jeon S, Hong SH. Scalable exfoliation process for highly soluble boron nitride nanoplatelets by hydroxide-assisted ball milling. Nano Letters. 2015;**15**:1238- 1244. DOI: 10.1021/nl504397h

[46] Harley-Trochimczyk A, Pham T, Chang J, Chen E, Worsley MA, Zettl A, et al. Platinum nanoparticle loading of boron nitride aerogel and its use as a novel material for low-power catalytic gas sensing. Advanced Functional Materials. 2016;**26**:433-439. DOI:

10.1002/adfm.201503605

10.1039/C5NR05625J

[47] Dai PC, Xue YM, Wang XB, Weng QH, Zhang C, Jiang XF, et al. Pollutant capturing SERS substrate: Porous boron nitride microfibers with uniform silver nanoparticle decoration. Nanoscale. 2014;**118**:21110-21118. DOI:

[48] Elumalai G, Noguchi H, Lyalin A,

nanoparticle decoration of insulating boron nitride nanosheet on inert gold electrode toward an efficient electrocatalyst for the reduction of oxygen to water. Electrochemistry Communications. 2016;**66**:53-57. DOI:

Taketsugu T, Uosaki K. Gold

10.1016/j.elecom.2016.02.021

elecom.2016.02.021

[50] Griffin A, Harvey A,

Cunningham B, Scullion D, Tian T, Shih CJ, et al. Spectroscopic size and thickness metrics for liquid-exfoliated

[49] Yang GH, Abulizi A, Zhu JJ. Sonochemical fabrication of gold nanoparticles-boron nitride sheets nanocomposites for enzymeless hydrogen peroxide detection. Ultrasonics Sonochemistry. 2014;**21**:1958-1963. DOI: 10.1016/j.

10.1021/nn5041729

**172**

**175**

**Chapter 10**

**Abstract**

Nanosafety

guidelines, environment safety

**1. Introduction**

*Muthuraman Yuvaraj, Venkatesan Yuvaraj,* 

*Venugopal Arunkumar, Muthaiyan Pandiyan* 

*and Kizhaeral Sevathapandian Subramanian*

The nanomaterials resembling nanotubes, nanospheres, nanofertilizer, nanoherbicide, nanoinsecticide, and nanosheets have the physical, chemical, biological, mechanical, electrical and thermal properties. Still, the nanoparticles have very minute dimensions, enormous area and high reactivity they need the potential ability to penetrate in living cells quite rapidly. The petite size nanoparticles contain lofty surface area may cause higher reactivity with nearby particles. It is broadly predictable that there is a critical need for more information and facts about the implications of manufactured nanomaterials on personal fitness and surroundings. Concerns about potential risks to health that may arise during the making, management, use, and discarding of these nanomaterials have been spoken over the past few years. Consequently, strong research action is being undertaken in various institutions, and industries across the world to appraise their toxicity and spread of nanoparticle.

**Keywords:** nanoparticle, issues of nanoparticles size, hazardous nanoparticles,

The possible risks to health from nanomaterials can be cheap by safe management and organization of the disclosure. Even as no sole part of direction can offer an ultimate, step-by-step advance to safe usage of all nanomaterials in all situations, there are some universal and exact best carry out guides that can be used in nearly all applications [1]. Nanotechnologies have speedy promoted the occasion a substitute making of smart innovative goods and processes have created an unbelievable increase latent for a huge number of industry sectors. The current dispute on the risks of nanotechnologies tends to specialize in the potential dangers of nanoparticles. A growing interest in the production and application of nanoparticles has

The protection issues with nanoparticles are not incredibly fine identified but they are possible for threat is obvious owing to the high exterior area-to-volume ratio, which can create the particles especially hasty or catalytic movement. The understanding of in what way nanomaterials relate to the alive system is imperfect [4, 5]. A toxicological study has been generated a great contract in order on the affiliation among the physical and chemical properties of nanoparticles and their difficult effect on our fitness. These artificial nanoparticles comprise nanotubes, fullerenes, nanowires, quantum dots and diverse nanoparticles used for drug release and analysis. Due to their odd shapes and high reactivity, their effect on the metabolism cannot simply be predicted [6].

been generated the need for appropriate safety measures [2, 3].

#### **Chapter 10**

## Nanosafety

*Muthuraman Yuvaraj, Venkatesan Yuvaraj, Venugopal Arunkumar, Muthaiyan Pandiyan and Kizhaeral Sevathapandian Subramanian*

#### **Abstract**

The nanomaterials resembling nanotubes, nanospheres, nanofertilizer, nanoherbicide, nanoinsecticide, and nanosheets have the physical, chemical, biological, mechanical, electrical and thermal properties. Still, the nanoparticles have very minute dimensions, enormous area and high reactivity they need the potential ability to penetrate in living cells quite rapidly. The petite size nanoparticles contain lofty surface area may cause higher reactivity with nearby particles. It is broadly predictable that there is a critical need for more information and facts about the implications of manufactured nanomaterials on personal fitness and surroundings. Concerns about potential risks to health that may arise during the making, management, use, and discarding of these nanomaterials have been spoken over the past few years. Consequently, strong research action is being undertaken in various institutions, and industries across the world to appraise their toxicity and spread of nanoparticle.

**Keywords:** nanoparticle, issues of nanoparticles size, hazardous nanoparticles, guidelines, environment safety

#### **1. Introduction**

The possible risks to health from nanomaterials can be cheap by safe management and organization of the disclosure. Even as no sole part of direction can offer an ultimate, step-by-step advance to safe usage of all nanomaterials in all situations, there are some universal and exact best carry out guides that can be used in nearly all applications [1]. Nanotechnologies have speedy promoted the occasion a substitute making of smart innovative goods and processes have created an unbelievable increase latent for a huge number of industry sectors. The current dispute on the risks of nanotechnologies tends to specialize in the potential dangers of nanoparticles. A growing interest in the production and application of nanoparticles has been generated the need for appropriate safety measures [2, 3].

The protection issues with nanoparticles are not incredibly fine identified but they are possible for threat is obvious owing to the high exterior area-to-volume ratio, which can create the particles especially hasty or catalytic movement. The understanding of in what way nanomaterials relate to the alive system is imperfect [4, 5]. A toxicological study has been generated a great contract in order on the affiliation among the physical and chemical properties of nanoparticles and their difficult effect on our fitness. These artificial nanoparticles comprise nanotubes, fullerenes, nanowires, quantum dots and diverse nanoparticles used for drug release and analysis. Due to their odd shapes and high reactivity, their effect on the metabolism cannot simply be predicted [6].

The inhaled nanoparticle can be deposited all through the human respiratory tract and lungs. Nanoparticles can be transferred in the lungs to added organs such as the brain, liver and maybe the fetus in pregnant women [7, 8]. Nanoparticles can get into the body during the surface of the skin, lungs, and gastrointestinal system. This force helps make free radicals, which may cause a cell to injure and break to deoxyribonucleic acid. Besides, these can pass through cell membranes in organisms and may interact with biological systems [9, 10].

### **2. Nano hazard identification**

The detection of nano hazards is that the starts to decide risk and contact. This step involves typical nanomaterials and their connected processes that source destructive. When assessing the risks coupled with nanomaterials, particular care must be taken to spot the specific achievement of surface chemistry, shape, size and morphology on toxicity caused to diverse organs. The successive key hazard categories could also be measured when assessing risk linked to nanomaterials [11, 12] (**Figure 1**).

#### **2.1 Surface charge**

The leading exciting chemistry characteristic of nanoparticle about toxicity is that the surface charge, with toxicity rising contained by the subsequent way: neutral < anionic < cationic.

#### **2.2 Surface chemistry**

The surface chemistry of nanoparticles may require a duty inside the age group of free radicals, which influences the broad surface reactivity and toxicity of ingested particles [13].

**177**

*Nanosafety*

**2.3 Particle shape**

**2.4 Particle size**

**3.1 Hygiene**

**3.2 Labeling and signage**

*DOI: http://dx.doi.org/10.5772/intechopen.92290*

Studies have recognized that contact with leathery particles like amphibole will boost the carcinogenic effect. Correspondingly the tubular formation of carbon nanotubes is supposed to cause inflammation and lesions in the lungs [14].

Nanoparticles will go through the membrane barriers ensuing in critical compensation for occurrence particularly silver nanoparticles with size <9 nm can enter the nuclear membrane of certain human cells nucleus and cause major deoxyribonucleic acid mutation [15, 16]. Weakly soluble inhaled nanoparticles will basis aerobic stress, leading to inflammation, fibrosis, and cancer. Several research reported that considerably higher toxicity of nano metals as compared to nanoceramics that

Do not eat or store food and beverages in a nanotechnology laboratory. Do not use mouth suction for pipetting or siphoning. Wash hands regularly to reduce nanoparticle exposure during intake and dermal contact. Remove gloves when exiting the laboratory, so as not to infect doorknobs, or when handling common use

Store in a well-sealed container, preferably one that can be opened with minimal

Specifically, watch out for exposure during cleaning operations. Wear gloves and work in a fume hood while handling nanoparticle and clean the fume hood afterward. If needed, monitor the lab air nanomaterial concentrations during clean-up. Wear respiration protection when working outside a fume hood or in an open fume hood and consider overall protection [20, 21]. Materials and surfaces can be cleaned by following techniques like wiped with a wet cloth where possible, rinsing off the cloth with water or disposing of it. The vacuum cleaner is equipped with a high-efficiency particulate air filter. Monitor the exhaust of the vacuum cleaner during operation. A malfunctioning filter can increase the exposure by dispersing the nanomaterial in the air. High-efficiency particulate air filtered vacuum cleaners with a combination of wet wiping is more suitable for most nanomaterial clean-up [22, 23]. Energetic cleaning methods such as dry cleaning or the use of compressed air should be prohibited [24]. Collect spill cleanup materials in a tightly closed container. The nanoparticle spill kit containing the

agitation of the contents. Label all chemical containers with the identity of the contents. Hazard warning and chemical concentration information should also be included if known. Apply cautious decision when leaving operations unattended: (i) Post signs to communicate appropriate warnings and precautions, (ii) anticipate potential equipment and facility failures, and (iii) provide appropriate suppression

have been recognized to higher suspension rate in water [17, 18].

**3. Finest practice to be pursued while using nanoparticles**

objects such as phones, multiuser computers, etc.

for chance release of hazardous chemicals [19].

**3.3 Clean-up measures and spills**

#### **2.3 Particle shape**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

and may interact with biological systems [9, 10].

**2. Nano hazard identification**

nanomaterials [11, 12] (**Figure 1**).

**2.1 Surface charge**

neutral < anionic < cationic.

**2.2 Surface chemistry**

ingested particles [13].

The inhaled nanoparticle can be deposited all through the human respiratory tract and lungs. Nanoparticles can be transferred in the lungs to added organs such as the brain, liver and maybe the fetus in pregnant women [7, 8]. Nanoparticles can get into the body during the surface of the skin, lungs, and gastrointestinal system. This force helps make free radicals, which may cause a cell to injure and break to deoxyribonucleic acid. Besides, these can pass through cell membranes in organisms

The detection of nano hazards is that the starts to decide risk and contact. This step involves typical nanomaterials and their connected processes that source destructive. When assessing the risks coupled with nanomaterials, particular care must be taken to spot the specific achievement of surface chemistry, shape, size and morphology on toxicity caused to diverse organs. The successive key hazard categories could also be measured when assessing risk linked to

The leading exciting chemistry characteristic of nanoparticle about toxicity is that the surface charge, with toxicity rising contained by the subsequent way:

The surface chemistry of nanoparticles may require a duty inside the age group

of free radicals, which influences the broad surface reactivity and toxicity of

**176**

**Figure 1.**

*Diagram of the projected safety plan.*

Studies have recognized that contact with leathery particles like amphibole will boost the carcinogenic effect. Correspondingly the tubular formation of carbon nanotubes is supposed to cause inflammation and lesions in the lungs [14].

#### **2.4 Particle size**

Nanoparticles will go through the membrane barriers ensuing in critical compensation for occurrence particularly silver nanoparticles with size <9 nm can enter the nuclear membrane of certain human cells nucleus and cause major deoxyribonucleic acid mutation [15, 16]. Weakly soluble inhaled nanoparticles will basis aerobic stress, leading to inflammation, fibrosis, and cancer. Several research reported that considerably higher toxicity of nano metals as compared to nanoceramics that have been recognized to higher suspension rate in water [17, 18].

### **3. Finest practice to be pursued while using nanoparticles**

#### **3.1 Hygiene**

Do not eat or store food and beverages in a nanotechnology laboratory. Do not use mouth suction for pipetting or siphoning. Wash hands regularly to reduce nanoparticle exposure during intake and dermal contact. Remove gloves when exiting the laboratory, so as not to infect doorknobs, or when handling common use objects such as phones, multiuser computers, etc.

#### **3.2 Labeling and signage**

Store in a well-sealed container, preferably one that can be opened with minimal agitation of the contents. Label all chemical containers with the identity of the contents. Hazard warning and chemical concentration information should also be included if known. Apply cautious decision when leaving operations unattended: (i) Post signs to communicate appropriate warnings and precautions, (ii) anticipate potential equipment and facility failures, and (iii) provide appropriate suppression for chance release of hazardous chemicals [19].

#### **3.3 Clean-up measures and spills**

Specifically, watch out for exposure during cleaning operations. Wear gloves and work in a fume hood while handling nanoparticle and clean the fume hood afterward. If needed, monitor the lab air nanomaterial concentrations during clean-up. Wear respiration protection when working outside a fume hood or in an open fume hood and consider overall protection [20, 21]. Materials and surfaces can be cleaned by following techniques like wiped with a wet cloth where possible, rinsing off the cloth with water or disposing of it. The vacuum cleaner is equipped with a high-efficiency particulate air filter. Monitor the exhaust of the vacuum cleaner during operation. A malfunctioning filter can increase the exposure by dispersing the nanomaterial in the air. High-efficiency particulate air filtered vacuum cleaners with a combination of wet wiping is more suitable for most nanomaterial clean-up [22, 23]. Energetic cleaning methods such as dry cleaning or the use of compressed air should be prohibited [24]. Collect spill cleanup materials in a tightly closed container. The nanoparticle spill kit containing the

following items Barricade tape, Latex or nitrile gloves, Adsorbent material, Wipes, Sealable plastic bags, Walk-off mat [25].

#### **4. Strategy for functioning with nanomaterials**

Use sensible general laboratory safety practices as found in your chemical hygiene set up. Do not handle nanoparticles with your bare skin. If it is necessary to handle nanoparticle powders outside of a high-efficiency particulate air filtered to maintain exhaust streamline flow hood. Lab equipment and exhaust systems should also be evaluated before removal, remodeling, or repair [26, 27]. Given the differing artificial ways and experimental goals, no blanket recommendation will be created concerning aerosol emissions controls. Consideration should tend to the high reactivity of some nanopowder materials about potential fire and explosion hazards [28].

#### **5. Constant monitoring of lab air**

The nanoparticle detector should be installed in every lab, in which gas-phase work on nanoparticles is passed out and where the capacity of nanoparticulate material exceeds a certain limit. We advocate a limit of 1 μg/h. An instrument of this kind is commercially available as a Joint Length Monitor. This unit contains a size parting mechanism so particles <0, 1 μm area unit mostly detected [29].

#### **6. Discarding of nanoparticles**

The quantities of nanoparticles like powders, colloids exceeding the milligram range should be treated as chemical if the particle solubility in water is very small (inorganics like gold, titanium oxide). If the solubility is higher, the principles consistent with the toxicity class of the macroscopic material apply. Nanoparticle residues in water from cleaning can be poured down the drain [30] (**Table 1**).

**179**

*Nanosafety*

**Waste nanomaterial**

Dust nanoparticles

Nanomaterial bound in resin or polymer

Nanomaterial in a solid matrix but

Nanomaterial in a solid matrix not

friable

friable

**Table 1.**

*DOI: http://dx.doi.org/10.5772/intechopen.92290*

Fluid solutions Process solvent-soluble

ions

Infected solids Moisten if necessary Double bag

with the solvent waste stream. Aggregate nanoparticle will get dissolved and form

As for liquid solutions or packages a nanomaterial in a solid matrix not friable

Moisten Double

None Single

**8. Working place with nanoparticle**

*Handling and Disposal of Waste Nanomaterial.*

spills after using suitable safety [33, 34].

**9. Development and usage of nanomaterials**

When handle nanomaterials in solutions or close substrates to reduce airborne release. While working with nanomaterials in liquids it must avoid dispersal of the liquid by operating through a spill instrumentality. Wear gloves that are suited for the liquid being handled. Avoid the dispersion of liquid droplets within the workplace air and directly close up spills, before evaporation or further spreading occurs. While working with nanomaterials in gas phase reactors add a closed reaction vessel, preferably around atmospheric or lower than atmospheric pressure [31, 32]. Make aware leak checks among runs once operating with systems under positive pressure adjust the quality safety rules for controlled vessels and place the vessel into an interior safety vessel. Clean all parts that are in touch with nanoparticles and

**Pre-treatment Containment Level of** 

Moisten Double Inside a local

plastic, sealable

Drip tray or funnel vial or container or drip tray

Single containment or double containment if liquid.

containment

containment

**engineering controls**

exhausted aeration enclosure or glove box

Inside a local exhausted aeration enclosure or glove box

Inside a local exhausted aeration enclosure

General aeration

Inside a local exhausted aeration enclosure

General aeration

**Disposal method**

Burning

Burning

Burning mix with solid waste. Burn either mix with other soluble waste or dilute to drain if appropriate

Burning or licensed landfill

Burning or licensed landfill

Burning or licensed landfill

Nanomaterials must be stored and transported in sealed shatter-resistant containers. The containers must be labeled with nanomaterial or composition and near

#### **7. Transport of nanoparticle**


*Biochemical Toxicology - Heavy Metals and Nanomaterials*

**4. Strategy for functioning with nanomaterials**

Sealable plastic bags, Walk-off mat [25].

**5. Constant monitoring of lab air**

**6. Discarding of nanoparticles**

**7. Transport of nanoparticle**

• Exposure monitoring

• Applicable regulation

following items Barricade tape, Latex or nitrile gloves, Adsorbent material, Wipes,

Use sensible general laboratory safety practices as found in your chemical hygiene set up. Do not handle nanoparticles with your bare skin. If it is necessary to handle nanoparticle powders outside of a high-efficiency particulate air filtered to maintain exhaust streamline flow hood. Lab equipment and exhaust systems should also be evaluated before removal, remodeling, or repair [26, 27]. Given the differing artificial ways and experimental goals, no blanket recommendation will be created concerning aerosol emissions controls. Consideration should tend to the high reactivity of some nanopowder materials about potential fire and explosion hazards [28].

The nanoparticle detector should be installed in every lab, in which gas-phase work on nanoparticles is passed out and where the capacity of nanoparticulate material exceeds a certain limit. We advocate a limit of 1 μg/h. An instrument of this kind is commercially available as a Joint Length Monitor. This unit contains a size

The quantities of nanoparticles like powders, colloids exceeding the milligram range should be treated as chemical if the particle solubility in water is very small (inorganics like gold, titanium oxide). If the solubility is higher, the principles consistent with the toxicity class of the macroscopic material apply. Nanoparticle residues in water from cleaning can be poured down the drain [30] (**Table 1**).

parting mechanism so particles <0, 1 μm area unit mostly detected [29].

• Safe handling of nanomaterials and normal operation procedures

• To check the hazards and toxicity of nanoparticle

• Engineering controls and equipment maintenance

• Environmental release, shipping, customer protection

• Description of nanoparticles should be known

• Labeling and handling of nanomaterials waste

• Personal protective equipment to be kept

**178**


#### **Table 1.**

*Handling and Disposal of Waste Nanomaterial.*

#### **8. Working place with nanoparticle**

When handle nanomaterials in solutions or close substrates to reduce airborne release. While working with nanomaterials in liquids it must avoid dispersal of the liquid by operating through a spill instrumentality. Wear gloves that are suited for the liquid being handled. Avoid the dispersion of liquid droplets within the workplace air and directly close up spills, before evaporation or further spreading occurs. While working with nanomaterials in gas phase reactors add a closed reaction vessel, preferably around atmospheric or lower than atmospheric pressure [31, 32]. Make aware leak checks among runs once operating with systems under positive pressure adjust the quality safety rules for controlled vessels and place the vessel into an interior safety vessel. Clean all parts that are in touch with nanoparticles and spills after using suitable safety [33, 34].

#### **9. Development and usage of nanomaterials**

Nanomaterials must be stored and transported in sealed shatter-resistant containers. The containers must be labeled with nanomaterial or composition and near particle size, along with any known hazard warnings. Weighing and measuring of dry powders where aerosolization and discharge of nanomaterials are possible should be conducted in clear and closed areas [25]. Different processing steps such as dispersing, mixing, spraying, machining, gas-phase processing have the potential to make nanoparticles with a high concentration. Employing a closed facility to process nanomaterials will considerably reduce occupational exposure during the assembly and processing stages. Particular care to be taken to avoid disturbance of the closed liquid medium to avoid dust scattering and thus disclosure through inhalation [35, 36]. Removal of waste and by-products generated at the assembly facility should be administered with minimum exposure to humans and therefore the environment.

#### **10. The behavior of nanopowders in the food industry**

Nanotechnologies propose a diversity of possible for relevance in different areas of food technology that comprise packaging, processing, quality and shelf life, ingredients and additives. The complexity in characterizing assorted nanomaterials used the food industry and biological systems incomplete information on toxicology and lack optimal test methods the risk appraisal and supervision of nanotechnologies harder [37, 38]. A preventive advance with detailed life cycle estimation and strongly required procedures to stakeholders connecting for diverse activities as formulating best practices that will support the growth of nanotechnology in the food sector for regulating the potential risk to humans and the environment. Humans are exposed to nanomaterials using oral way during the residues present in cultivated crops, meat, and milk produced for consumption further oral contact is most significant in food processing technology and functional foods. Nanomaterial exposure from food packaging is mostly dermal, arising from the usage of such materials [39, 40].

#### **11. Conclusions**

The nanotechnology is an enables gifted technology its vast marvelous future applications. Conversely, there are a few drawbacks for example toxicity of the soil, ecological harm and human organ damage caused by nanoparticles. The vital significance provides to contain nanosafety into the occasion of novel nanotechnologies and find products of nanosafety before making a design. It has been at present identified that some engineered nanomaterials will present new and abnormal risks, but there is very little information on how these risks can be recognized, assessed and proscribed. In distinction, good working hygiene practices and existing knowledge on operational with dangerous substances provide a useful source for working safely with nanomaterials. Further, investigate to be conducted for nanosafety and discarding of the nanoparticle. Finally, in the future, many types of nanoparticles may turn out to be of less toxicity but preventative measures should be used while handling particle.

**181**

**Author details**

Muthuraman Yuvaraj1

Muthaiyan Pandiyan1

Cheyyar, Tamil Nadu, India

\*, Venkatesan Yuvaraj2

2 Department of Physics, Arignar Anna Government Arts College,

3 Tamil Nadu Agricultural University, Coimbatore, India

\*Address all correspondence to: yuvasoil@gmail.com

provided the original work is properly cited.

and Kizhaeral Sevathapandian Subramanian3

1 Agricultural College and Research Institute, Vazhavachanur, Tamil Nadu, India

© 2020 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,

, Venugopal Arunkumar1

,

*Nanosafety*

*DOI: http://dx.doi.org/10.5772/intechopen.92290*

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

**10. The behavior of nanopowders in the food industry**

the environment.

materials [39, 40].

**11. Conclusions**

be used while handling particle.

particle size, along with any known hazard warnings. Weighing and measuring of dry powders where aerosolization and discharge of nanomaterials are possible should be conducted in clear and closed areas [25]. Different processing steps such as dispersing, mixing, spraying, machining, gas-phase processing have the potential to make nanoparticles with a high concentration. Employing a closed facility to process nanomaterials will considerably reduce occupational exposure during the assembly and processing stages. Particular care to be taken to avoid disturbance of the closed liquid medium to avoid dust scattering and thus disclosure through inhalation [35, 36]. Removal of waste and by-products generated at the assembly facility should be administered with minimum exposure to humans and therefore

Nanotechnologies propose a diversity of possible for relevance in different areas

The nanotechnology is an enables gifted technology its vast marvelous future applications. Conversely, there are a few drawbacks for example toxicity of the soil, ecological harm and human organ damage caused by nanoparticles. The vital significance provides to contain nanosafety into the occasion of novel nanotechnologies and find products of nanosafety before making a design. It has been at present identified that some engineered nanomaterials will present new and abnormal risks, but there is very little information on how these risks can be recognized, assessed and proscribed. In distinction, good working hygiene practices and existing knowledge on operational with dangerous substances provide a useful source for working safely with nanomaterials. Further, investigate to be conducted for nanosafety and discarding of the nanoparticle. Finally, in the future, many types of nanoparticles may turn out to be of less toxicity but preventative measures should

of food technology that comprise packaging, processing, quality and shelf life, ingredients and additives. The complexity in characterizing assorted nanomaterials used the food industry and biological systems incomplete information on toxicology and lack optimal test methods the risk appraisal and supervision of nanotechnologies harder [37, 38]. A preventive advance with detailed life cycle estimation and strongly required procedures to stakeholders connecting for diverse activities as formulating best practices that will support the growth of nanotechnology in the food sector for regulating the potential risk to humans and the environment. Humans are exposed to nanomaterials using oral way during the residues present in cultivated crops, meat, and milk produced for consumption further oral contact is most significant in food processing technology and functional foods. Nanomaterial exposure from food packaging is mostly dermal, arising from the usage of such

**180**

### **Author details**

Muthuraman Yuvaraj1 \*, Venkatesan Yuvaraj2 , Venugopal Arunkumar1 , Muthaiyan Pandiyan1 and Kizhaeral Sevathapandian Subramanian3

1 Agricultural College and Research Institute, Vazhavachanur, Tamil Nadu, India

2 Department of Physics, Arignar Anna Government Arts College, Cheyyar, Tamil Nadu, India

3 Tamil Nadu Agricultural University, Coimbatore, India

\*Address all correspondence to: yuvasoil@gmail.com

© 2020 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.

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[17] Mulhopt S, Dilger M, Diabate S, Schlager C, Krebs T, Zimmermann R, et al. Toxicity testing of combustion aerosols at the air-liquid interface with a self-contained and easy-to-use exposure system. Journal of Aerosol Science. 2016;**96**:38-55

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[19] Prasad R, Bhattacharyya A, Nguyen QD. Nanotechnology in sustainable agriculture: Recent developments, challenges, and perspectives. Frontiers in Microbiology. 2017;**8**:1014-1019

[20] Wong JKL, Mohseni R, Hamidieh AA, MacLaren RE, Habib N, Seifalian AM. Will nanotechnology bring new hope for gene delivery. Trends in Biotechnology. 2017;**35**:434-451

[21] Malik S, Krasheninnikov AV, Marchesan S. Advances in nanocarbon composite materials. Beilstein Journal of Nanotechnology. 2018;**9**:2-10

[22] Thiruvengadam M, Rajakumar G, Chung IM. Nanotechnology: Current uses and future applications in the food industry. Biotech. 2018;**8**:74

[23] Kim DY, Kadam A, Shinde S, Saratale RG, Patra J, Ghodake G. Recent developments in nanotechnology transforming the agricultural sector: A transition replete with opportunities. Journal of the Science of Food and Agriculture. 2018;**98**:849-864

[24] Jahan S, Yusoff IB, Alias YB, Bakar AFBA. Reviews of the toxicity behavior of five potential engineered nanomaterials (ENMs) into the aquatic ecosystem. Toxicology Reports. 2017;**4**:211-220

[25] Wright MV, Matson CW, Baker LS, Castellon BT, Watkins PS, King RS. Titanium dioxide nanoparticle exposure reduces algal biomass and alters algal assemblage composition in wastewater effluent-dominated stream mesocosms. Science of the Total Environment. 2018;**626**:336-356

[26] Saint-Cricq M, Carrete J, Gaboriaud C, Gravel E, Doris E, Thielens N, et al. Human immune protein C1q selectively disaggregates carbon nanotubes. Nano Letters. 2017;**17**:3409-3415

[27] Naatz H, Lin S, Li R, Jiang W, Ji Z, Chang CH, et al. Safe-by-design CuO nanoparticles via Fe-doping, Cu-O bond length variation, and biological assessment in cells and zebrafish embryos. ACS Nano. 2017;**11**:501-515

[28] Iqbal SA, Wallach JD, Khoury MJ, Schully SD, Ioannidis JP. Reproducible research practices and transparency across the biomedical literature. PLoS Biology. 2016;**14**:12-19

[29] Truffier Boutry D, Fiorentino B, Bartolomei V, Soulas R, Sicardy O, Benayad A, et al. Characterization of photocatalytic paints: A relationship between the photocatalytic properties release of nanoparticles and volatile organic compounds. Environmental Science. Nano. 2017;**4**:1998-2009

[30] Jain N, Bhargava A, Pareek V, Akhtar MS, Panwar J. Does seed size and surface anatomy play role in combating phytotoxicity of nanoparticles. Ecotoxicology. 2017;(26):238-249

[31] Mantecca P, Kasemets K, Deokar A, Perelshtein I, Gedanken A, Bahk YK, et al. Airborne nanoparticle release and toxicological risk from metaloxide-coated textiles: Toward a multiscale safe-by-design approach.

**182**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

[9] Liu Z, Jiang W, Nam J, Moon J, Kim B. Immuno modulating nanomedicine for cancer therapy. Nano Letters.

[10] Kraegeloh A, Suarez-Merino B, Sluijters T, Micheletti C. Implementation of safe-by-design for nanomaterial development and safe innovation: Why we need a comprehensive approach.

Nanomaterials. 2018;**14**:4-8

[11] Pavan C, Fubini B. Unveiling the variability of quartz hazard in light of recent toxicological findings. Chemical Research in Toxicology.

[12] Jeon Y-R, Yu J, Choi S-J. Fate determination of ZnO in commercial foods and human intestinal cells. International Journal of Molecular

[13] Hjorth R, van Hove L, Wickson F. What can nanosafety learn from drug development. The feasibility of "safety by design". Nanotoxicology.

[14] Holman C, Piper SK, Grittner U, Diamantaras AA, Kimmelman J. Where have all the rodents gone? The effects of attrition in experimental research on cancer and stroke. PLoS Biology.

[15] Lewandowsky S, Bishop D. Research integrity: Don't let

[16] Trinh TX, Cho J-S, Jeon H, Byun H-G, Yoon T-H, Kim J. Quasi-SMILES-based nano-quantitative structure–Activity relationship model to predict the cytotoxicity of multiwalled carbon nanotubes to human lung cells. Chemical Research in Toxicology.

transparency damage science. Nature.

Sciences. 2020;**21**:433-439

2018;**18**:6655-6659

2016;**30**:469-485

2017;**113**:305-312

2016;**14**:25-31

2016;**529**:459-461

2018;**31**:183-190

[1] Lin S, Yu T, Yu Z, Hu X, Yin D. Nanomaterials safer-by-design: An environmental safety perspective. Advanced Materials. 2018;**17**:56-91

**References**

[2] Vithanage M, Seneviratne M, Ahmad M, Sarkar B. Contrasting effects of engineered carbon nanotubes on plants: A review. Environmental Geochemistry and Health. 2017;**39**:21-39

[3] Hol PJ, Gjerdet NR. Novel nanoparticulate and ionic titanium antigens for hypersensitivity testing. International Journal of Molecular

[4] Vietti G, Lison D, van den Brul S. Mechanisms of lung fibrosis induced by carbon nanotubes:

Towards an Adverse Outcome Pathway (AOP). Particle and Fibre Toxicology.

[5] Naatz S, Lin R, Li W, Jiang Z, Ji CH, Chang J, et al. Safe-by-design of CuO nanoparticles via Fe-doping, Cu-O bond lengths variation and biological assessment in cells and zebra fish embryos. ACS Nano. 2017;**11**:501-510

[6] Thomm E, Bromme R. How source information shapes lay interpretations of science conflicts: Interplay between sourcing, conflict explanation, source evaluation, and claim evaluation. Reading and Writing. 2016;**29**:1629-1652

Development of theoretical descriptors for cytotoxicity evaluation of metallic nanoparticles. Chemical Research in Toxicology. 2017;**30**:1549-1555

[8] Wang W, Sedykh A, Sun H, Zhao L, Russo DP, Zhou H, et al. Predicting nano–bio interactions by integrating

quantitative nanostructure activity relationship modeling. ACS Nano.

[7] Boukhvalov DW, Yoon TH.

nanoparticle libraries and

2017;**11**:12641-12649

Sciences. 2018;**19**:11-21

2016;**13**:1-11

Environmental Science & Technology. 2017;**51**:9305-9317

[32] Starost K, Frijns E, Van Laer J, Faisal N, Egizabal A, Elizetxea C, et al. The effect of nanosilica (SiO2) and nanoalumina (Al2O3) reinforced polyester nanocomposites on aerosol nanoparticle emissions into the environment during automated drilling. Aerosol Science and Technology. 2017;**51**:1035-1046

[33] Vlasova II, Kapralov AA, Michael ZP, Burkert SC, Shurin MR, Star A, et al. Enzymatic oxidative biodegradation of nanoparticles: Mechanisms, significance and applications. Toxicology and Applied Pharmacology. 2016;**299**:58-69

[34] Cobaleda-Siles M, Guillamon AP, Delpivo C, Vazquez-Campos S, Puntes VF. Safer by design strategies. Journal of Physics Conference Series. 2017;**838**:12-16

[35] Singh D, Sotiriou GA, Zhang F, Mead J, Bello D, Wohlleben W, et al. End-of-life thermal decomposition of nano-enabled polymers: Effect of nanofiller loading and polymer matrix on by-products. Environmental Science. Nano. 2016;**3**:1293-1305

[36] Lopez De Ipina JM, Hernan A, Cenigaonaindia X, Insunza M, Florez S, Seddon R, et al. Implementation of a safe-by design approach in the development of new open pilot lines for the manufacture of carbon nanotube-based nano-enabled products. Journal of Physics Conference Series. 2017;**838**:12-18

[37] Fageria L, Pareek V, Dilip V, Bhargava A, Pasha SS, Laskar IR, et al. Biosynthesized protein-capped silver nanoparticles induce ros-dependent proapoptotic signals and prosurvival autophagy in cancer cells. ACS Omega. 2017;**2**:1489-1504

[38] Bruno D, Mattos B, Tardy L, Washington LE, Magalhaes Orlando J, Rojas E. Controlled release for crop and wood protection: Recent progress toward sustainable and safe nanostructured biocidal systems. Journal of Controlled Release. 2017;**262**:139-150

**Chapter 11**

*and Kavita Rani*

**Abstract**

ascertained.

**1. Introduction**

**185**

Biological Role of *Withania*

*somnifera* against Promiscuity of

Interaction with Macrophages

*Jitendra Kumar, Chander Datt, Surya Kant Verma*

**Keywords:** agriculture, immunotoxicity, macrophages, nanofertilizer,

Nanotechnology is an emerging technology which can lead to a new revolution in many fields of science [1]. Nanoparticles (NPs) are gaining importance recently due to their exciting applications in different fields like biomedical, pharmaceutical, agriculture, etc. The properties of the materials change as their size approaches the nanoscale, and nanoparticles have a very high surface area to volume ratio and high energy. Application of nanoparticle in the agriculture and food sectors is relatively

nanoparticles, *Withania somnifera*, zinc oxide

new as compared to their use in health sector.

Zinc Oxide Nano Particles and Its

In agriculture and food industry, nanotechnology can be utilized to improve crop yield, food quality, shelf life, safety, cost and nutritional benefits. Zinc is a trace element and its deficiency causes health problems in human beings and animals. The use of zinc oxide nanoparticles (ZnO NPs) is growing exponentially in food industry, biomedicine and nanofertilizer segment. A remarkable presence of nanomaterials in ecosystem and consumer products can cause adverse effects. Hence, it is an important challenge for the use of nanoparticles in agriculture as fertilizer to enhance plant yield on one hand and their interaction with the cells of the innate immune system in animals on the other hand. So, public concern about their potential toxicity is increasing. ZnO NPs interact with cells and produce harmful effects in a dose dependent manner. The reactive oxygen species generation might be a reason for the toxicity of ZnO NPs. The toxicity is caused due to dissolved Zn++ ions by absorption which causes adverse effect on phagocytosis and oxidative stress by free radical while *Withania somnifera* induced the phagocytosis activity by antioxidant mechanism thus having protective effects. It is emphasized that further research is needed on the use of nanoparticles in agriculture, animal husbandry, and human health sector so that their safer levels for use could be

[39] Reinosa JJ, Docio CMA, Ramirez VZ, Lozano JFF. Hierarchical nano ZnO-micro TiO2 composites: High UV protection yield lowering photodegradation in sunscreens. Ceramics International. 2018;**44**:2827-2834

[40] Feliu N, Pelaz B, Zhang Q, del Pino P, Nystrom A, Parak W. Nanoparticle dosage-a nontrivial task of utmost importance for quantitative nanosafety research. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 2016;**8**:479-492

#### **Chapter 11**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

[38] Bruno D, Mattos B, Tardy L, Washington LE, Magalhaes Orlando J, Rojas E. Controlled release for crop and wood protection: Recent progress

toward sustainable and safe nanostructured biocidal systems. Journal of Controlled Release.

[39] Reinosa JJ, Docio CMA,

High UV protection yield lowering photodegradation in sunscreens. Ceramics International.

2018;**44**:2827-2834

Ramirez VZ, Lozano JFF. Hierarchical nano ZnO-micro TiO2 composites:

[40] Feliu N, Pelaz B, Zhang Q, del Pino P, Nystrom A, Parak W. Nanoparticle dosage-a nontrivial task of utmost importance for quantitative nanosafety research. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 2016;**8**:479-492

2017;**262**:139-150

Environmental Science & Technology.

environment during automated drilling.

[34] Cobaleda-Siles M, Guillamon AP, Delpivo C, Vazquez-Campos S, Puntes VF. Safer by design strategies. Journal of Physics Conference Series.

[35] Singh D, Sotiriou GA, Zhang F, Mead J, Bello D, Wohlleben W, et al. End-of-life thermal decomposition of nano-enabled polymers: Effect of nanofiller loading and polymer matrix on by-products. Environmental Science.

[36] Lopez De Ipina JM, Hernan A, Cenigaonaindia X, Insunza M, Florez S, Seddon R, et al. Implementation of a safe-by design approach in the development of new open pilot lines for the manufacture of carbon nanotube-based nano-enabled products. Journal of Physics Conference Series.

[37] Fageria L, Pareek V, Dilip V, Bhargava A, Pasha SS, Laskar IR, et al. Biosynthesized protein-capped silver nanoparticles induce ros-dependent proapoptotic signals and prosurvival autophagy in cancer cells. ACS Omega.

Aerosol Science and Technology.

[33] Vlasova II, Kapralov AA, Michael ZP, Burkert SC, Shurin MR, Star A, et al. Enzymatic oxidative biodegradation of nanoparticles: Mechanisms, significance and applications. Toxicology and Applied Pharmacology. 2016;**299**:58-69

[32] Starost K, Frijns E, Van Laer J, Faisal N, Egizabal A, Elizetxea C, et al. The effect of nanosilica (SiO2) and nanoalumina (Al2O3) reinforced polyester nanocomposites on aerosol nanoparticle emissions into the

2017;**51**:9305-9317

2017;**51**:1035-1046

2017;**838**:12-16

Nano. 2016;**3**:1293-1305

2017;**838**:12-18

2017;**2**:1489-1504

**184**

## Biological Role of *Withania somnifera* against Promiscuity of Zinc Oxide Nano Particles and Its Interaction with Macrophages

*Jitendra Kumar, Chander Datt, Surya Kant Verma and Kavita Rani*

#### **Abstract**

In agriculture and food industry, nanotechnology can be utilized to improve crop yield, food quality, shelf life, safety, cost and nutritional benefits. Zinc is a trace element and its deficiency causes health problems in human beings and animals. The use of zinc oxide nanoparticles (ZnO NPs) is growing exponentially in food industry, biomedicine and nanofertilizer segment. A remarkable presence of nanomaterials in ecosystem and consumer products can cause adverse effects. Hence, it is an important challenge for the use of nanoparticles in agriculture as fertilizer to enhance plant yield on one hand and their interaction with the cells of the innate immune system in animals on the other hand. So, public concern about their potential toxicity is increasing. ZnO NPs interact with cells and produce harmful effects in a dose dependent manner. The reactive oxygen species generation might be a reason for the toxicity of ZnO NPs. The toxicity is caused due to dissolved Zn++ ions by absorption which causes adverse effect on phagocytosis and oxidative stress by free radical while *Withania somnifera* induced the phagocytosis activity by antioxidant mechanism thus having protective effects. It is emphasized that further research is needed on the use of nanoparticles in agriculture, animal husbandry, and human health sector so that their safer levels for use could be ascertained.

**Keywords:** agriculture, immunotoxicity, macrophages, nanofertilizer, nanoparticles, *Withania somnifera*, zinc oxide

#### **1. Introduction**

Nanotechnology is an emerging technology which can lead to a new revolution in many fields of science [1]. Nanoparticles (NPs) are gaining importance recently due to their exciting applications in different fields like biomedical, pharmaceutical, agriculture, etc. The properties of the materials change as their size approaches the nanoscale, and nanoparticles have a very high surface area to volume ratio and high energy. Application of nanoparticle in the agriculture and food sectors is relatively new as compared to their use in health sector.

In India, more than 60% of the population survive on agriculture, but unfortunately this sector is facing various global challenges. Therefore, nanotechnology has a dominant position in transforming agriculture and food industry. Nanotechnology has a great ability to transform conventional agricultural practices and boost yield and growth of corps. Zinc oxide NPs (ZnO NPs) are used as fertilizer which support their growth and improves production [2].

Zinc oxide NPs may be used as a source of Zn in supplements and functional foods [3]. ZnO NPs also act as antimicrobial agents against harmful bacteria. The antimicrobial activity of ZnO NPs has been partly attributed to their ability to penetrate into microbial cells and animal cells and generate reactive oxygen species (ROS) that damage cellular components thereby leading to cytotoxicity [4]. A single oral dose of ZnO NPs caused hepatic cell injury, kidney toxicity, and lung damage [5]**.** The studies in frogs showed that ZnO NPs exhibited more toxicity than a dissolved form of Zn which was attributed to their greater ability to induce oxidative damage in cells [6]. The administration of ZnO NPs increased all liver enzymes [7]. In this chapter, we attempted to explore the potential use of nanoparticles in agriculture, biological, and pharmacological significance of *Withania somnifera* against the promiscuity of zinc oxide ZnO NPs and their interaction with macrophages.

#### **2. Use of zinc oxide nanoparticles in agriculture and animal husbandry**

roots *of W. somnifera.* The biological properties of crude root extracts have been largely reported and only a few are related to the pure compound (withaferin A) as

*Plant uptake, transport and environmental transformation mechanism of nanofertilizer (ZnO NPs) into*

*Biological Role of* Withania somnifera *against Promiscuity of Zinc Oxide Nano Particles…*

*DOI: http://dx.doi.org/10.5772/intechopen.90128*

Zinc is crucial for normal development and function of cells mediating innate immunity, neutrophils, and natural killer cells (NKs). Phagocytosis, intracellular killing, and cytokine production are affected by Zn deficiency. Zinc is a micronutrient required by organisms and plays a vital role in maintaining immune and macrophage function. There is a progressive decline in immune response with the advance in aging due to the deficiency of Zn [17]. There is impairment of monocytes, reduced cytotoxicity in NK cells, and reduced phagocytosis in neutrophils [18]. Zinc is also a major intracellular regulator of lymphocyte apoptosis [19]. Impaired immune function in elderly subjects due to Zn deficiency has been shown to be reversed by an adequate Zn supplementation [18]. The beneficial effects of lower doses of Zn (≤ 50 mg/d) on immune function have been reported while very

The defense system is the bedrock of living systems and innate immunity is an integral part of health. It is the first line of the defense mechanism of the body from lower organism to mammals. Any alteration in innate immunity leads to disease conditions; however, adaptive immunity plays a great role in defense mechanism. Innate immunity has two arms, i.e., the afferent and efferent. The afferent arm is lipopolysaccharide (LPS) or endotoxin [21]. As to the effector arm of innate immunity, Hunter (1774) first recognized leukocytes at the site of inflammation. The cellular theory of immunity was given by Metchnikoff, 1884 [22] and must be recognized in the functional analysis of innate immune cells. Massart and Bordet

**3. Immunological health importance of zinc as microelement**

*ecosystem and entry in food chain [14, 15]. Designed by first author using Google as tool.*

high doses of Zn (≥ 150 mg/d) may impair cellular immunity [20].

**4. Mechanism of innate immunity in animals**

immunomodulatory function.

**Figure 1.**

**187**

#### **2.1 ZnO NPs and their potential use in agriculture**

Once nanomaterials (NMs) are released to the environment, they accumulate in ecosystems and pose threats to living organisms; therefore, it is important to understand the behavior of NMs in soil and to assess the risks for arable soil ecosystems [8]. About 260,000–309,000 metric tons (MT) of NMs were produced globally in 2010 [9], and worldwide consumption of NMs is likely to grow from 225,060 to 585,000 MT during 2014–2019 [10]. The third most commonly used metal-containing NMs are ZnO NPs with an estimated global annual production between 550 and 33,400 tons [11]. The concentration of ZnO NPs in the environment was found to be 3.1–31 μg kg<sup>1</sup> and 76–760 μg L<sup>1</sup> in soil and water, respectively [12]. ZnO NPs can strongly attach to soil particles. They exhibit low mobility at various ionic strengths [13] and show higher sorption compared to ionic zinc, and possible uptake mechanism has been illustrated in **Figure 1.**

#### **2.2 Effects of ZnO nanoparticles on animal health**

Unplanned use of ZnO NPs as nanofertilizer in agriculture leads to their entry in the food chain, and ultimately nanofertilizers enter in the body directly or indirectly, and their interaction with immune cells may have deleterious effects. The effects of ZnO NPs on the immune system are not completely understood. Some researchers postulated that increased cytosolic Zn2+ and the generation of ROS play important roles [16]. In innate immune, cells recognize ZnO NPs via toll-like receptors (TLR) which bind to corresponding antigens ZnO NPs and activate signal transduction pathway and inflammatory response. The ZnO NPs induce apoptosis and necrosis in macrophages in relation to their important role in the clearance of entered particulates and the regulation of immune responses during inflammation.

*Withania somnifera* (L.) Dual (Solanaceae) Indian ginseng or Indian winter cherry is a medicinal plant. Different parts of the plant have been used in Ayurvedic medicine formulations. Withaferin A is a steroidal lactone found in the leaves and

*Biological Role of* Withania somnifera *against Promiscuity of Zinc Oxide Nano Particles… DOI: http://dx.doi.org/10.5772/intechopen.90128*

#### **Figure 1.**

In India, more than 60% of the population survive on agriculture, but unfortunately this sector is facing various global challenges. Therefore, nanotechnology has a dominant position in transforming agriculture and food industry. Nanotechnology has a great ability to transform conventional agricultural practices and boost yield and growth of corps. Zinc oxide NPs (ZnO NPs) are used as fertilizer which

Zinc oxide NPs may be used as a source of Zn in supplements and functional foods [3]. ZnO NPs also act as antimicrobial agents against harmful bacteria. The antimicrobial activity of ZnO NPs has been partly attributed to their ability to penetrate into microbial cells and animal cells and generate reactive oxygen species (ROS) that damage cellular components thereby leading to cytotoxicity [4]. A single oral dose of ZnO NPs caused hepatic cell injury, kidney toxicity, and lung damage [5]**.** The studies in frogs showed that ZnO NPs exhibited more toxicity than a dissolved form of Zn which was attributed to their greater ability to induce oxidative damage in cells [6]. The administration of ZnO NPs increased all liver enzymes [7]. In this chapter, we attempted to explore the potential use of nanoparticles in agriculture, biological, and pharmacological significance of *Withania somnifera* against the promiscuity of zinc oxide ZnO NPs and their

**2. Use of zinc oxide nanoparticles in agriculture and animal husbandry**

Once nanomaterials (NMs) are released to the environment, they accumulate in

Unplanned use of ZnO NPs as nanofertilizer in agriculture leads to their entry in the food chain, and ultimately nanofertilizers enter in the body directly or indirectly, and their interaction with immune cells may have deleterious effects. The effects of ZnO NPs on the immune system are not completely understood. Some researchers postulated that increased cytosolic Zn2+ and the generation of ROS play important roles [16]. In innate immune, cells recognize ZnO NPs via toll-like receptors (TLR) which bind to corresponding antigens ZnO NPs and activate signal transduction pathway and inflammatory response. The ZnO NPs induce apoptosis and necrosis in macrophages in relation to their important role in the clearance of entered particulates and the regulation of immune responses during inflammation. *Withania somnifera* (L.) Dual (Solanaceae) Indian ginseng or Indian winter cherry is a medicinal plant. Different parts of the plant have been used in Ayurvedic medicine formulations. Withaferin A is a steroidal lactone found in the leaves and

ecosystems and pose threats to living organisms; therefore, it is important to understand the behavior of NMs in soil and to assess the risks for arable soil ecosystems [8]. About 260,000–309,000 metric tons (MT) of NMs were produced globally in 2010 [9], and worldwide consumption of NMs is likely to grow from 225,060 to 585,000 MT during 2014–2019 [10]. The third most commonly used metal-containing NMs are ZnO NPs with an estimated global annual production between 550 and 33,400 tons [11]. The concentration of ZnO NPs in the environment was found to be 3.1–31 μg kg<sup>1</sup> and 76–760 μg L<sup>1</sup> in soil and water, respectively [12]. ZnO NPs can strongly attach to soil particles. They exhibit low mobility at various ionic strengths [13] and show higher sorption compared to ionic zinc,

and possible uptake mechanism has been illustrated in **Figure 1.**

**2.2 Effects of ZnO nanoparticles on animal health**

**186**

support their growth and improves production [2].

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

**2.1 ZnO NPs and their potential use in agriculture**

interaction with macrophages.

*Plant uptake, transport and environmental transformation mechanism of nanofertilizer (ZnO NPs) into ecosystem and entry in food chain [14, 15]. Designed by first author using Google as tool.*

roots *of W. somnifera.* The biological properties of crude root extracts have been largely reported and only a few are related to the pure compound (withaferin A) as immunomodulatory function.

#### **3. Immunological health importance of zinc as microelement**

Zinc is crucial for normal development and function of cells mediating innate immunity, neutrophils, and natural killer cells (NKs). Phagocytosis, intracellular killing, and cytokine production are affected by Zn deficiency. Zinc is a micronutrient required by organisms and plays a vital role in maintaining immune and macrophage function. There is a progressive decline in immune response with the advance in aging due to the deficiency of Zn [17]. There is impairment of monocytes, reduced cytotoxicity in NK cells, and reduced phagocytosis in neutrophils [18]. Zinc is also a major intracellular regulator of lymphocyte apoptosis [19]. Impaired immune function in elderly subjects due to Zn deficiency has been shown to be reversed by an adequate Zn supplementation [18]. The beneficial effects of lower doses of Zn (≤ 50 mg/d) on immune function have been reported while very high doses of Zn (≥ 150 mg/d) may impair cellular immunity [20].

#### **4. Mechanism of innate immunity in animals**

The defense system is the bedrock of living systems and innate immunity is an integral part of health. It is the first line of the defense mechanism of the body from lower organism to mammals. Any alteration in innate immunity leads to disease conditions; however, adaptive immunity plays a great role in defense mechanism. Innate immunity has two arms, i.e., the afferent and efferent. The afferent arm is lipopolysaccharide (LPS) or endotoxin [21]. As to the effector arm of innate immunity, Hunter (1774) first recognized leukocytes at the site of inflammation. The cellular theory of immunity was given by Metchnikoff, 1884 [22] and must be recognized in the functional analysis of innate immune cells. Massart and Bordet

had showed that injured cells secrete chemicals that attract phagocytes in 1917. The myeloid cells in invertebrates' are precursors of the innate immunity. Macrophages are professional immune cells that engulf and destroy foreign particles. Myeloid cells include mononuclear phagocytes and polymorph nuclear phagocytes. Macrophages are mononuclear phagocytes derived from blood monocytes. Macrophages are distributed in all parts of the body of the host and also present within the parenchyma of the heart, lungs, liver, brain, and peritoneal cavity. Pathogens invading the host body through any route are killed by macrophages. Macrophages have the potential ability of supervisory of innate immunity. Reactive oxygen intermediates are produced in phagosomes of neutrophils and macrophages. Superoxide radicals (O2 �) are generated by the p91 subunit of cytochrome form (O2) [23]. Superoxide (H2O2) is produced from O2 � anions where superoxide dismutase is the catalytic enzyme.

$$\text{2O}\_2 + \text{NADPH} \left( \text{oxidase} \right) \rightarrow \text{2O}\_2\text{\bullet}^- + \text{NADP} + \text{H}^+ \tag{1}$$

$$\text{2H}^+ + \text{2O}\_2\text{\textbullet}^- = \text{H}\_2\text{O}\_2 + \text{O}\_2 \tag{2}$$

There are three main subtypes of macrophages: one is classically activated M1 macrophages which play an important role in host defense and antitumor immunity, while another one is M2 macrophages, the suppressor and regulator of wound healing. Third type is M3 which are phagocytic cells that continuously express different types of receptors that facilitate removal of necrotic tissue, aged red blood cells, and toxin molecules from the circulation. Macrophages maintain tissue homeostasis, while macrophages and DCs act as sentinel cells for the immune response [26]. Neutrophils are recruited and inflammation is promoted by mediators as indicated by macrophages [28]. Last main classes of macrophages are known as regulatory macrophages and are similar to suppressive M2 macrophages [29]. Regulatory macrophages are induced by toll-like receptor (TLR) agonist in the presence of prostaglandin, apoptotic cells, and immunoglobulin G (IgG) immune complexes and defined the release of the immunosuppressive cytokines IL-10 and TGF-1 [29]. Regulatory macrophages are poor antigen-presenting cells (APCs) and have the inclination to induce T**H**<sup>2</sup> and regulatory T cell responses that can further

*Biological Role of* Withania somnifera *against Promiscuity of Zinc Oxide Nano Particles…*

The macrophages are important cells of immune system that function in innate and adaptive immunity and can play major role in the protective and pathogenic activity. Different types of pattern recognition receptors including biosensor like C-type lectin receptors, helicase RIG like receptors, NOD-like receptors and TLRs

The invading pathogens, foreign substances (ZNFs, silica, and stone dust particle), microbes and dead and dying cells are recognized by these receptors as a danger signal [30]. Adaptors induced signaling causes myeloid differentiation and regulation of inflammatory vesicle formation activity. This cascade further triggers antimicrobial activity of M1 macrophages by stimulating the production of cytokines TNF and IL-1 [31]. Apart from innate immunity, macrophages also play

**5.2 Promiscuity and interaction of zinc oxide nanoparticles with macrophages**

The ZnO NPs have been engineered, synthesized, and commonly used in products including sunscreens, cosmetic products, food and medical materials. These play a significant role in the biomedical area for disease diagnosis and therapy [33].

suppress antitumor and chronic inflammatory responses.

*Physiological characters and significant roles of animal macrophages.*

*DOI: http://dx.doi.org/10.5772/intechopen.90128*

**5.1 Significance of macrophages in innate immunity**

are expressed in macrophages.

**189**

**Table 1.**

an important role in wound healing [32].

Hypochlorous acid (HO Cl), a reactive halide, is produced from H2O2 by the action of myeloperoxidase. These radicals not only kill microbes directly but also generate other metabolites for this purpose, and singlet oxygen can be generated by O Cl�, the former being strangely reactive with C: C double bands. Hydroxyl radicals can be produced where HO Cl react with superoxide.

$$\text{C}\text{Cl}^- + \text{H}\_2\text{O}\_2 + \text{H}^+ = \text{HO}\text{Cl} + \text{H}\_2\text{O} \tag{3}$$

$$\text{O}\_2\text{\textbullet}^- + \text{HO}\text{ Cl} = \text{O}\_2\text{\textbullet}^- + \text{OH}\text{\textbullet}^-\text{Cl}^-\tag{4}$$

Therefore, hydroxyl radical (OH•) could be produced were agent using upper oxide as substrate, reactive nitrogen species (NO•) can be produced.

$$\text{O}\_2\text{\textbullet}^- + \text{H}\_2\text{O}\_2 = \text{OH}^\bullet + \text{OH} + \text{O}\_2 \tag{5}$$

$$\text{O}\_2\text{\textbullet}^- + \text{NO}\text{\textbullet} = \text{ONOO} \tag{6}$$

Complement, lactoferrin, lysozyme, and antimicrobial peptides are the humoral component of innate immunity. Lysozyme present in the saliva and tear inhibits the cell wall synthesis in bacteria. Complement is an enzymatic proton which plays an important role in innate immunity. Antimicrobial peptides and C-reactive proteins (CRP) are also having defense ability by disrupting plasma membrane.

#### **5. Macrophages and their roles in animal health**

The macrophages are mononuclear phagocytes and are committed progenitor cells in the bone marrow [24]. There are mainly two types of phagocytic cells, namely, macrophages and dendritic cells (DCs), which have similar cell surface receptors but different functional activities which are short-lived, and their life span depends on the nature of immune response [25]. All types of macrophages are differentiated from circulating monocyte and DCs by their expression of Fc, F4/80, and CD11b receptors. Macrophages are the main inducers of the adaptive T cell responses. Macrophages are skilled in scavenging dead cells, cellular debris, phagocytosis, and remodeling after tissue injury [26]. Their names and phenotypes vary based on their anatomical location. Physiological characters and significant roles of macrophages are listed in **Table 1** [27].

#### *Biological Role of* Withania somnifera *against Promiscuity of Zinc Oxide Nano Particles… DOI: http://dx.doi.org/10.5772/intechopen.90128*


#### **Table 1.**

had showed that injured cells secrete chemicals that attract phagocytes in 1917. The myeloid cells in invertebrates' are precursors of the innate immunity. Macrophages are professional immune cells that engulf and destroy foreign particles. Myeloid cells include mononuclear phagocytes and polymorph nuclear phagocytes. Macrophages are mononuclear phagocytes derived from blood monocytes. Macrophages are distributed in all parts of the body of the host and also present within the parenchyma of the heart, lungs, liver, brain, and peritoneal cavity. Pathogens invading the host body through any route are killed by macrophages. Macrophages have the potential ability of supervisory of innate immunity. Reactive oxygen intermediates are produced in phagosomes of neutrophils and macrophages.

�) are generated by the p91 subunit of cytochrome

Cl� þ H2O2 þ H<sup>þ</sup> ¼ HO Cl þ H2O (3)

� <sup>þ</sup> H2O2 <sup>¼</sup> OH• <sup>þ</sup> OH <sup>þ</sup> O2 (5)

� þ NO• ¼ ONOO (6)

� anions where superoxide

� þ NADP þ H<sup>þ</sup> (1)

� ¼ H2O2 þ O2 (2)

� þ OH• þ Cl� (4)

Superoxide radicals (O2

dismutase is the catalytic enzyme.

form (O2) [23]. Superoxide (H2O2) is produced from O2

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

2O2 þ NADPH oxidase ð Þ! 2O2•

2H<sup>þ</sup> þ 2O2•

� þ HO Cl ¼ O2•

oxide as substrate, reactive nitrogen species (NO•) can be produced.

O2•

(CRP) are also having defense ability by disrupting plasma membrane.

radicals can be produced where HO Cl react with superoxide.

O2•

O2•

**5. Macrophages and their roles in animal health**

macrophages are listed in **Table 1** [27].

**188**

Hypochlorous acid (HO Cl), a reactive halide, is produced from H2O2 by the action of myeloperoxidase. These radicals not only kill microbes directly but also generate other metabolites for this purpose, and singlet oxygen can be generated by O Cl�, the former being strangely reactive with C: C double bands. Hydroxyl

Therefore, hydroxyl radical (OH•) could be produced were agent using upper

Complement, lactoferrin, lysozyme, and antimicrobial peptides are the humoral component of innate immunity. Lysozyme present in the saliva and tear inhibits the cell wall synthesis in bacteria. Complement is an enzymatic proton which plays an important role in innate immunity. Antimicrobial peptides and C-reactive proteins

The macrophages are mononuclear phagocytes and are committed progenitor cells in the bone marrow [24]. There are mainly two types of phagocytic cells, namely, macrophages and dendritic cells (DCs), which have similar cell surface receptors but different functional activities which are short-lived, and their life span depends on the nature of immune response [25]. All types of macrophages are differentiated from circulating monocyte and DCs by their expression of Fc, F4/80, and CD11b receptors. Macrophages are the main inducers of the adaptive T cell responses. Macrophages are skilled in scavenging dead cells, cellular debris, phagocytosis, and remodeling after tissue injury [26]. Their names and phenotypes vary based on their anatomical location. Physiological characters and significant roles of

*Physiological characters and significant roles of animal macrophages.*

There are three main subtypes of macrophages: one is classically activated M1 macrophages which play an important role in host defense and antitumor immunity, while another one is M2 macrophages, the suppressor and regulator of wound healing. Third type is M3 which are phagocytic cells that continuously express different types of receptors that facilitate removal of necrotic tissue, aged red blood cells, and toxin molecules from the circulation. Macrophages maintain tissue homeostasis, while macrophages and DCs act as sentinel cells for the immune response [26]. Neutrophils are recruited and inflammation is promoted by mediators as indicated by macrophages [28]. Last main classes of macrophages are known as regulatory macrophages and are similar to suppressive M2 macrophages [29]. Regulatory macrophages are induced by toll-like receptor (TLR) agonist in the presence of prostaglandin, apoptotic cells, and immunoglobulin G (IgG) immune complexes and defined the release of the immunosuppressive cytokines IL-10 and TGF-1 [29]. Regulatory macrophages are poor antigen-presenting cells (APCs) and have the inclination to induce T**H**<sup>2</sup> and regulatory T cell responses that can further suppress antitumor and chronic inflammatory responses.

#### **5.1 Significance of macrophages in innate immunity**

The macrophages are important cells of immune system that function in innate and adaptive immunity and can play major role in the protective and pathogenic activity. Different types of pattern recognition receptors including biosensor like C-type lectin receptors, helicase RIG like receptors, NOD-like receptors and TLRs are expressed in macrophages.

The invading pathogens, foreign substances (ZNFs, silica, and stone dust particle), microbes and dead and dying cells are recognized by these receptors as a danger signal [30]. Adaptors induced signaling causes myeloid differentiation and regulation of inflammatory vesicle formation activity. This cascade further triggers antimicrobial activity of M1 macrophages by stimulating the production of cytokines TNF and IL-1 [31]. Apart from innate immunity, macrophages also play an important role in wound healing [32].

#### **5.2 Promiscuity and interaction of zinc oxide nanoparticles with macrophages**

The ZnO NPs have been engineered, synthesized, and commonly used in products including sunscreens, cosmetic products, food and medical materials. These play a significant role in the biomedical area for disease diagnosis and therapy [33]. ZnO NPs are also widely used in the food industry and as nanofertilizer in agriculture. The wide application of ZnO NPs increased the chance of human and animal's exposure [34]. They can be absorbed into the body and redistributed into various organs after environmental exposure [34]. Hence, the safety assessment of nanoparticle is mandatory. The ZnO NPs can influence the immune system and affect the process of diseases and the emergency responses of immunity governed by macrophages [35]. ZnO NPs are foreign particles, and the macrophages play an important role in the recognition, processing, and removal of ZnO NPs [36]. The ZnO NPs interact with soluble proteins to form a halo corona that affects NP activity. The composition of protein coronas varies according to the size of NPs [37]. Protein coronas of NP surface have two layers including hard corona nearer to the NP surface and soft corona composed of reversibly adsorbed materials and largesize NPs phagocytized by macrophages through nanoparticle protein complexes corona. ZnO NPs deflate phagocytosis of macrophages and show cytotoxic and bactericidal activities by enhancing oxidative stress which may disrupt bacterial outer cell membrane and causes cell apoptosis. The ZnO NPs also inhibit nitric oxide (NO) production through the NF-Kβ signaling. NO reduces ZnONPs toxicity in rice seedlings by regulating oxidative damage and antioxidant defense systems [38].

**7.1 Effect of NPs on TLR signaling of innate immunity health**

**8. Indian ayurvedic medicinal plant:** *Withania somnifera*

**8.1 Pharmacological and medicinal activities of withaferin a**

Withaferin A is the key withanolide prototype which has been shown to have anti-inflammatory [54], antitumor [55], anti-angiogenesis [56], radio-sensitizing activity, and chemopreventive [57] and immunosuppressive [58] properties. Withaferin A is highly reactive because of the ketone containing unsaturated

proportional to the size of NPs.

and diabetes).

phagocytic activities [14].

**191**

**7.2 Effects of NPs on phagocytic cells**

*DOI: http://dx.doi.org/10.5772/intechopen.90128*

The TLR is a type I transmembrane receptors which contain an N-terminal domain (leucine-rich repeat) and a C-terminal toward cytoplasm. When macrophage receptors recognized PAMP, TLRs recruit a TIR domain such as MyD88 and TRIF and initiate signaling events called downstream signaling by the secretion of different inflammatory molecules (chemokines, inflammatory cytokines, and IFNs I) [47]. The TLR signaling is responsible for the transcription of inflammatory and immune responses genes [48]. ZnO nanoparticles induced MyD88-dependent proinflammatory cytokines via a TLR signal pathway [49]. The TLRs may have important roles in NP uptake and for their cellular response which is directly

*Biological Role of* Withania somnifera *against Promiscuity of Zinc Oxide Nano Particles…*

Macrophages and dendritic cells have phagocytic activity; hence they readily uptake nanoparticles. Therefore, magnetic nanoparticles and nanoparticles-based PET agents were usually used for the visualization of macrophages in human diseases (cancer, atherosclerosis, myocardial infarction, aortic aneurysm,

*Withania somnifera (*Ashwagandha), an Indian Ayurvedic medicinal plant, is a green shrub and belongs to the Solanaceae family. For over 3000 years, Indians cultivated and applied its whole plant extract or separate constituents in Ayurvedic and indigenous medicine [50]. It was shown to have anti-inflammatory, antitumor, anti-stress, antioxidant, immuno-modulatory, hematopoietic, and rejuvenating properties thus benefiting the endocrine, cardiopulmonary, and central nervous systems [51]. It inhibits immunologically induced inflammation and a variety of pharmacological effects in *Babl/c mice* [52]. Various mechanisms have been proposed to explain the antitumor activity of Ashwagandha including potent anti-inflammatory, anti-angiogenic, anti-metastatic, pro-apoptotic, and radiosensitizing properties [53]. An extract of *W. somnifera* showed immunological activity in *Balb/c mice*. Treatment with different doses of *W. somnifera* root extract (20 mg/dose/animal; i.p.) enhanced the total WBC counts. *W. somnifera* extract along with sheep red blood cell antigen (SRBC) increased antibody titer in circulation and plaque-forming cell numbers (PFC) in the spleen and reduced the delayedtype hypersensitivity reaction in mice model. Withania extract improved in phagocytic activity of macrophages when compared to untreated mice. The immunomodulatory effects of *W. somnifera* against ZnO NP-mediated toxicity in *Balb*/*c* mice study showed a dose-dependent reduction in phagocytosis, an increase in the levels of NO production along with upregulation of *TLR6, and arginase* gene. However, the adverse effect of ZnO NP on macrophages was reduced by *W. somnifera* extract and withaferin A with decreased *TLR6* overexpression and improved

#### **5.3 Mechanism of ZnO NPs induced toxic effects on cells of immune system**

The effects of ZnO NPs on the immune system depend on their physicochemical properties [39]. A nanotoxicological effect of NPs depends on the size, size distribution, surface area, electrostatic charge, and solubility [40]. The ZnO NPs are more water-soluble which result in more dissolution of toxic ions and ROS production [41]. ZnO NPs undergo endocytosis into the macrophages cells, dissolve into bioavailable zinc ion, and increase oxidative stress through ROS which cause immunotoxicity. The important factor for the immunotoxicity of ZnO NPs is ROS. Intracellular ROS production has at least some contribution in cell death induced by ZnO NPs [42, 43].

#### **6. Effect on epithelial barriers of innate immunity**

The important components of the innate immune system are epithelial barriers, phagocytic cells (dendritic cells, polymorphonuclear leukocytes, monocytes/macrophages), phagocytic leukocytes, basophils, mast cells, eosinophils, natural killer cells, circulating plasma proteins. TLR is the main signaling in the innate immunity which induce expression of inflammatory gene. Metal oxide nanoparticles trigger the TLR signaling pathway**.**

#### **7. The innate immune system and role of TLRS signaling pathway**

The innate immune system relies on the recognition of pathogen-associated microbial particles (PAMPs) through a limited number of germ line-encoded pattern recognition receptors belonging to the family of TLRs [44]. The activation of TLR signaling induces cytokines production and phagocytosis of macrophages along with catalytic activity of NK cells. More importantly, TLR signaling activation can also enhance antigen presentation via upregulating the expression of major histocompatibility complex (MHC) and co-stimulatory molecules (CD80 and CD86) on dendritic cells leading to adaptive immunity activations. Nanoparticles enhanced TLR signaling pathways which act as adjuvants [45]. The TLR antagonists or inhibitors that reduced the inflammatory response would have beneficial therapeutic effects in autoimmune diseases and sepsis [46].

*Biological Role of* Withania somnifera *against Promiscuity of Zinc Oxide Nano Particles… DOI: http://dx.doi.org/10.5772/intechopen.90128*

#### **7.1 Effect of NPs on TLR signaling of innate immunity health**

The TLR is a type I transmembrane receptors which contain an N-terminal domain (leucine-rich repeat) and a C-terminal toward cytoplasm. When macrophage receptors recognized PAMP, TLRs recruit a TIR domain such as MyD88 and TRIF and initiate signaling events called downstream signaling by the secretion of different inflammatory molecules (chemokines, inflammatory cytokines, and IFNs I) [47]. The TLR signaling is responsible for the transcription of inflammatory and immune responses genes [48]. ZnO nanoparticles induced MyD88-dependent proinflammatory cytokines via a TLR signal pathway [49]. The TLRs may have important roles in NP uptake and for their cellular response which is directly proportional to the size of NPs.

#### **7.2 Effects of NPs on phagocytic cells**

ZnO NPs are also widely used in the food industry and as nanofertilizer in agriculture. The wide application of ZnO NPs increased the chance of human and animal's exposure [34]. They can be absorbed into the body and redistributed into various organs after environmental exposure [34]. Hence, the safety assessment of nanoparticle is mandatory. The ZnO NPs can influence the immune system and affect the process of diseases and the emergency responses of immunity governed by macrophages [35]. ZnO NPs are foreign particles, and the macrophages play an important role in the recognition, processing, and removal of ZnO NPs [36]. The ZnO NPs interact with soluble proteins to form a halo corona that affects NP activity. The composition of protein coronas varies according to the size of NPs [37]. Protein coronas of NP surface have two layers including hard corona nearer to the NP surface and soft corona composed of reversibly adsorbed materials and largesize NPs phagocytized by macrophages through nanoparticle protein complexes corona. ZnO NPs deflate phagocytosis of macrophages and show cytotoxic and bactericidal activities by enhancing oxidative stress which may disrupt bacterial outer cell membrane and causes cell apoptosis. The ZnO NPs also inhibit nitric oxide (NO) production through the NF-Kβ signaling. NO reduces ZnONPs toxicity in rice seedlings by regulating oxidative damage and antioxidant defense systems [38].

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

**5.3 Mechanism of ZnO NPs induced toxic effects on cells of immune system**

**6. Effect on epithelial barriers of innate immunity**

effects in autoimmune diseases and sepsis [46].

the TLR signaling pathway**.**

**190**

The effects of ZnO NPs on the immune system depend on their physicochemical properties [39]. A nanotoxicological effect of NPs depends on the size, size distribution, surface area, electrostatic charge, and solubility [40]. The ZnO NPs are more water-soluble which result in more dissolution of toxic ions and ROS production [41]. ZnO NPs undergo endocytosis into the macrophages cells, dissolve into bioavailable zinc ion, and increase oxidative stress through ROS which cause immunotoxicity. The important factor for the immunotoxicity of ZnO NPs is ROS. Intracellular ROS production has at least some contribution in cell death induced by ZnO NPs [42, 43].

The important components of the innate immune system are epithelial barriers, phagocytic cells (dendritic cells, polymorphonuclear leukocytes, monocytes/macrophages), phagocytic leukocytes, basophils, mast cells, eosinophils, natural killer cells, circulating plasma proteins. TLR is the main signaling in the innate immunity which induce expression of inflammatory gene. Metal oxide nanoparticles trigger

**7. The innate immune system and role of TLRS signaling pathway**

The innate immune system relies on the recognition of pathogen-associated microbial particles (PAMPs) through a limited number of germ line-encoded pattern recognition receptors belonging to the family of TLRs [44]. The activation of TLR signaling induces cytokines production and phagocytosis of macrophages along with catalytic activity of NK cells. More importantly, TLR signaling activation can also enhance antigen presentation via upregulating the expression of major histocompatibility complex (MHC) and co-stimulatory molecules (CD80 and CD86) on dendritic cells leading to adaptive immunity activations. Nanoparticles enhanced TLR signaling pathways which act as adjuvants [45]. The TLR antagonists or inhibitors that reduced the inflammatory response would have beneficial therapeutic

Macrophages and dendritic cells have phagocytic activity; hence they readily uptake nanoparticles. Therefore, magnetic nanoparticles and nanoparticles-based PET agents were usually used for the visualization of macrophages in human diseases (cancer, atherosclerosis, myocardial infarction, aortic aneurysm, and diabetes).

#### **8. Indian ayurvedic medicinal plant:** *Withania somnifera*

*Withania somnifera (*Ashwagandha), an Indian Ayurvedic medicinal plant, is a green shrub and belongs to the Solanaceae family. For over 3000 years, Indians cultivated and applied its whole plant extract or separate constituents in Ayurvedic and indigenous medicine [50]. It was shown to have anti-inflammatory, antitumor, anti-stress, antioxidant, immuno-modulatory, hematopoietic, and rejuvenating properties thus benefiting the endocrine, cardiopulmonary, and central nervous systems [51]. It inhibits immunologically induced inflammation and a variety of pharmacological effects in *Babl/c mice* [52]. Various mechanisms have been proposed to explain the antitumor activity of Ashwagandha including potent anti-inflammatory, anti-angiogenic, anti-metastatic, pro-apoptotic, and radiosensitizing properties [53]. An extract of *W. somnifera* showed immunological activity in *Balb/c mice*. Treatment with different doses of *W. somnifera* root extract (20 mg/dose/animal; i.p.) enhanced the total WBC counts. *W. somnifera* extract along with sheep red blood cell antigen (SRBC) increased antibody titer in circulation and plaque-forming cell numbers (PFC) in the spleen and reduced the delayedtype hypersensitivity reaction in mice model. Withania extract improved in phagocytic activity of macrophages when compared to untreated mice. The immunomodulatory effects of *W. somnifera* against ZnO NP-mediated toxicity in *Balb*/*c* mice study showed a dose-dependent reduction in phagocytosis, an increase in the levels of NO production along with upregulation of *TLR6, and arginase* gene. However, the adverse effect of ZnO NP on macrophages was reduced by *W. somnifera* extract and withaferin A with decreased *TLR6* overexpression and improved phagocytic activities [14].

#### **8.1 Pharmacological and medicinal activities of withaferin a**

Withaferin A is the key withanolide prototype which has been shown to have anti-inflammatory [54], antitumor [55], anti-angiogenesis [56], radio-sensitizing activity, and chemopreventive [57] and immunosuppressive [58] properties. Withaferin A is highly reactive because of the ketone containing unsaturated

A ring, the epoxide in B ring, and unsaturated lactone ring. In another study, withaferin A inhibited NF-κB at a very low concentration by targeting the ubiquitin-mediated proteasome pathway in endothelial cells. In vitro experiments demonstrated that withaferin A interfered with TNF-induced NF-κB activation at the level or upstream of IKKβ [57]. Withaferin A inhibited the expression of iNOS in the lipopolysaccharide (LPS)-stimulated murine macrophage cell line [59]. Withaferin-A inhibited, LPS-induced COX-2 expression and PGE2 production in BV2 murine microglial cells [60]. Both pre and post-treatment of astrocytes with Withaferin-A attenuated LPS-induced production of tumor necrosis factor-α and the expression of COX-2 with expression of induced nitric oxide synthase by blocking the NF-κB activity [61]. Treatment with withaferin A increased SOD, catalase, and glutathione peroxidase activity in rat brain frontal cortex and striatal concentrations [62].

#### **9. Conclusions**

Nanoparticles are gaining importance recently due to their exciting applications in different fields like agriculture, human health, and livestock sector. Increased application of ZnO NPs is clearly indicating the adverse effect on immunity. It is, therefore, necessary to explore safety level of ZnO NPs and their role in humans and animals. The future work must be placed in the context of current risk assessments which must be associated with ZnO NPs toxicity and safety level and their uses. Further research work is emphasized for elucidating the nature of ZnO nanoparticles and their fate in living and non living matrices which can serve as to safeguard the ecosystem functioning. The ecosystem strengthening should be in term of agriculture and livestock particularly concerns production, food resource, immune and health status of animals. The role of *W. somnifera* as an antidote to immune complication induced by ZnO NPs exposure needs further research.

#### **Conflict of interest**

The authors declare no competing interest.

#### **Notes/thanks/other declarations**

We would like to acknowledge the support from the Director, ICAR-National Dairy Research Institute, Karnal-132,001, Haryana, India.

**Author details**

Jitendra Kumar<sup>1</sup>

Haryana, India

Haryana, India

**193**

\*, Chander Datt<sup>2</sup>

\*Address all correspondence to: jitoperon@gmail.com

provided the original work is properly cited.

, Surya Kant Verma<sup>1</sup> and Kavita Rani<sup>1</sup>

1 Division of Animal Biochemistry, ICAR-National Dairy Research Institute, Karnal,

*Biological Role of* Withania somnifera *against Promiscuity of Zinc Oxide Nano Particles…*

*DOI: http://dx.doi.org/10.5772/intechopen.90128*

2 Division of Animal Nutrition, ICAR-National Dairy Research Institute, Karnal,

© 2020 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,

#### **Author contributions**

The authors' responsibilities were as follows: Dr. Jitendra Kumar, Dr. Chander Datt, Suryakant Verma and Kavita Rani conceived and designed the chapter.

*Biological Role of* Withania somnifera *against Promiscuity of Zinc Oxide Nano Particles… DOI: http://dx.doi.org/10.5772/intechopen.90128*

#### **Author details**

A ring, the epoxide in B ring, and unsaturated lactone ring. In another study, withaferin A inhibited NF-κB at a very low concentration by targeting the

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

the lipopolysaccharide (LPS)-stimulated murine macrophage cell line [59]. Withaferin-A inhibited, LPS-induced COX-2 expression and PGE2 production in BV2 murine microglial cells [60]. Both pre and post-treatment of astrocytes with Withaferin-A attenuated LPS-induced production of tumor necrosis factor-α and the expression of COX-2 with expression of induced nitric oxide synthase by blocking the NF-κB activity [61]. Treatment with withaferin A increased SOD, catalase, and glutathione peroxidase activity in rat brain frontal cortex and striatal

concentrations [62].

**9. Conclusions**

**Conflict of interest**

**Author contributions**

**192**

The authors declare no competing interest.

Dairy Research Institute, Karnal-132,001, Haryana, India.

**Notes/thanks/other declarations**

ubiquitin-mediated proteasome pathway in endothelial cells. In vitro experiments demonstrated that withaferin A interfered with TNF-induced NF-κB activation at the level or upstream of IKKβ [57]. Withaferin A inhibited the expression of iNOS in

Nanoparticles are gaining importance recently due to their exciting applications in different fields like agriculture, human health, and livestock sector. Increased application of ZnO NPs is clearly indicating the adverse effect on immunity. It is, therefore, necessary to explore safety level of ZnO NPs and their role in humans and animals. The future work must be placed in the context of current risk assessments which must be associated with ZnO NPs toxicity and safety level and their uses.

nanoparticles and their fate in living and non living matrices which can serve as to safeguard the ecosystem functioning. The ecosystem strengthening should be in term of agriculture and livestock particularly concerns production, food resource, immune and health status of animals. The role of *W. somnifera* as an antidote to immune complication induced by ZnO NPs exposure needs further research.

We would like to acknowledge the support from the Director, ICAR-National

The authors' responsibilities were as follows: Dr. Jitendra Kumar, Dr. Chander

Datt, Suryakant Verma and Kavita Rani conceived and designed the chapter.

Further research work is emphasized for elucidating the nature of ZnO

Jitendra Kumar<sup>1</sup> \*, Chander Datt<sup>2</sup> , Surya Kant Verma<sup>1</sup> and Kavita Rani<sup>1</sup>

1 Division of Animal Biochemistry, ICAR-National Dairy Research Institute, Karnal, Haryana, India

2 Division of Animal Nutrition, ICAR-National Dairy Research Institute, Karnal, Haryana, India

\*Address all correspondence to: jitoperon@gmail.com

© 2020 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.

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[57] Manoharan S, Panjamurthy K, Balakrishnan S, Vasudevan K, Vellaichamy L. Circadian timedependent chemopreventive potential of withaferin-a in 7, 12-dimethyl-benz [a] anthracene-induced oral carcinogenesis. Pharmacological Reports. 2009b;**61**(4):719-726

[58] Shohat B, Kirson I, Lavie D. Immunosuppressive activity of two plant steroidal lactones withaferin a and withanolide E. Biomedicine. 1978;**28**(1): 18-24

[59] Oh JH, Lee TJ, Park JW, Kwon TK. Withaferin a inhibits iNOS expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-κB in RAW 264.7 cells. European Journal of Pharmacology. 2008;**599**(1):11-17

[60] Choi MJ, Park EJ, Min KJ, Park JW, Kwon TK. Endoplasmic reticulum stress mediates withaferin A-induced apoptosis in human renal carcinoma cells. Toxicology In Vitro. 2011;**25**(3): 692-698

[61] Martorana F, Guidotti G, Brambilla L, Rossi D. Withaferin a inhibits nuclear factor-κB-dependent pro-inflammatory and stress response pathways in the astrocytes. Neural Plasticity. 2015;**38**:1964

[62] Bhattacharya SK, Satyan KS, Ghosal S. Antioxidant activity of glycowithanolides from Withania somnifera. Indian Journal of Experimental Biology. 1997;**35**:236-239

**199**

**Chapter 12**

**Abstract**

species for monitoring PAHs.

European eel

decades [2].

**1. Introduction**

Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants generated primarily during the incomplete combustion of organic materials (e.g., coal, oil, petrol, and wood). Many PAHs have toxic, mutagenic, and/or carcinogenic functions. PAHs are highly lipid soluble which lead to a fast absorption by the gastrointestinal tract of marine mammals. They are immediately distributed in a vast variety of tissues with a notable tendency for localization in body fat. Metabolism of PAHs is obtained via the cytochrome P450-mediated mixed function oxidase system with oxidation or hydroxylation as the first step. PAHs are environmental contaminants that pose significant risk to health of fish. The effect of PAHs on fish is a topic of rising attention in a lot of countries. Different studies using the bile metabolites separated by high-performance liquid chromatography with fluorescence detection were presented. The aim is to compare the levels of PAH metabolites in fish from different areas and fish species. The major metabolite present in all fish was 1-hydroxypyrene. The data confirm the importance of 1-hydroxypyrene as the key PAH metabolite in fish bile and suggest that the European eel is an ideal

**Keywords:** PAHs, organic pollutants, metabolism, fish, 1-hydroxypyrene,

Aquatic ecosystems are susceptible to receiving and accumulating contaminants [1]. In particular, polycyclic aromatic hydrocarbons (PAHs) have been identified as general causes of the deterioration of aquatic ecosystems in recent

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and persistent environmental contaminants found in sediments and associated waters of urbanized estuaries and coastal areas [3–5]. They are a class of compounds found in crude oil and are everywhere in the aquatic ecosystem [6–12]. PAHs are the most toxic pollutants of crude oil and are remembered by the United States Environmental Protection Agency (EPA) as priority toxic components because of its persistence in the environment and are toxic to fishes [13, 14]; thus, PAHs are of special interest following oil spills and in environmental control. They come from natural and anthropogenic sources. The latter can be associated to pyrolysis and incomplete combustion of

(PAHs) and Their Influence to

Some Aquatic Species

*Ayoub Baali and Ahmed Yahyaoui*

#### **Chapter 12**

## Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species

*Ayoub Baali and Ahmed Yahyaoui*

#### **Abstract**

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants generated primarily during the incomplete combustion of organic materials (e.g., coal, oil, petrol, and wood). Many PAHs have toxic, mutagenic, and/or carcinogenic functions. PAHs are highly lipid soluble which lead to a fast absorption by the gastrointestinal tract of marine mammals. They are immediately distributed in a vast variety of tissues with a notable tendency for localization in body fat. Metabolism of PAHs is obtained via the cytochrome P450-mediated mixed function oxidase system with oxidation or hydroxylation as the first step. PAHs are environmental contaminants that pose significant risk to health of fish. The effect of PAHs on fish is a topic of rising attention in a lot of countries. Different studies using the bile metabolites separated by high-performance liquid chromatography with fluorescence detection were presented. The aim is to compare the levels of PAH metabolites in fish from different areas and fish species. The major metabolite present in all fish was 1-hydroxypyrene. The data confirm the importance of 1-hydroxypyrene as the key PAH metabolite in fish bile and suggest that the European eel is an ideal species for monitoring PAHs.

**Keywords:** PAHs, organic pollutants, metabolism, fish, 1-hydroxypyrene, European eel

#### **1. Introduction**

Aquatic ecosystems are susceptible to receiving and accumulating contaminants [1]. In particular, polycyclic aromatic hydrocarbons (PAHs) have been identified as general causes of the deterioration of aquatic ecosystems in recent decades [2].

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and persistent environmental contaminants found in sediments and associated waters of urbanized estuaries and coastal areas [3–5]. They are a class of compounds found in crude oil and are everywhere in the aquatic ecosystem [6–12]. PAHs are the most toxic pollutants of crude oil and are remembered by the United States Environmental Protection Agency (EPA) as priority toxic components because of its persistence in the environment and are toxic to fishes [13, 14]; thus, PAHs are of special interest following oil spills and in environmental control. They come from natural and anthropogenic sources. The latter can be associated to pyrolysis and incomplete combustion of

organic element [15]. Wastewater, atmospheric deposition, and petroleum spillage are some of the most important PAH sources. PAHs and their intermediate degradation products have the potential to generate toxic or mutagenic effects in fish [16–18] and humans [19]. PAH metabolites in the bile fluid are generally accepted as measures for PAH exposure in fish because of the rapid metabolism of PAH in most vertebrates [3]. Therefore, PAH metabolites in fish are recommended as monitoring parameters in European seas [20, 21].

In this chapter, we briefly review the origin, toxicity, and transformation of PAHs in the aquatic environment, highlighting their efficient metabolism in fish. We also review the presence of PAHs on fish bile and the works reported on that.

#### **2. Organic contamination by polycyclic aromatic hydrocarbons (PAH)**

#### **2.1 PAH origin**

PAHs are mainly formed during the incomplete combustion of organic matter and during the slow maturation of organic matter accumulated in deep sedimentary environments. These two origins present distinct formation mechanisms that are realized with different kinetics and induce variable molecular distributions (related to stability) [22].

#### *2.1.1 Pyrolytic origin*

Pyrolytic PAHs are generated by processes of incomplete combustion of organic matter at high temperatures. The mechanisms involved in their formation involve the production of free radicals by pyrolysis at high temperature (≥500°C) of the fossil material (oils, fuel oil, organic matter, etc.) under oxygen-deficient conditions. PAHs of pyrolytic origin come from the combustion of automotive fuel, domestic combustion (coal, wood, etc.), industrial production (steelworks, aluminum smelters, etc.), and energy production (power stations operating on oil or coal) or incinerators [23].

#### *2.1.2 Petrogenic origin*

The process of diagenesis can give rise to petroleum and other fossil fuels containing the so-called petrogenic PAHs. These PAHs are formed at low temperatures (150°C) over long periods of time. They result from exposure of organic matter to adequate conditions of temperature and pressure. The proportion of PAHs in oils varies according to their origin and level of refinement. In general, petrogenic PAH mixtures are marked by a predominance of low molecular weight PAHs, three cycles or less, and substituted PAHs [22].

PAHs represent between 20 and 40% by weight of crude oils which are mainly composed of saturated hydrocarbons. However, they are less than a few percent of the composition of refined gasoline (<0.5% by mass) or kerosene [24].

#### *2.1.3 Biological origin*

PAHs can also be formed by microorganisms from biogenic precursors such as diterpenes and triterpenes, steroids, pigments, or quinones in sediments or recent soils [25, 26]; these precursors can come from terrestrial or aquatic biological tissues (plants, animals, bacteria, macro- and microalgae) [27, 28].

**201**

*Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species*

The toxicity of several PAHs is a phenomenon that is well-known. Research has been conducted by several environmental groups such as the US Environmental Protection Agency-Toxic Substances Control Act (US EPA-TSCA) and the International Agency for Research on Cancer (IARC). The toxicity of PAHs to aquatic species is affected by metabolism and photooxidation. They are generally harmful in the presence of ultraviolet light. PAHs have moderate to high acute toxicity to aquatic organisms and birds. Mammals can absorb PAHs by various routes, e.g., dermal contact, inhalation, and ingestion [14, 29, 30]. The concentrations of PAHs found in fish are expected to be much higher than in the environment from which they were taken because of their bioaccumulation. Withal, metabolism of

Teleost fish have an immense capacity to metabolize PAHs because of the enzymes cytochrome P450 in their tissues that oxidatively biotransform PAHs to

The half-life times of PAHs in various biological tissues (bivalves, crustaceans, and fish) are of the order of a few days to 10 days and are about five times higher for

The environmental matrices are moreover complex, containing numerous endogenous or exogenous, mineral or organic molecules between which interactions can take place. Synergistic toxic effects have been observed in particular

Indeed, the carcinogenic nature of some of these molecules alone or in mixtures is proven. Twelve of these are classified by the International Agency for Research on Cancer (IARC) as probably carcinogenic to humans [35]. After contamination by these substances, they are biotransformed in the liver into different (poly)hydroxy-PAHs. The risks of PAHs to fish and other aquatic organisms in natural systems are highly uncertain due to the occurrence of complex, incompletely characterized mixtures of these chemicals, large spatial heterogeneity in exposure concentrations, incomplete understanding of the importance of UV-activated PAH toxicity, the biological and physical controls on fish exposure to UV light, and the bioaccumulation of PAHs. These uncertainties are especially great for early-life-stage fish, which might be particularly susceptible to UV-activated because of their small size, lack of protective pigmentation and gill coverings, and ready accumulation of PAHs.

Sediments contaminated with PAHs pose a real threat to all living organisms,

PAHs with a low molecular weight can be found in all environmental matrices, since higher molecular weight compounds are more deeply associated (physically and chemically) with sediments/soils and particles than the other abiotic sample types. PAHs in air can be modified via chemical oxidation and photochemical processes [8], whereas in sediments/soils and the uppermost portion of the water column, degradation of PAHs, particularly lower molecular weight PAHs, occurs via photooxidation [6, 36]. In addition to parent PAHs, oxygenated PAH metabolites formed during these degradation processes can persist associating with sediments up to 6 months after initial addition to the water column and thus can endure in the environment for extended periods of time [37]. In water samples and sediment,

*DOI: http://dx.doi.org/10.5772/intechopen.86213*

PAHs is sufficient to prevent biomagnifications [31, 32].

**3. Transformations of PAHs in marine ecosystem**

**3.1 Physical and chemical degradation**

even those that feed on the benthic prey.

hydroxylated metabolites [33].

heavy PAHs relative to lower PAHs.

between metals and PAH quinones [34].

**2.2 PAH toxicity**

*Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species DOI: http://dx.doi.org/10.5772/intechopen.86213*

#### **2.2 PAH toxicity**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

parameters in European seas [20, 21].

on that.

**2.1 PAH origin**

to stability) [22].

*2.1.1 Pyrolytic origin*

coal) or incinerators [23].

or less, and substituted PAHs [22].

*2.1.2 Petrogenic origin*

*2.1.3 Biological origin*

organic element [15]. Wastewater, atmospheric deposition, and petroleum spillage are some of the most important PAH sources. PAHs and their intermediate degradation products have the potential to generate toxic or mutagenic effects in fish [16–18] and humans [19]. PAH metabolites in the bile fluid are generally accepted as measures for PAH exposure in fish because of the rapid metabolism of PAH in most vertebrates [3]. Therefore, PAH metabolites in fish are recommended as monitoring

In this chapter, we briefly review the origin, toxicity, and transformation of PAHs in the aquatic environment, highlighting their efficient metabolism in fish. We also review the presence of PAHs on fish bile and the works reported

**2. Organic contamination by polycyclic aromatic hydrocarbons (PAH)**

PAHs are mainly formed during the incomplete combustion of organic matter and during the slow maturation of organic matter accumulated in deep sedimentary environments. These two origins present distinct formation mechanisms that are realized with different kinetics and induce variable molecular distributions (related

Pyrolytic PAHs are generated by processes of incomplete combustion of organic matter at high temperatures. The mechanisms involved in their formation involve the production of free radicals by pyrolysis at high temperature (≥500°C) of the fossil material (oils, fuel oil, organic matter, etc.) under oxygen-deficient conditions. PAHs of pyrolytic origin come from the combustion of automotive fuel, domestic combustion (coal, wood, etc.), industrial production (steelworks, aluminum smelters, etc.), and energy production (power stations operating on oil or

The process of diagenesis can give rise to petroleum and other fossil fuels containing the so-called petrogenic PAHs. These PAHs are formed at low temperatures (150°C) over long periods of time. They result from exposure of organic matter to adequate conditions of temperature and pressure. The proportion of PAHs in oils varies according to their origin and level of refinement. In general, petrogenic PAH mixtures are marked by a predominance of low molecular weight PAHs, three cycles

PAHs represent between 20 and 40% by weight of crude oils which are mainly composed of saturated hydrocarbons. However, they are less than a few percent of

PAHs can also be formed by microorganisms from biogenic precursors such as diterpenes and triterpenes, steroids, pigments, or quinones in sediments or recent soils [25, 26]; these precursors can come from terrestrial or aquatic biological tissues

the composition of refined gasoline (<0.5% by mass) or kerosene [24].

(plants, animals, bacteria, macro- and microalgae) [27, 28].

**200**

The toxicity of several PAHs is a phenomenon that is well-known. Research has been conducted by several environmental groups such as the US Environmental Protection Agency-Toxic Substances Control Act (US EPA-TSCA) and the International Agency for Research on Cancer (IARC). The toxicity of PAHs to aquatic species is affected by metabolism and photooxidation. They are generally harmful in the presence of ultraviolet light. PAHs have moderate to high acute toxicity to aquatic organisms and birds. Mammals can absorb PAHs by various routes, e.g., dermal contact, inhalation, and ingestion [14, 29, 30]. The concentrations of PAHs found in fish are expected to be much higher than in the environment from which they were taken because of their bioaccumulation. Withal, metabolism of PAHs is sufficient to prevent biomagnifications [31, 32].

Teleost fish have an immense capacity to metabolize PAHs because of the enzymes cytochrome P450 in their tissues that oxidatively biotransform PAHs to hydroxylated metabolites [33].

The half-life times of PAHs in various biological tissues (bivalves, crustaceans, and fish) are of the order of a few days to 10 days and are about five times higher for heavy PAHs relative to lower PAHs.

The environmental matrices are moreover complex, containing numerous endogenous or exogenous, mineral or organic molecules between which interactions can take place. Synergistic toxic effects have been observed in particular between metals and PAH quinones [34].

Indeed, the carcinogenic nature of some of these molecules alone or in mixtures is proven. Twelve of these are classified by the International Agency for Research on Cancer (IARC) as probably carcinogenic to humans [35]. After contamination by these substances, they are biotransformed in the liver into different (poly)hydroxy-PAHs.

The risks of PAHs to fish and other aquatic organisms in natural systems are highly uncertain due to the occurrence of complex, incompletely characterized mixtures of these chemicals, large spatial heterogeneity in exposure concentrations, incomplete understanding of the importance of UV-activated PAH toxicity, the biological and physical controls on fish exposure to UV light, and the bioaccumulation of PAHs. These uncertainties are especially great for early-life-stage fish, which might be particularly susceptible to UV-activated because of their small size, lack of protective pigmentation and gill coverings, and ready accumulation of PAHs.

#### **3. Transformations of PAHs in marine ecosystem**

#### **3.1 Physical and chemical degradation**

Sediments contaminated with PAHs pose a real threat to all living organisms, even those that feed on the benthic prey.

PAHs with a low molecular weight can be found in all environmental matrices, since higher molecular weight compounds are more deeply associated (physically and chemically) with sediments/soils and particles than the other abiotic sample types. PAHs in air can be modified via chemical oxidation and photochemical processes [8], whereas in sediments/soils and the uppermost portion of the water column, degradation of PAHs, particularly lower molecular weight PAHs, occurs via photooxidation [6, 36]. In addition to parent PAHs, oxygenated PAH metabolites formed during these degradation processes can persist associating with sediments up to 6 months after initial addition to the water column and thus can endure in the environment for extended periods of time [37]. In water samples and sediment,

some microorganisms (e.g., fungi, bacteria) have been demonstrated to mineralize PAHs under aerobic conditions, particularly those compounds that contain twoand three-fused rings (e.g., fluorene, naphthalene), to their basic elements or to biodegrade these compounds to more polar degradation products [3, 8]. More information on PAH microbial degradation pathways and identification of degradation can be found in Cerniglia and Heitkamp [38], Juhasz and Naidu [39], and Bamforth and Singleton [40]. Part of research studies have proved that pyrene can be mineralized by certain strains of bacteria (e.g., *Mycobacterium*) under optimum growing conditions in the laboratory, but it is uncertain if this occurs in the natural environment [41, 42]. In contrast, other higher molecular weight PAHs (e.g., five- and six-ring compounds) are not readily degraded by microbes and thus are more likely to accumulate in these environmental media (particularly in fine-grained sediments with high organic carbon content) [3, 39]. Under anoxic conditions, PAHs persist in sediments, particularly in organic sediments [42].

#### **3.2 Biotransformation in the aquatic food web**

Pyrolytic PAHs are the most common in aquatic environments. After the emission of pyrolytic PAHs into the atmosphere, the molecules fall back and settle on the surface of the water or soil [23]. Under the action of soil leaching, PAHs are transported to water bodies. These hydrophobic molecules then associate with other particles of the column of water and accumulate in the sediment [23].

In aquatic organisms, exposure to PAHs can occur through dermal exposure, respiration, or consumption of contaminated prey (e.g., annelids, crustaceans) or sediment [43]. Biotransformation of PAHs in aquatic organisms occurs to varying degrees depending on a number of factors, including the rate of uptake, metabolic capability, physical condition, feeding strategy, and age [43, 44]. Invertebrates are capable of PAH uptake from their environment and have been shown to have varying levels of PAH-metabolizing capability [44]. Mollusks generally have lower PAH-metabolizing capability than certain species of polychaetes, crustaceans, and fish [3].

PAH metabolism in fish is mainly conducted by inducible enzymes of the cytochrome P450 family, in particular CYP1A. These enzymes are localized in the membranes of the smooth endoplasmic reticulum, located mainly in the liver, but are also present in other organs. They are expressed and functional from the earliest stages of fish development. These enzymes catalyze the addition of an oxygen atom to the PAH molecule through an NADPH-dependent reaction. CYP1A protein is induced during exposure of the body to PAHs. CYP1A induction is rapid, and activity levels are often increased by a factor of 100 a few hours after exposure. These highly polar conjugated metabolites are then excreted into urine or the bile for rapid rejection over the gastrointestinal tract [14, 43, 45, 46]. Concerning the result of this rapid metabolism in fish, concentrations of parent PAHs are insignificant in muscle and other tissues. Thus, to determinate a recent exposure to PAHs in teleost fish, bile and urine are used, with the preference of bile because of its easy sampling. Otherwise, differences in the metabolism of benzo[a]pyrene (BaP), including differences in the types and proportions of metabolites formed, have been shown between two fish of freshwater [47, 48]. These differences could contribute to variations in susceptibility to these carcinogenic compounds among fish species. Differences in glutathione-S-transferases may also help explain differential susceptibility to chemically induced carcinogenesis among fish species [46]. A number of analytical methods have been developed to measure PAH metabolites in fish bile and are reviewed in [14]. Oil spills, including the Deepwater Horizon (DWH) spill in the Gulf of Mexico in 2010, show the necessity of the need for additional

**203**

*Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species*

methods to determine PAH exposure in seafood or protected species (e.g., marine mammals) where species cannot be lethally collected. For example, a rapid, sensitive HPLC-fluorescence method was developed by the US Food and Drug Administration [49] during the DWH spill and was used by federal and Gulf state analytical laboratories as part of the seafood safety response [50]. The development of new analytical methods like those can provide important information on PAH

PAHs are an important factor of contamination in the environment but also a risk to human health. In fact, the dangers related to PAHs vary according to their toxicity on the one hand and the many sources of exposure on the other. It has also been proven that carcinogenic and mutagenic effects related to a single compound of PAHs were found. A large number of effects have been identified [51]. In fact, genotoxicity and tumorigenesis observed in fish are linked to the presence of metabolites. Beyond genotoxicity, there are many other effects observed, for

Benzo[a]pyrene, for example, which is highly studied, leads to a decrease in weight [52] and growth [53], an increase in the gonado-somatic index (GSI) in Japanese medaka (*Oryzias latipes*) [52], DNA breaks in oysters (*Crassostrea gigas*) [54], and DNA adducts in zebrafish and on human liver cells (HepG2) [55]. Teratogenic effects in particular on the heart of sardine (*Clupea pallasii*) [56] and zebrafish [57, 58] have been observed as well as anemia in scorpion fish (*Sebastes schlegelii*) [59]. Benzo[a]pyrene affects the reproduction of isopods (*Oniscus asellus* and *Asellus porcellio scaber*) [53] and accumulates in oocytes in catfish (*Ictalurus punctatus*) [60]; it disrupts the expression of the aromatase (enzyme necessary for the conversion of androgen such as estrogen isosterone) in female mummichog (*Fundulus heteroclitus*) [61] and inhibits the synthesis of testosterone and estradiol

The toxicity of a compound can be enhanced or reduced by endogenous and exogenous factors. For example, in fish, hypoxia [63], geographical origin and fish life history [64], and/or the various PAH compounds [65] can cause variations. The penetration time in the fish embryo and the depuration time can vary considerably. In addition, the effects produced by these molecules, tested individually, do not

Survival is a commonly used variable. This variable has made it possible to develop standardized tests in toxicology such as LD50 (lethal dose) or LC50 (lethal concentration) calculations. Although protected in their chorions, fish eggs and then larvae are particularly exposed because, in most cases, they are unable to flee contaminated areas during their early-life stages [66]. It is during development, during the establishment of all organs and systems, that contaminants can pass this barrier. They can affect the development and have long-term consequences. Exposure to PAHs can lead to decreased survival in aquatic organisms during acute exposures. For example, there has been a decrease in survival after early exposure

In the case of chronic exposure in the early stages, similar effects can be observed. Impairment in survival has been observed in salmon (*Oncorhynchus gorbuscha*) exposed to crude oil [68], in minnow (*Pimephales promelas*) exposed

*DOI: http://dx.doi.org/10.5772/intechopen.86213*

**4. Effects of PAHs on aquatic species**

example, in behavior, reproduction, and growth.

in flounder (*Platichthys flesus* L.) [62].

**4.1 Effects of PAHs on the survival**

necessarily follow a dose-effect relationship [52].

of salmon (*Oncorhynchus gorbuscha*) to dissolve PAHs [67].

exposure in aquatic organisms.

methods to determine PAH exposure in seafood or protected species (e.g., marine mammals) where species cannot be lethally collected. For example, a rapid, sensitive HPLC-fluorescence method was developed by the US Food and Drug Administration [49] during the DWH spill and was used by federal and Gulf state analytical laboratories as part of the seafood safety response [50]. The development of new analytical methods like those can provide important information on PAH exposure in aquatic organisms.

#### **4. Effects of PAHs on aquatic species**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

sediments, particularly in organic sediments [42].

**3.2 Biotransformation in the aquatic food web**

chaetes, crustaceans, and fish [3].

some microorganisms (e.g., fungi, bacteria) have been demonstrated to mineralize PAHs under aerobic conditions, particularly those compounds that contain twoand three-fused rings (e.g., fluorene, naphthalene), to their basic elements or to biodegrade these compounds to more polar degradation products [3, 8]. More information on PAH microbial degradation pathways and identification of degradation can be found in Cerniglia and Heitkamp [38], Juhasz and Naidu [39], and Bamforth and Singleton [40]. Part of research studies have proved that pyrene can be mineralized by certain strains of bacteria (e.g., *Mycobacterium*) under optimum growing conditions in the laboratory, but it is uncertain if this occurs in the natural environment [41, 42]. In contrast, other higher molecular weight PAHs (e.g., five- and six-ring compounds) are not readily degraded by microbes and thus are more likely to accumulate in these environmental media (particularly in fine-grained sediments with high organic carbon content) [3, 39]. Under anoxic conditions, PAHs persist in

Pyrolytic PAHs are the most common in aquatic environments. After the emission of pyrolytic PAHs into the atmosphere, the molecules fall back and settle on the surface of the water or soil [23]. Under the action of soil leaching, PAHs are transported to water bodies. These hydrophobic molecules then associate with other

In aquatic organisms, exposure to PAHs can occur through dermal exposure, respiration, or consumption of contaminated prey (e.g., annelids, crustaceans) or sediment [43]. Biotransformation of PAHs in aquatic organisms occurs to varying degrees depending on a number of factors, including the rate of uptake, metabolic capability, physical condition, feeding strategy, and age [43, 44]. Invertebrates are capable of PAH uptake from their environment and have been shown to have varying levels of PAH-metabolizing capability [44]. Mollusks generally have lower PAH-metabolizing capability than certain species of poly-

PAH metabolism in fish is mainly conducted by inducible enzymes of the cytochrome P450 family, in particular CYP1A. These enzymes are localized in the membranes of the smooth endoplasmic reticulum, located mainly in the liver, but are also present in other organs. They are expressed and functional from the earliest stages of fish development. These enzymes catalyze the addition of an oxygen atom to the PAH molecule through an NADPH-dependent reaction. CYP1A protein is induced during exposure of the body to PAHs. CYP1A induction is rapid, and activity levels are often increased by a factor of 100 a few hours after exposure. These highly polar conjugated metabolites are then excreted into urine or the bile for rapid rejection over the gastrointestinal tract [14, 43, 45, 46]. Concerning the result of this rapid metabolism in fish, concentrations of parent PAHs are insignificant in muscle and other tissues. Thus, to determinate a recent exposure to PAHs in teleost fish, bile and urine are used, with the preference of bile because of its easy sampling. Otherwise, differences in the metabolism of benzo[a]pyrene (BaP), including differences in the types and proportions of metabolites formed, have been shown between two fish of freshwater [47, 48]. These differences could contribute to variations in susceptibility to these carcinogenic compounds among fish species. Differences in glutathione-S-transferases may also help explain differential susceptibility to chemically induced carcinogenesis among fish species [46]. A number of analytical methods have been developed to measure PAH metabolites in fish bile and are reviewed in [14]. Oil spills, including the Deepwater Horizon (DWH) spill in the Gulf of Mexico in 2010, show the necessity of the need for additional

particles of the column of water and accumulate in the sediment [23].

**202**

PAHs are an important factor of contamination in the environment but also a risk to human health. In fact, the dangers related to PAHs vary according to their toxicity on the one hand and the many sources of exposure on the other. It has also been proven that carcinogenic and mutagenic effects related to a single compound of PAHs were found. A large number of effects have been identified [51]. In fact, genotoxicity and tumorigenesis observed in fish are linked to the presence of metabolites. Beyond genotoxicity, there are many other effects observed, for example, in behavior, reproduction, and growth.

Benzo[a]pyrene, for example, which is highly studied, leads to a decrease in weight [52] and growth [53], an increase in the gonado-somatic index (GSI) in Japanese medaka (*Oryzias latipes*) [52], DNA breaks in oysters (*Crassostrea gigas*) [54], and DNA adducts in zebrafish and on human liver cells (HepG2) [55]. Teratogenic effects in particular on the heart of sardine (*Clupea pallasii*) [56] and zebrafish [57, 58] have been observed as well as anemia in scorpion fish (*Sebastes schlegelii*) [59]. Benzo[a]pyrene affects the reproduction of isopods (*Oniscus asellus* and *Asellus porcellio scaber*) [53] and accumulates in oocytes in catfish (*Ictalurus punctatus*) [60]; it disrupts the expression of the aromatase (enzyme necessary for the conversion of androgen such as estrogen isosterone) in female mummichog (*Fundulus heteroclitus*) [61] and inhibits the synthesis of testosterone and estradiol in flounder (*Platichthys flesus* L.) [62].

The toxicity of a compound can be enhanced or reduced by endogenous and exogenous factors. For example, in fish, hypoxia [63], geographical origin and fish life history [64], and/or the various PAH compounds [65] can cause variations. The penetration time in the fish embryo and the depuration time can vary considerably. In addition, the effects produced by these molecules, tested individually, do not necessarily follow a dose-effect relationship [52].

#### **4.1 Effects of PAHs on the survival**

Survival is a commonly used variable. This variable has made it possible to develop standardized tests in toxicology such as LD50 (lethal dose) or LC50 (lethal concentration) calculations. Although protected in their chorions, fish eggs and then larvae are particularly exposed because, in most cases, they are unable to flee contaminated areas during their early-life stages [66]. It is during development, during the establishment of all organs and systems, that contaminants can pass this barrier. They can affect the development and have long-term consequences. Exposure to PAHs can lead to decreased survival in aquatic organisms during acute exposures. For example, there has been a decrease in survival after early exposure of salmon (*Oncorhynchus gorbuscha*) to dissolve PAHs [67].

In the case of chronic exposure in the early stages, similar effects can be observed. Impairment in survival has been observed in salmon (*Oncorhynchus gorbuscha*) exposed to crude oil [68], in minnow (*Pimephales promelas*) exposed to contaminated sediment [69], in *Chanos chanos* and capelin (*Mallotus villosus*) exposed to dissolved PAHs (anthracene, B[a]P, pyrene and heavy fuel oil) [66, 70], and in shrimp (*Palaemonetes pugio*) exposed to the pyrene feed which also shows reduced survival [71]. In other studies, survival is not affected by PAHs. This is the case, for example, in terrestrial isopods, where oral administration does not have significant effects on survival [53]. It is also not affected after exposure to BaP in mummichog (*Fundulus heteroclitus*) [72]. PAHs can affect survival in some cases and not in others. This extreme variable may not be the most sensitive for all species or all types of exposure.

#### **4.2 Malformations and growth**

PAHs induce malformations during development. They lead to a decrease in skeletal mineralization in bass (*Dicentrarchus labrax*) [73] and craniofacial deformities in scorpion fish (*Sebastiscus marmoratus*) [74]. Jaw malformations [75] in this same fish as well as in zebrafish [58] were also observed. The number of edemas is also increased in scorpion fish (*Sebastes schlegelii*), salmon (*Oncorhynchus gorbuscha*) [67, 76], and medaka (*Oryzias latipes*) [77], as well as the occurrence of hemorrhages in trout (*Oncorhynchus mykiss*) [78]. The impact of PAHs on growth is frequently reflected in a reduction in size and/or weight [67, 79, 80]. This reduction in growth is observed regardless of the mode of administration of PAHs, the concentrations used, and the duration of exposure [59, 70, 81]. Weight reduction is often proportional to contamination [81]. Unfortunately, these are not the only visible damage. A decrease in lipid reserves may be observed and result in a decrease in energy reserves [10, 79].

#### **4.3 Metabolism and osmoregulation**

In a PAH study, fish were exposed to the soluble fraction of a crude oil mixture. Structural lesions and morphological differences are noted on the gill [82]. These differences would be related to a metabolism aimed at reducing contact with the pollutant, which would reduce the gill surface and oxygen supply. A reduction in oxygen uptake could compromise the fish metabolism [82].

Osmoregulation problems following exposure of *Sebastiscus marmoratus* to dissolve PAHs have been observed [75]. PAHs would inhibit Na+ /K+ activity in a dose-dependent manner and play a role in osmoregulation.

#### **4.4 Effect on behavior**

The behavioral response of an animal following exposure to stress and/or contaminant (s) is increasingly studied [83–87].

The behavior makes it possible to discriminate a large number of integrating variables from the changes induced by PAHs. Swimming activity can be assessed as well as other aspects such as lethargy, anxiety, social communication, eating behavior, flight response, learning, or reproductive behavior.

A reduced swimming activity in seabream (*Sparus aurata*) was observed following a 4 days of exposure to dissolved PAHs [88, 89]: phenanthrene, fluorine, and pyrene [89]. An increase in lethargy and a reduction in the number of surface surges have also been observed following exposure to dissolved PAHs in this species [88, 89].

These variables can also be used to evaluate the neurotoxic effects of a contaminant. The reduction of social interactions following exposure to phenanthrene [89] is proven.

The escape response, in the presence of fluoranthene, has been demonstrated in control fish [90]. The fish are placed in a double flow aquarium. A flow of control

**205**

*Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species*

water and a flow of water containing fluoranthene are present. Fish that have never been exposed are fleeing fluoranthene. On the other hand, fish that have been previously exposed to a high dose of fluoranthene no longer leak the molecule [90]. Learning and exploration abilities are diminished after exposure to PAHs. For example, discrimination of a familiar object is altered in mice exposed to BaP. In the same vein, dietary exposure of the mother to a mixture containing the 16 priority PAHs of the USEPA leads to behavioral alteration in the next generation, especially in a new environment. The reproductive behavior can also be disturbed. The ability of a male to find a female can be altered, as is the case in amphipods, for example [91].

PAHs are lipophilic molecules that are transported and found in the ovaries via vitellogenin and/or lipovitellin [60]. They can also result in inhibition of vitellogenin synthesis, as has already been shown in trout after exposure to β-naphthoflavone [92]. This exposure compromises the maturation of the ovaries and causes an increase in apoptosis in gonadal cells [91]. These pollutants lead, for example, to reproductive inhibition in shrimp exposed to pyrene [71]. A decrease in fecundity, number of breeding cycles, and larval survival is observed in different fish species.

In mussels, gametes are deformed and are present in small numbers [91].

Females are not the only ones to be affected. Male sperm quality can also be altered after exposure to benzo[b]fluoranthene, as is the case in mice exposed via breast milk [59]. Sperm quality is reduced, and there is also an increase in testicular

High-performance liquid chromatography (HPLC) is generally used for the determination of PAH metabolites in considerable fish species [93–98] and has been

Although bile metabolites have been measured in many species of fish [13], those selected for biomonitoring programs tend to be common, long-lived species at the top of the food chain, with relatively sedentary life styles and benthic habits [99]. Consequently, the common eel (*Anguilla anguilla*) has been used in studies of PAH contamination [100, 101]. Pleuronectid flatfish are also well-suited to biomonitoring, and the European flounder (*Pleuronectes flesus*), an abundant flatfish in most European

The study conducted by Ruddock et al. [104] in the Severn Estuary showed that from the six metabolites of polycyclic aromatic hydrocarbons (PAHs) identified and quantified from the bile of *Anguilla anguilla*, *Pleuronectes flesus*, and *Conger conger* collected during 1997, the main metabolite present in all fish was 1-hydroxypyrene with lower proportions of 1-hydroxychrysene and 1-hydroxyphenanthrene and small concentration of three benzo[a]pyrene derivatives. The results approve the importance of 1-hydroxypyrene as the important PAH metabolite in fish bile and suggest that the *A. anguilla* is an excellent species for monitoring PAHs in estuarine ecosystems. 1-Hydroxypyrene is invariably the major metabolite present in the bile of fish exposed to PAH-contaminated sediments [105], and this was confirmed by the results of this work for fish in the Severn Estuary. Pyrene is produced by many petrogenic and pyrolytic processes [43] and has been detected in significant number in sewage outfalls [106]. It is regarded the best general indicator of PAH exposure in fish [100, 107]. The contribution of 1-OH-Phen to the total metabolites detected ranged from approximately 2% in flounders and conger eels to 8% in common eels.

estuaries, has been frequently chosen to assess PAH contamination [102, 103].

*DOI: http://dx.doi.org/10.5772/intechopen.86213*

**4.5 Effect on reproduction**

apoptosis.

**5. PAHs in eels**

covered by an intercalibration exercise [22].

#### *Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species DOI: http://dx.doi.org/10.5772/intechopen.86213*

water and a flow of water containing fluoranthene are present. Fish that have never been exposed are fleeing fluoranthene. On the other hand, fish that have been previously exposed to a high dose of fluoranthene no longer leak the molecule [90].

Learning and exploration abilities are diminished after exposure to PAHs. For example, discrimination of a familiar object is altered in mice exposed to BaP. In the same vein, dietary exposure of the mother to a mixture containing the 16 priority PAHs of the USEPA leads to behavioral alteration in the next generation, especially in a new environment. The reproductive behavior can also be disturbed. The ability of a male to find a female can be altered, as is the case in amphipods, for example [91].

#### **4.5 Effect on reproduction**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

or all types of exposure.

energy reserves [10, 79].

**4.4 Effect on behavior**

**4.3 Metabolism and osmoregulation**

oxygen uptake could compromise the fish metabolism [82].

dose-dependent manner and play a role in osmoregulation.

behavior, flight response, learning, or reproductive behavior.

contaminant (s) is increasingly studied [83–87].

dissolve PAHs have been observed [75]. PAHs would inhibit Na+

**4.2 Malformations and growth**

to contaminated sediment [69], in *Chanos chanos* and capelin (*Mallotus villosus*) exposed to dissolved PAHs (anthracene, B[a]P, pyrene and heavy fuel oil) [66, 70], and in shrimp (*Palaemonetes pugio*) exposed to the pyrene feed which also shows reduced survival [71]. In other studies, survival is not affected by PAHs. This is the case, for example, in terrestrial isopods, where oral administration does not have significant effects on survival [53]. It is also not affected after exposure to BaP in mummichog (*Fundulus heteroclitus*) [72]. PAHs can affect survival in some cases and not in others. This extreme variable may not be the most sensitive for all species

PAHs induce malformations during development. They lead to a decrease in skeletal mineralization in bass (*Dicentrarchus labrax*) [73] and craniofacial deformities in scorpion fish (*Sebastiscus marmoratus*) [74]. Jaw malformations [75] in this same fish as well as in zebrafish [58] were also observed. The number of edemas is also increased in scorpion fish (*Sebastes schlegelii*), salmon (*Oncorhynchus gorbuscha*) [67, 76], and medaka (*Oryzias latipes*) [77], as well as the occurrence of hemorrhages in trout (*Oncorhynchus mykiss*) [78]. The impact of PAHs on growth is frequently reflected in a reduction in size and/or weight [67, 79, 80]. This reduction in growth is observed regardless of the mode of administration of PAHs, the concentrations used, and the duration of exposure [59, 70, 81]. Weight reduction is often proportional to contamination [81]. Unfortunately, these are not the only visible damage. A decrease in lipid reserves may be observed and result in a decrease in

In a PAH study, fish were exposed to the soluble fraction of a crude oil mixture. Structural lesions and morphological differences are noted on the gill [82]. These differences would be related to a metabolism aimed at reducing contact with the pollutant, which would reduce the gill surface and oxygen supply. A reduction in

Osmoregulation problems following exposure of *Sebastiscus marmoratus* to

The behavioral response of an animal following exposure to stress and/or

The behavior makes it possible to discriminate a large number of integrating variables from the changes induced by PAHs. Swimming activity can be assessed as well as other aspects such as lethargy, anxiety, social communication, eating

A reduced swimming activity in seabream (*Sparus aurata*) was observed following a 4 days of exposure to dissolved PAHs [88, 89]: phenanthrene, fluorine, and pyrene [89]. An increase in lethargy and a reduction in the number of surface surges have also been observed following exposure to dissolved PAHs in this species [88, 89]. These variables can also be used to evaluate the neurotoxic effects of a contaminant. The reduction of social interactions following exposure to phenanthrene [89] is proven. The escape response, in the presence of fluoranthene, has been demonstrated in control fish [90]. The fish are placed in a double flow aquarium. A flow of control

/K+

activity in a

**204**

PAHs are lipophilic molecules that are transported and found in the ovaries via vitellogenin and/or lipovitellin [60]. They can also result in inhibition of vitellogenin synthesis, as has already been shown in trout after exposure to β-naphthoflavone [92]. This exposure compromises the maturation of the ovaries and causes an increase in apoptosis in gonadal cells [91]. These pollutants lead, for example, to reproductive inhibition in shrimp exposed to pyrene [71]. A decrease in fecundity, number of breeding cycles, and larval survival is observed in different fish species. In mussels, gametes are deformed and are present in small numbers [91].

Females are not the only ones to be affected. Male sperm quality can also be altered after exposure to benzo[b]fluoranthene, as is the case in mice exposed via breast milk [59]. Sperm quality is reduced, and there is also an increase in testicular apoptosis.

#### **5. PAHs in eels**

High-performance liquid chromatography (HPLC) is generally used for the determination of PAH metabolites in considerable fish species [93–98] and has been covered by an intercalibration exercise [22].

Although bile metabolites have been measured in many species of fish [13], those selected for biomonitoring programs tend to be common, long-lived species at the top of the food chain, with relatively sedentary life styles and benthic habits [99]. Consequently, the common eel (*Anguilla anguilla*) has been used in studies of PAH contamination [100, 101]. Pleuronectid flatfish are also well-suited to biomonitoring, and the European flounder (*Pleuronectes flesus*), an abundant flatfish in most European estuaries, has been frequently chosen to assess PAH contamination [102, 103].

The study conducted by Ruddock et al. [104] in the Severn Estuary showed that from the six metabolites of polycyclic aromatic hydrocarbons (PAHs) identified and quantified from the bile of *Anguilla anguilla*, *Pleuronectes flesus*, and *Conger conger* collected during 1997, the main metabolite present in all fish was 1-hydroxypyrene with lower proportions of 1-hydroxychrysene and 1-hydroxyphenanthrene and small concentration of three benzo[a]pyrene derivatives. The results approve the importance of 1-hydroxypyrene as the important PAH metabolite in fish bile and suggest that the *A. anguilla* is an excellent species for monitoring PAHs in estuarine ecosystems. 1-Hydroxypyrene is invariably the major metabolite present in the bile of fish exposed to PAH-contaminated sediments [105], and this was confirmed by the results of this work for fish in the Severn Estuary. Pyrene is produced by many petrogenic and pyrolytic processes [43] and has been detected in significant number in sewage outfalls [106]. It is regarded the best general indicator of PAH exposure in fish [100, 107]. The contribution of 1-OH-Phen to the total metabolites detected ranged from approximately 2% in flounders and conger eels to 8% in common eels.

Phenanthrenes are released to the atmosphere during the combustion of fossil fuels, particularly coal, oil, and its refined products [43]. Like all PAHs with two to four benzene rings, phenanthrenes can remain suspended in airborne particles for long periods [108]. Compared to the other PAHs detected, BaP has a very low solubility in water and low bioavailability, but metabolites of BaP are especially important because of their potent mutagenic and carcinogenic properties [109–111].

A recent study conducted by Baali et al. [2] on bile metabolites of PAHs in 18 European eels (*Anguilla anguilla*), 7 moray (Muraenidae), and 28 conger eels (*Conger conger*) from Moroccan waters (Moulay Bousselham lagoon and Boujdour) shows the presence of two polycyclic aromatic hydrocarbon (PAH) metabolites, 1-hydroxypyrene (1-OH-Pyr) and 1-hydroxyphenanthrene (1-OH-Phen). The highperformance liquid chromatography with fluorescence detection method was used to separate the bile metabolites.

The goals of the present study were to compare the levels of PAH metabolites in eels from the lagoon and sea and also to compare levels of PAH metabolites between the different eels.

In this study the PAH metabolites (1-OH-Pyr and 1-OH-Phen) were detected in all species. The results of this investigation show that the concentration of 1-OH-Pyr was high for *Anguilla anguilla* than the other species (**Figure 1**). The conger eels represent the species with the lower concentration of 1-OH-Pyr. This result reflects the low degree of contamination in Boujdour coast (**Figure 1**). Thus, the presence of high concentration of 1-OH-Pyr and 1-OH-Phen in the bile of the European eels and morays reflects the high degree of contamination in the lagoon which is due to the anthropogenic activity in Moulay Bousselham lagoon. From the comparison between the contamination of the European eels and morays belonging to Moulay Bousselham lagoon, the results show that the first species present a higher concentration of PAH metabolites than the second one (**Figure 1**). This conclusion confirms that the *Anguilla anguilla* is more suitable species for monitoring PAH contamination. The European eels spend most of their life in muddy sediment which usually present a high PAH concentration levels. The pollutants in sediment are easily accumulated [112–114]. Accordingly, it is recognized that sediment contamination has a particular interest with regard to aquatic ecosystem quality. Sediment is an important source of pollutants and the factor with the high impact on the deterioration of the water quality. Although the feeding habit of the European eels may result in higher exposure to PAHs whence the high concentration of 1-OH-Pyr and 1-OH-Phen in the bile of this species [115], the accumulation of PAHs from the surrounding water is considered more efficient than impacted food [116]. The level of PAH metabolites in fish bile varies according to the area. The results show that Boujdour Sea is not a polluted site [117]. Moulay Bousselham lagoon is a semi-closed area; the concentration of pollutants in this site is higher than Boujdour Sea because of its lower water circulation. In the lagoon PAHs are easily accumulated than that in the sea [112]. Our results confirm that 1-OH-Pyr is the major metabolite present in fish bile [104, 105] and the best indicator of PAH exposure in fish [100, 107]. It was found that 1-OH-Pyr is the dominant compound in eel bile [118–120]. The results show that the levels of PAHs in Morocco are lower than those obtained in the other regions. As a conclusion of this study, the possible health risk of PAH contamination in Boujdour coast and Moulay Bousselham lagoon might be low compared to the other European sites.

The concentration of 1-OH-Pyr varies significantly with length (p < 0.05) for each species. The results obtained in this study [2] show that the concentration of PAH metabolites does not always increase with the size; there are obviously factors which can affect the exposure of this pollutant such as species differences, age, sex, maturity, and diet.

**207**

**6. Conclusion**

*mean (triangles) and range (panels).*

**Figure 1.**

inorganic minerals.

*Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species*

PAHs are originally organic compounds that are created from the partial combustion of organic elements or pyrolysis of organic material. These compounds are associated to the treatment of wood, oil, coal, and gas in order to produce the energy. PAHs are transferred in the air in gas or particle aspect, and they are accumulated by wet and dry deposition. The transported elements play important role in the chemistry of the atmosphere. These particles also have significant impact in human health, because many PAHs are classified as probable human carcinogens. The other faculty of PAHs is the capacity of degrading microorganism such as bacteria, fungi, and algae. It concerns the failure of organic compounds through biotransformation into less complex metabolites and through mineralization into

*Bile metabolite 1-hydroxypyrene (a) and 1-hydroxyphenanthrene (b) concentrations detected in European eels (Anguilla anguilla) collected from different areas and eels from Morocco (conger, moray, and European eel) as* 

In this chapter, many effects on the biology of species following exposure to PAHs have been demonstrated. At the end of these organic studies on fish, it has been shown that the PAH biliary metabolites studied have the potential to describe

The study of a possible contamination of eels from different countries shows that 1-hydroxypyrene (1-OH-Pyr) is the dominant pollutant present in fish bile and

the state of exposure of fish to organic pollutants (PAHs).

is the best general indicator of PAH contamination.

*DOI: http://dx.doi.org/10.5772/intechopen.86213*

*Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species DOI: http://dx.doi.org/10.5772/intechopen.86213*

#### **Figure 1.**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

to separate the bile metabolites.

the different eels.

Phenanthrenes are released to the atmosphere during the combustion of fossil fuels, particularly coal, oil, and its refined products [43]. Like all PAHs with two to four benzene rings, phenanthrenes can remain suspended in airborne particles for long periods [108]. Compared to the other PAHs detected, BaP has a very low solubility in water and low bioavailability, but metabolites of BaP are especially important because of their potent mutagenic and carcinogenic properties [109–111]. A recent study conducted by Baali et al. [2] on bile metabolites of PAHs in 18 European eels (*Anguilla anguilla*), 7 moray (Muraenidae), and 28 conger eels (*Conger conger*) from Moroccan waters (Moulay Bousselham lagoon and Boujdour) shows the presence of two polycyclic aromatic hydrocarbon (PAH) metabolites, 1-hydroxypyrene (1-OH-Pyr) and 1-hydroxyphenanthrene (1-OH-Phen). The highperformance liquid chromatography with fluorescence detection method was used

The goals of the present study were to compare the levels of PAH metabolites in eels from the lagoon and sea and also to compare levels of PAH metabolites between

In this study the PAH metabolites (1-OH-Pyr and 1-OH-Phen) were detected in all species. The results of this investigation show that the concentration of 1-OH-Pyr was high for *Anguilla anguilla* than the other species (**Figure 1**). The conger eels represent the species with the lower concentration of 1-OH-Pyr. This result reflects the low degree of contamination in Boujdour coast (**Figure 1**). Thus, the presence of high concentration of 1-OH-Pyr and 1-OH-Phen in the bile of the European eels and morays reflects the high degree of contamination in the lagoon which is due to the anthropogenic activity in Moulay Bousselham lagoon. From the comparison between the contamination of the European eels and morays belonging to Moulay Bousselham lagoon, the results show that the first species present a higher concentration of PAH metabolites than the second one (**Figure 1**). This conclusion confirms that the *Anguilla anguilla* is more suitable species for monitoring PAH contamination. The European eels spend most of their life in muddy sediment which usually present a high PAH concentration levels. The pollutants in sediment are easily accumulated [112–114]. Accordingly, it is recognized that sediment contamination has a particular interest with regard to aquatic ecosystem quality. Sediment is an important source of pollutants and the factor with the high impact on the deterioration of the water quality. Although the feeding habit of the European eels may result in higher exposure to PAHs whence the high concentration of 1-OH-Pyr and 1-OH-Phen in the bile of this species [115], the accumulation of PAHs from the surrounding water is considered more efficient than impacted food [116]. The level of PAH metabolites in fish bile varies according to the area. The results show that Boujdour Sea is not a polluted site [117]. Moulay Bousselham lagoon is a semi-closed area; the concentration of pollutants in this site is higher than Boujdour Sea because of its lower water circulation. In the lagoon PAHs are easily accumulated than that in the sea [112]. Our results confirm that 1-OH-Pyr is the major metabolite present in fish bile [104, 105] and the best indicator of PAH exposure in fish [100, 107]. It was found that 1-OH-Pyr is the dominant compound in eel bile [118–120]. The results show that the levels of PAHs in Morocco are lower than those obtained in the other regions. As a conclusion of this study, the possible health risk of PAH contamination in Boujdour coast and Moulay Bousselham lagoon

**206**

maturity, and diet.

might be low compared to the other European sites.

The concentration of 1-OH-Pyr varies significantly with length (p < 0.05) for each species. The results obtained in this study [2] show that the concentration of PAH metabolites does not always increase with the size; there are obviously factors which can affect the exposure of this pollutant such as species differences, age, sex,

*Bile metabolite 1-hydroxypyrene (a) and 1-hydroxyphenanthrene (b) concentrations detected in European eels (Anguilla anguilla) collected from different areas and eels from Morocco (conger, moray, and European eel) as mean (triangles) and range (panels).*

#### **6. Conclusion**

PAHs are originally organic compounds that are created from the partial combustion of organic elements or pyrolysis of organic material. These compounds are associated to the treatment of wood, oil, coal, and gas in order to produce the energy.

PAHs are transferred in the air in gas or particle aspect, and they are accumulated by wet and dry deposition. The transported elements play important role in the chemistry of the atmosphere. These particles also have significant impact in human health, because many PAHs are classified as probable human carcinogens.

The other faculty of PAHs is the capacity of degrading microorganism such as bacteria, fungi, and algae. It concerns the failure of organic compounds through biotransformation into less complex metabolites and through mineralization into inorganic minerals.

In this chapter, many effects on the biology of species following exposure to PAHs have been demonstrated. At the end of these organic studies on fish, it has been shown that the PAH biliary metabolites studied have the potential to describe the state of exposure of fish to organic pollutants (PAHs).

The study of a possible contamination of eels from different countries shows that 1-hydroxypyrene (1-OH-Pyr) is the dominant pollutant present in fish bile and is the best general indicator of PAH contamination.

Of the different eels investigated, European eels (*Anguilla anguilla*) contained the highest metabolite concentrations. This species looks like the most suitable for monitoring PAH contamination in the environment.

Using the studies conducted by several authors, we found that the rivers and lagoon contain PAH concentrations much higher than the coastal waters. These results appear normal in view that there is low water exchange in the rivers and lagoon ecosystems.

Finally we conclude that the quantification and identification of the metabolites in fish bile can give a rapid indication on the level of PAH contamination.

### **Conflict of interest**

The authors declare that they have no competing interests.

### **Author details**

Ayoub Baali\* and Ahmed Yahyaoui Laboratory of Biodiversity, Ecology and Genome, Faculty of Science, Mohammed V University in Rabat, Rabat, Morocco

\*Address all correspondence to: ayoubbaali22@gmail.com

© 2019 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.

**209**

*Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species*

[8] Lima ALC, Farrington JW, Reddy CM. Combustion-derived polycyclic aromatic hydrocarbons in the

[9] Hylland K. Polycyclic aromatic hydrocarbon (PAH) ecotoxicology in marine ecosystems. Journal of Toxicology and Environmental Health, Part A. 2006;**69**:109-123

[10] Meador JP, Sommers FC, Ylitalo GM, Sloan CA. Altered growth and related physiological responses in juvenile Chinook salmon (*Oncorhynchus tshawytscha*) from dietary exposure to polycyclic aromatic hydrocarbons (PAHs). Canadian Journal of Fisheries and Aquatic Sciences.

[11] Meador JP, Buzitis J, Bravo CF. Using fluorescent aromatic compounds in bile from juvenile salmonids to predict exposure to polycyclic aromatic hydrocarbons. Environmental Toxicology and Chemistry.

Forensics. 2005;**6**:109-131

2006;**63**:2364-2376

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2010;**30**:224-244

[12] Yanagida GK, Anulacion BF, Bolton JL, Boyd D, Lomax DP, Olson OP, et al. Polycyclic aromatic hydrocarbons and risk to threatened and endangered Chinook salmon in the lower Columbia River estuary. Archives of Environmental Contamination and Toxicology. 2012;**62**(2):282-295

[13] Tuvikene A. Responses of fish to aromatic hydrocarbons. Annales Zoologici Fennici. 1995;**32**:295-309

[14] Beyer J, Jonsson G, Porte C, Krahn MM, Ariese F. Analytical methods for determining metabolites of polycyclic aromatic hydrocarbon (PAH) pollutants in fish bile: A review. Environmental Toxicology and Pharmacology.

environment—A review. Environmental

*DOI: http://dx.doi.org/10.5772/intechopen.86213*

[1] Ahmad I, Pacheco M, Santos A. *Anguilla anguilla* L. oxidative stress biomarkers: An in situ study of freshwater wetland ecosystem (Pateira de Fermentelos, Portugal). Chemosphere. 2006;**65**:952-962

[2] Baali A, Kammann U, Hanel R, El Qoraychy I, Yahyaoui A. Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in three species of fish from Morocco. Environmental Sciences Europe. 2016;**28**:25. DOI: 10.1186/

[3] Meador J, Stein J, Reichert W, Varanasi U. Bioaccumulation of polycyclic aromatic hydrocarbons by marine organisms. Reviews of Environmental Contamination and

Toxicology. 1995;**143**:79-165

[4] Woodhead RJ, Law RJ, Matthiessen P. Polycyclic aromatic hydrocarbons in surface sediments around England and Wales, and their possible biological significance. Marine Pollution Bulletin.

[5] Blahová J, Havelková M, Kružíková

[6] McElroy AE, Farrington JW, Teal JM. Bioavailability of PAH in the aquatic environment. In: Varanasi U, editor. Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. Boca Raton, FL: CRC

[7] Ramesh A, Walker SA, Hood DB, Guillén MD, Schneider K, Weyand EH. Bioavailability and risk assessment of orally ingested polycyclic aromatic hydrocarbons. International Journal of

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1999;**38**:773-790

*Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species DOI: http://dx.doi.org/10.5772/intechopen.86213*

#### **References**

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

monitoring PAH contamination in the environment.

lagoon ecosystems.

**Conflict of interest**

Of the different eels investigated, European eels (*Anguilla anguilla*) contained the highest metabolite concentrations. This species looks like the most suitable for

Using the studies conducted by several authors, we found that the rivers and lagoon contain PAH concentrations much higher than the coastal waters. These results appear normal in view that there is low water exchange in the rivers and

Finally we conclude that the quantification and identification of the metabolites

in fish bile can give a rapid indication on the level of PAH contamination.

The authors declare that they have no competing interests.

**208**

**Author details**

provided the original work is properly cited.

Ayoub Baali\* and Ahmed Yahyaoui

University in Rabat, Rabat, Morocco

\*Address all correspondence to: ayoubbaali22@gmail.com

© 2019 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,

Laboratory of Biodiversity, Ecology and Genome, Faculty of Science, Mohammed V

[1] Ahmad I, Pacheco M, Santos A. *Anguilla anguilla* L. oxidative stress biomarkers: An in situ study of freshwater wetland ecosystem (Pateira de Fermentelos, Portugal). Chemosphere. 2006;**65**:952-962

[2] Baali A, Kammann U, Hanel R, El Qoraychy I, Yahyaoui A. Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in three species of fish from Morocco. Environmental Sciences Europe. 2016;**28**:25. DOI: 10.1186/ s12302-016-0093-6

[3] Meador J, Stein J, Reichert W, Varanasi U. Bioaccumulation of polycyclic aromatic hydrocarbons by marine organisms. Reviews of Environmental Contamination and Toxicology. 1995;**143**:79-165

[4] Woodhead RJ, Law RJ, Matthiessen P. Polycyclic aromatic hydrocarbons in surface sediments around England and Wales, and their possible biological significance. Marine Pollution Bulletin. 1999;**38**:773-790

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Press, Inc.; 1989. pp. 2-33

Technology. 1987;**21**:648-653

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[39] Juhasz AL, Naidu R. Extraction and recovery of organochlorine pesticides from fungal mycelia. Journal of Microbiological Methods.

[40] Bamforth SM, Singleton I.

[41] Heitkamp MA, Franklin W, Cerniglia CE. Microbial metabolism of polycyclic aromatic hydrocarbons: Isolation and characterization of a pyrene-degrading bacterium. Applied and Environmental Microbiology.

[42] Heitkamp MA, Cerniglia CE. Polycyclic aromatic hydrocarbon degradation by a *Mycobacterium* sp. in microcosms containing sediment and water from a pristine ecosystem.

Applied and Environmental Microbiology. 1989;**55**:1968-1973

FL: CRC Press; 1989. pp. 93-149

[43] Varanasi U, Stein JE, Nishimoto M. Biotransformation and disposition of polycyclic aromatic hydrocarbons in fish. In: Varanasi U, editor. Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. Boca Raton,

Bioremediation of polycyclic aromatic hydrocarbons: Current knowledge and future directions. Journal of Chemical Technology and Biotechnology.

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[30] Veltman K, Huijbregts MAJ, Rye H, Hertwich EG. Including impacts of particulate emissions on marine ecosystems in life cycle assessment: The case of offshore oil and gas production. Integrated Environmental Assessment and Management. 2011;**7**:678-686

sediments by GC-MS. International Journal of Environmental Research and

Public Health. 2012;**9**:2175-2188

[31] Inomata Y, Kajino M, Sato K, Ohara T, Kurokawa J, Ueda H, et al. Emission and atmospheric transport of particulate PAHs in Northeast Asia. Environmental Science & Technology. 2012;**46**(9):4941-4949. DOI: 10.1021/

[32] Tudoran MA, Putz MV. Polycyclic aromatic hydrocarbons: From in cerebro to in silico eco-toxicity fate. Chemical Bulletin of Politehnica

[33] Choi MS. Effects of tributyltin (TBT) on the expression of cytochrome P4501A, aryl hydrocarbon receptor and vitellogenin genes[Master's thesis].

[34] El Morhit M, Fekhaoui M, Elie P, Girard P, Yahyaoui A, El Abidi A, et al. Heavy metals in sediment, water and the European glass eel *Anguilla anguilla* (Osteichthyes: Anguillidae),

(Morocco, eastern Atlantic). Cybium.

[35] AESN (Agence de l'Eau Seine Normandie). Guide des profils de vulnérabilité des eaux de baignade.

[36] McElroy AE, Farrington JW, Teal JM. Bioavailability of polycyclic aromatic hydrocarbons in the aquatic environment. In: Varanasi U, editor. Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic

University of Timisoara. 2012;**57**(71):50-53

Sunmoon University; 2012

from Loukkos River estuary

2009;**33**:219-228

2009. p. 84

es300391w

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sediments by GC-MS. International Journal of Environmental Research and Public Health. 2012;**9**:2175-2188

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

[22] Wariaghly F. Etude écotoxicologique et parasitologique chez l'Anguille (*Anguilla anguilla* L.) dans les

estuaires marocains : Sebou et Loukkos (Atlantique) [thesis]. Rabat: Université Mohammed V-Agdal, Faculté des

[23] Abdel-shafy HI, Mansour MSM. A

hydrocarbons: Source, environmental impact, effect on human health and remediation. Egyptian Journal of Petroleum. 2016;**25**:107-123

[25] Laflamme RE, Hites RA. The global distribution of polycyclic aromatic hydrocarbons in recent sediments. Geochimica et Cosmochimica Acta.

[26] Wakeham SG, Schaffner C, Giger W. Polycyclic aromatic hydrocarbons in recent lake sediments-I. Compounds

having anthropogenic origins. Geochimica et Cosmochimica Acta.

[27] Ventakesan MI. Occurrence and possible sources of perylene in marine sediments—A review. Marine

[28] Wang X, Hong HS, Mu JL, Lin JQ,

Chemistry. 1988;**25**(1):1-27

Wang SH. Polycyclic aromatic hydrocarbon (PAH) metabolites in marine fishes as a specific biomarker to indicate PAH pollution in the marine coastal environment. Journal of Environmental Science and Health, Part A. Toxic/Hazardous Substances and Environmental Engineering.

[29] Dong C, Chen C, Chen C.

Determination of polycyclic aromatic hydrocarbons in industrial harbor

review on polycyclic aromatic

[24] Cole GM. Assessment and Remediation of Petroleum Contaminated Sites. 1st ed. Lewis

Publishers; 1994. 384 p

1978;**42**(3):289-303

1980;**44**:403-413

2008;**43**:219-226

Sciences; 2013

[15] Eisler R. Eisler's Encyclopedia of Environmentally Hazardous Priority Chemicals. 1st ed. Amsterdam: Elsevier;

[16] Brinkmann M, Hudjetz S, Cofalla C, Roger S, Kammann U, Zhang X, et al. A combined hydraulic and toxicological approach to assess re-suspended sediments during simulated flood events. Part I-multiple biomarkers in rainbow trout. Journal of Soils and Sediments.

2007. 986 p

2010;**10**:1347-1361

2014;**152**:38-42

2000a;**49**:453-467

2006;**366**:112-123

[17] Brinkmann M, Eichbaum K, Kammann U, Hudjetz S, Cofalla C, Buchinger S, et al. Physiologicallybased toxicokinetic models help identifying the key factors affecting

contaminant uptake during flood events. Aquatic Toxicology.

[18] Monteiro PRR, Reis-Henriques MA, Coimbra J. Plasma steroid levels in female flounder (*Platichthys flesus*) after chronic dietary exposure to single polycyclic aromatic hydrocarbons. Marine Environmental Research.

[19] Chen SC, Liao CM. Health risk assessment on human exposed to environmental polycyclic aromatic hydrocarbons pollution sources. Science of the Total Environment.

[20] OSPAR Commission. Co-ordinated environmental monitoring programme (CEMP). 2008. Available from: http:// www.ospar.org/content/content. asp?menu=00900301400000 [Accessed: 27 December 2015]

[21] HELCOM. Core indicators final report of the HELCOM CORESET project. Baltic Sea Environment Proceedings No. 136. 2013. 71 p. http:// www.helcom.fi/Lists/Publications/ BSEP136.pdf#search=core%20set [Accessed: 26 December 2015]

**210**

[30] Veltman K, Huijbregts MAJ, Rye H, Hertwich EG. Including impacts of particulate emissions on marine ecosystems in life cycle assessment: The case of offshore oil and gas production. Integrated Environmental Assessment and Management. 2011;**7**:678-686

[31] Inomata Y, Kajino M, Sato K, Ohara T, Kurokawa J, Ueda H, et al. Emission and atmospheric transport of particulate PAHs in Northeast Asia. Environmental Science & Technology. 2012;**46**(9):4941-4949. DOI: 10.1021/ es300391w

[32] Tudoran MA, Putz MV. Polycyclic aromatic hydrocarbons: From in cerebro to in silico eco-toxicity fate. Chemical Bulletin of Politehnica University of Timisoara. 2012;**57**(71):50-53

[33] Choi MS. Effects of tributyltin (TBT) on the expression of cytochrome P4501A, aryl hydrocarbon receptor and vitellogenin genes[Master's thesis]. Sunmoon University; 2012

[34] El Morhit M, Fekhaoui M, Elie P, Girard P, Yahyaoui A, El Abidi A, et al. Heavy metals in sediment, water and the European glass eel *Anguilla anguilla* (Osteichthyes: Anguillidae), from Loukkos River estuary (Morocco, eastern Atlantic). Cybium. 2009;**33**:219-228

[35] AESN (Agence de l'Eau Seine Normandie). Guide des profils de vulnérabilité des eaux de baignade. 2009. p. 84

[36] McElroy AE, Farrington JW, Teal JM. Bioavailability of polycyclic aromatic hydrocarbons in the aquatic environment. In: Varanasi U, editor. Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic

Environment. Boca Raton, Florida: CRC Press, Inc.; 1989. pp. 2-33

[37] Hinga KR, Pilson MEQ. Persistence of benz[a]anthracene degradation products in an enclosed marine ecosystem. Environmental Science & Technology. 1987;**21**:648-653

[38] Cerniglia CE, Heitkamp MA. Microbial degradation of polycyclic aromatic hydrocarbons (PAH) in the aquatic environment. In: Varanasi U, editor. Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. Boca Raton, FL: CRC Press; 1989. pp. 41-68

[39] Juhasz AL, Naidu R. Extraction and recovery of organochlorine pesticides from fungal mycelia. Journal of Microbiological Methods. 2000;**39**:149-158

[40] Bamforth SM, Singleton I. Bioremediation of polycyclic aromatic hydrocarbons: Current knowledge and future directions. Journal of Chemical Technology and Biotechnology. 2005;**80**(7):723-736

[41] Heitkamp MA, Franklin W, Cerniglia CE. Microbial metabolism of polycyclic aromatic hydrocarbons: Isolation and characterization of a pyrene-degrading bacterium. Applied and Environmental Microbiology. 1988;**54**:2549-2555

[42] Heitkamp MA, Cerniglia CE. Polycyclic aromatic hydrocarbon degradation by a *Mycobacterium* sp. in microcosms containing sediment and water from a pristine ecosystem. Applied and Environmental Microbiology. 1989;**55**:1968-1973

[43] Varanasi U, Stein JE, Nishimoto M. Biotransformation and disposition of polycyclic aromatic hydrocarbons in fish. In: Varanasi U, editor. Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. Boca Raton, FL: CRC Press; 1989. pp. 93-149

[44] James MO. Biotransformation and disposition of PAH in aquatic invertebrates. In: Varanasi U, editor. Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. Boca Raton, FL: CRC Press; 1989. pp. 70-88

[45] Roubal WT, Collier TK, Malins DC. Accumulation and metabolism of carbon-14 labeled benzene, naphthalene, and anthracene by young coho salmon (*Oncorhynchus kisutch*). Archives of Environmental Contamination and Toxicology. 1977;**5**:513-529

[46] Carls MG, Holland L, Larsen M, Collier TK, Scholz NL, Incardona JP. Fish embryos are damaged by dissolved PAHs, not oil particles. Aquatic Toxicology. 2008;**88**:121-127

[47] Pangrekar J, Kandaswami C, Kole P, Kumar S, Sikka HC. Comparative metabolism of benzo(a)pyrene, chrysene and phenanthrene by brown bullhead liver microsomes. Marine Environmental Research. 1995;**39**:51-55

[48] Sikka HC, Steward AR, Kandaswami C, Rutkowski JP, Zaleski J, Kumar S, et al. Metabolism of benzo(a) pyrene and persistence of DNA adducts in the brown bullhead (*Ictalurus nebulosus*). Comparative Biochemistry & Physiology. 1991;**100C**(1-2):25-28

[49] Gratz S, Mohrhaus A, Gamble B, Gracie J, Jackson D, Roetting J, et al. Screen for the presence of polycyclic aromatic hydrocarbons in select seafoods using LC-fluorescence. Laboratory Information Bulletin. 2010;**4475**:1-39

[50] Ylitalo GM, Krahn MM, Dickhoff WW, Stein JE, Walker CC, Lassitter CL, et al. Federal seafood safety response to the Deepwater Horizon oil spill. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(50):20274-20279

[51] Hansen D, Di Toro D, McGrath J, Swartz R, Mount D, Burgess R, editors. Procedures for the Derivation of Equilibrium Partitioning Sediment Benchmarks (ESBs) for the Protection of Benthic Organisms: PAH Mixtures. Narragansett, RI: Duluth, MN: Newport, Or: USEPA; 2003. p. 175

[52] Chikae M, Hatano Y, Ikeda R, Morita Y, Hasan Q, Tamiya E. Effects of bis(2-ethylhexyl) phthalate and benzo[a]pyrene on the embryos of Japanese medaka (*Oryzias latipes*). Environmental Toxicology and Pharmacology. 2004;**16**:141-145

[53] Van Brummelen TC, Van Gestel CAM, Verweij RA. Long-term toxicity of five polycyclic aromatic hydrocarbons for the terrestrial isopods *Oniscus Asellus* and *Porcellio Scaber*. Environmental Toxicology and Chemistry. 1996;**15**:1199-1210

[54] Wessel N, Rousseau S, Caisey X, Quiniou F, Akcha F. Investigating the relationship between embryotoxic and genotoxic effects of benzo[a] pyrene, 17[alpha]-ethinylestradiol and endosulfan on *Crassostrea gigas* embryos. Aquatic Toxicology. 2007;**85**:133-142

[55] Miranda CL, Chung WG, Wang-Buhler JL, Musafia-Jeknic T, Baird WM, Buhler DR. Comparative in vitro metabolism of benzo[a]pyrene by recombinant zebrafish CYP1A and liver microsomes from [beta] naphthoflavone-treated rainbow trout. Aquatic Toxicology. 2006;**80**:101-108

[56] Incardona JP, Carls MG, Day HL, Sloan CA, Bolton JL, Collier TK, et al. Cardiac arrhythmia is the primary response of embryonic Pacific herring (*Clupea pallasi*) exposed to crude oil during weathering. Environmental Science & Technology. 2009;**43**:201-207

[57] Hicken CE, Linbo TL, Baldwin DH, Willis ML, Myers MS, Holland L, et al. Sublethal exposure to crude oil

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[64] Diekmann M, Nagel R. Different survival rates in zebrafish (*Danio rerio*) from different origins. Journal of Applied Ichthyology.

[65] Djomo JE, Garrigues P, Narbonn JF. Uptake and depuration of polycyclic aromatic hydrocarbons from sediment by the zebrafish (*Brachydanio rerio*). Environmental Toxicology and Chemistry. 1996;**15**:1177-1181

[66] Frantzen M, Falk-Petersen IB, Nahrgang J, Smith TJ, Olsen GH, Hangstad TA, et al. Toxicity of crude oil and pyrene to the embryos of beach spawning capelin (*Mallotus villosus*). Aquatic Toxicology. 2012;**108**:42-52

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1999;**18**:494-503

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during embryonic development alters cardiac morphology and reduces aerobic capacity in adult fish. Proceedings of the National Academy of Sciences of the United States of America.

[58] Incardona JP, Collier TK, Scholz NL. Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons. Toxicology

[59] Kim SG, Park DK, Jang SW, Lee JS,

[60] Montverdi GH, Di Giulio RT. In vitro and in vivo association of 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene with the yolkprecursor protein vitellogenin. Environmental Toxicology.

2000;**19**(10):2502-2511. DOI: 10.1002/

[61] Patel MR, Scheffler BE, Wang L, Willett KL. Effects of benzo(a)

pyrene exposure on killifish (*Fundulus heteroclitus*) aromatase activities and mRNA. Aquatic Toxicology.

[62] Rocha Monteiro PR, Reis-Henriques MA, Coimbra J. Polycyclic aromatic hydrocarbons inhibit in vitro ovarian steroidogenesis in the flounder

(*Platichthys flesus* L.). Aquat. Toxicology.

[63] Matson CW, Timme-Laragy AR, Di Giulio RT. Fluoranthene, but not benzo[a]pyrene, interacts with hypoxia resulting in pericardial effusion and lordosis in developing zebrafish. Chemosphere. 2008;**74**:149-154

and Applied Pharmacology.

Kim SS, Chung MH. Effects of dietary benzo[a]pyrene on growth and hematological parameters in juvenile rockfish, Sebastes schlegeli (Hilgendorf). Bulletin of Environmental Contamination and Toxicology. 2008;**81**:470-474

2011;**108**:7086-7090

2004;**196**:191-205

etc.5620191016

2006;**77**(3):267-278

2000;**48**:549-559

*Polycyclic Aromatic Hydrocarbons (PAHs) and Their Influence to Some Aquatic Species DOI: http://dx.doi.org/10.5772/intechopen.86213*

during embryonic development alters cardiac morphology and reduces aerobic capacity in adult fish. Proceedings of the National Academy of Sciences of the United States of America. 2011;**108**:7086-7090

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

[51] Hansen D, Di Toro D, McGrath J, Swartz R, Mount D, Burgess R, editors. Procedures for the Derivation of Equilibrium Partitioning Sediment Benchmarks (ESBs) for the Protection of Benthic Organisms: PAH Mixtures. Narragansett, RI: Duluth, MN: Newport, Or: USEPA; 2003. p. 175

[52] Chikae M, Hatano Y, Ikeda R, Morita Y, Hasan Q, Tamiya E. Effects of bis(2-ethylhexyl) phthalate and benzo[a]pyrene on the embryos of Japanese medaka (*Oryzias latipes*). Environmental Toxicology and Pharmacology. 2004;**16**:141-145

[53] Van Brummelen TC, Van Gestel CAM, Verweij RA. Long-term toxicity of five polycyclic aromatic hydrocarbons for the terrestrial isopods *Oniscus Asellus* and *Porcellio Scaber*. Environmental Toxicology and

Chemistry. 1996;**15**:1199-1210

[54] Wessel N, Rousseau S, Caisey X, Quiniou F, Akcha F. Investigating the relationship between embryotoxic and genotoxic effects of benzo[a] pyrene, 17[alpha]-ethinylestradiol and endosulfan on *Crassostrea gigas* embryos. Aquatic Toxicology. 2007;**85**:133-142

[55] Miranda CL, Chung WG, Wang-Buhler JL, Musafia-Jeknic T, Baird WM, Buhler DR. Comparative in vitro

[56] Incardona JP, Carls MG, Day HL, Sloan CA, Bolton JL, Collier TK, et al. Cardiac arrhythmia is the primary response of embryonic Pacific herring (*Clupea pallasi*) exposed to crude oil during weathering. Environmental Science & Technology. 2009;**43**:201-207

[57] Hicken CE, Linbo TL, Baldwin DH, Willis ML, Myers MS, Holland L, et al. Sublethal exposure to crude oil

metabolism of benzo[a]pyrene by recombinant zebrafish CYP1A and liver microsomes from [beta] naphthoflavone-treated rainbow trout. Aquatic Toxicology. 2006;**80**:101-108

[44] James MO. Biotransformation and disposition of PAH in aquatic invertebrates. In: Varanasi U, editor. Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. Boca Raton, FL: CRC

[45] Roubal WT, Collier TK, Malins DC.

[46] Carls MG, Holland L, Larsen M, Collier TK, Scholz NL, Incardona JP. Fish embryos are damaged by dissolved

[47] Pangrekar J, Kandaswami C, Kole P, Kumar S, Sikka HC. Comparative metabolism of benzo(a)pyrene, chrysene and phenanthrene by brown bullhead liver microsomes. Marine Environmental Research. 1995;**39**:51-55

Kandaswami C, Rutkowski JP, Zaleski J, Kumar S, et al. Metabolism of benzo(a) pyrene and persistence of DNA adducts

PAHs, not oil particles. Aquatic Toxicology. 2008;**88**:121-127

[48] Sikka HC, Steward AR,

in the brown bullhead (*Ictalurus nebulosus*). Comparative Biochemistry & Physiology. 1991;**100C**(1-2):25-28

[49] Gratz S, Mohrhaus A, Gamble B, Gracie J, Jackson D, Roetting J, et al. Screen for the presence of polycyclic aromatic hydrocarbons in select seafoods using LC-fluorescence. Laboratory Information Bulletin.

[50] Ylitalo GM, Krahn MM, Dickhoff WW, Stein JE, Walker CC, Lassitter CL, et al. Federal seafood safety response to the Deepwater Horizon oil spill. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(50):20274-20279

Accumulation and metabolism of carbon-14 labeled benzene, naphthalene, and anthracene by young coho salmon (*Oncorhynchus kisutch*). Archives of Environmental Contamination and Toxicology.

Press; 1989. pp. 70-88

1977;**5**:513-529

**212**

2010;**4475**:1-39

[58] Incardona JP, Collier TK, Scholz NL. Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons. Toxicology and Applied Pharmacology. 2004;**196**:191-205

[59] Kim SG, Park DK, Jang SW, Lee JS, Kim SS, Chung MH. Effects of dietary benzo[a]pyrene on growth and hematological parameters in juvenile rockfish, Sebastes schlegeli (Hilgendorf). Bulletin of Environmental Contamination and Toxicology. 2008;**81**:470-474

[60] Montverdi GH, Di Giulio RT. In vitro and in vivo association of 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene with the yolkprecursor protein vitellogenin. Environmental Toxicology. 2000;**19**(10):2502-2511. DOI: 10.1002/ etc.5620191016

[61] Patel MR, Scheffler BE, Wang L, Willett KL. Effects of benzo(a) pyrene exposure on killifish (*Fundulus heteroclitus*) aromatase activities and mRNA. Aquatic Toxicology. 2006;**77**(3):267-278

[62] Rocha Monteiro PR, Reis-Henriques MA, Coimbra J. Polycyclic aromatic hydrocarbons inhibit in vitro ovarian steroidogenesis in the flounder (*Platichthys flesus* L.). Aquat. Toxicology. 2000;**48**:549-559

[63] Matson CW, Timme-Laragy AR, Di Giulio RT. Fluoranthene, but not benzo[a]pyrene, interacts with hypoxia resulting in pericardial effusion and lordosis in developing zebrafish. Chemosphere. 2008;**74**:149-154

[64] Diekmann M, Nagel R. Different survival rates in zebrafish (*Danio rerio*) from different origins. Journal of Applied Ichthyology. 2005;**21**:451-454

[65] Djomo JE, Garrigues P, Narbonn JF. Uptake and depuration of polycyclic aromatic hydrocarbons from sediment by the zebrafish (*Brachydanio rerio*). Environmental Toxicology and Chemistry. 1996;**15**:1177-1181

[66] Frantzen M, Falk-Petersen IB, Nahrgang J, Smith TJ, Olsen GH, Hangstad TA, et al. Toxicity of crude oil and pyrene to the embryos of beach spawning capelin (*Mallotus villosus*). Aquatic Toxicology. 2012;**108**:42-52

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*Biochemical Toxicology - Heavy Metals and Nanomaterials*

life stages. Part I: Adverse effects in rainbow trout. Environmental Science and Pollution Research. 2014;**21**:13720-13731. DOI: 10.1007/

[78] Sundberg H, Ishaq R, Åkerman G, Tjärnlund U, Zebühr Y, Linderoth M, et al. A bio-effect directed fractionation study for toxicological and chemical characterization of organic compounds in bottom sediment. Toxicological

[79] Gilliers C, Claireaux G, Galois R, Loizeau V, Le Pape O. Influence of hydrocarbons exposure on survival, growth and condition of juvenile flatfish: A mesocosm experiment. Journal of Life Sciences. 2012;**4**:113-122

[80] Gundersen DT, Kristanto SW, Curtis LR, Al-Yakoob SN, Metwally MM, Al-Ajmi D. Subacute toxicity of the water-soluble fractions of Kuwait crude oil and partially combusted crude oil on *Menidia beryllina* and *Palaemonetes pugio*. Archives of Environmental Contamination and Toxicology.

[81] Moles A, Rice SD. Effects of crude oil and naphthalene on growth, caloric content, and fat content of pink salmon juveniles in seawater. Transactions of the American Fisheries Society.

[82] Agamy E. Impact of laboratory exposure to light Arabian crude oil, dispersed oil and dispersant on the gills of the juvenile brown spotted grouper (*Epinephelus chlorostigma*):

[83] Ali S, Champagne DL, Richardson MK. Behavioral profiling of zebrafish embryos exposed to a panel of 60 watersoluble compounds. Behavioural Brain Research. 2012;**228**(2):272-283. DOI:

A histopathological study. Marine Environmental Research.

10.1016/j.bbr.2011.11.020

s11356-014-2804-0

Sciences. 2005;**84**:63-72

1996;**31**:1-8

1983;**112**:205-211

2013;**86**:46-55

Environmental Contamination and

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[72] Dong W, Wang L, Thornton C, Scheffler BE, Willett KL. Benzo(a) pyrene decreases brain and ovarian aromatase mRNA expression in *Fundulus heteroclitus*. Aquatic Toxicology. 2008;**88**:289-300

[73] Danion M, Deschamps MH, Thomas-Guyon H, Bado-Nilles A, Le Floch S, Quentel C, et al. Effect of an experimental oil spill on vertebral bone tissue quality in European sea bass (*Dicentrarchus labrax* L.). Ecotoxicology and Environmental

Safety. 2011;**74**:1888-1895

Research. 2012;**77**:30-34

[74] Shi X, He C, Zuo Z, Li R, Chen D, Chen R, et al. Pyrene exposure influences the craniofacial cartilage development of *Sebastiscus marmoratus* embryos. Marine Environmental

[75] Li R, Zuo Z, Chen D, He C, Chen R, Chen Y, et al. Inhibition by polycyclic aromatic hydrocarbons of ATPase activities in *Sebastiscus marmoratus* larvae: Relationship with the

development of early life stages. Marine Environmental Research. 2011;**71**:86-90

[76] Jee JH, Park KH, Keum YH, Kang JC. Effects of 7,12 dimethylbenz(a)

haematological parameters in Korean rockfish, *Sebastes schlegeli* (Hilgendorf). Aquaculture Research. 2006;**37**:431-442

[77] Le Bihanic F, Clérandeau C, Morin B, Cousin X, Cachot J. Developmental toxicity of PAH mixtures in fish early

anthracene on growth and

Toxicology. 2013;**90**:60-68

**214**

[85] Egan RJ, Bergner CL, Hart PC, Cachat JM, Canavello PR, Elegante MF, et al. Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behavioural Brain Research. 2009;**205**:38-44

[86] López-Patiño MA, Yu L, Cabral H, Zhdanova IV. Anxiogenic effects of cocaine withdrawal in zebrafish. Physiology & Behavior. 2008;**93**:160-171

[87] Sackerman J, Donegan J, Cunningham C, Nguyen N, Lawless K, Long A, et al. Zebrafish behavior in novel environments: Effects of acute exposure to anxiolytic compounds and choice of *Danio rerio* line. Journal of Comparative Psychology. 2010;**23**:43-61

[88] Correia AD, Goncalves R, Scholze M, Ferreira M, Reis-Henriques MA. Biochemical and behavioral responses in gilthead seabream (*Sparus aurata*) to phenanthrene. Journal of Experimental Marine Biology and Ecology. 2007;**347**:109-122

[89] Goncalves R, Scholze M, Ferreira AM, Martins M, Correia AD. The joint effect of polycyclic aromatic hydrocarbons on fish behavior. Environmental Research. 2008;**108**:205-213

[90] Farr AJ, Chabot CC, Taylor DH. Behavioral avoidance of fluoranthene by fathead minnows (*Pimephales promelas*). Neurotoxicology and Teratology. 1995;**17**:265-271

[91] Vignet C. Altération de la physiologie des poissons exposés à des hydrocarbures aromatiques polycycliques (HAP): Comportement et reproduction [thesis]. France: Université de La Rochelle; 2014

[92] Anderson MJ, Miller MR, Hinton DE. In vitro modulation of 17-b-estradiolinduced vitellogenin synthesis: Effects of cytochrome P4501A1 inducing compounds on rainbow trout (*Oncorhynchus mykiss*) liver cells. Aquatic Toxicology. 1996;**34**:327-350

[93] Holth TF, Nourizadeh-Lillabadi R, Blaesbjerg M, Grung M, Holbech H, Petersen GI, et al. Differential gene expression and biomarkers in zebrafish (*Danio rerio*) following exposure to produced water components. Aquatic Toxicology. 2008;**90**:277-291

[94] Pikkarainen A. Ethoxyresorufin-O-deethylase (EROD) activity and bile metabolites as contamination indicators in Baltic Sea perch: Determination by HPLC. Chemosphere. 2006;**65**:1888-1897

[95] Pj V, Keinänen M, Vuontisjärvi H, Barsiene J, Broeg K, Förlin L, et al. Use of biliary PAH metabolites as a biomarker of pollution in fish from the Baltic Sea. Marine Pollution Bulletin. 2006;**53**:479-487

[96] Kammann U, Lang T, Vobach M, Wosniok W. Ethoxyresorufin-Odeethylase (EROD) activity in dab (*Limanda limanda*) as biomarker for marine monitoring. Environmental Science and Pollution Research. 2005;**12**:140-145

[97] Harman C, Thomas K, Tollefsen KE, Meier S, Bøyum O, Grung M. Monitoring the freely dissolved concentrations of polycyclic aromatic hydrocarbons (PAH) and alkylphenols (AP) around a Norwegian oil platform by holistic passive sampling. Marine Pollution Bulletin. 2009;**58**:1671-1679

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[111] Penning TM, Burczynski ME, Hung CF, McCoull KD, Palackal NT, Tsuruda L. Dihydrodiol dehydrogenases and polycyclic aromatic hydrocarbon activation: Generation of reactive and redox active O-quinones. Chemical Research in Toxicology. 1999;**12**:1-18

[112] Hisano T, Hayase T. Countermeasures against water pollution in enclosed coastal seas in Japan. Marine Pollution Bulletin. 1991;**23**:479-484

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[114] Van Schooten FJ, Maas LM, Moonen EJC, Kleinjans JCS, van der Oost R. DNA dosimetry in biological indicator species living on PAHcontaminated soils and sediments. Ecotoxicology and Environmental Safety. 1995;**30**(2):171-179

*Biochemical Toxicology - Heavy Metals and Nanomaterials*

of PAH exposure. Aquatic Toxicology.

[106] Zhou JL, Fileman TW, Evans S, Donkin P, Llewellyn C, Readman JW, et al. Fluoranthene and pyrene in the suspended particulate matter and surface sediments of the Humber Estuary, UK. Marine Pollution Bulletin.

[107] Lin ELC, Cormier SM, Racine RN. Synchronous fluorometric measurement of metabolites of

Toxicology and Chemistry.

Publishers; 1979. pp. 7-43

Perspectives. 1985;**64**:69-84

1989;**188**(1):561-566

[112] Hisano T, Hayase T. Countermeasures against water pollution in enclosed coastal seas in Japan. Marine Pollution Bulletin.

1991;**23**:479-484

Charles; 1978. 192 p

polycyclic aromatic hydrocarbons in the bile of brown bullhead. Environmental

[108] Neff JM. Sources of PAH in the aquatic environment. In: Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. London: Applied Science

[109] Cavalieri E, Rogan E. Role of radical cations in aromatic hydrocarbon carcinogenesis. Environmental Health

[110] Johnston EP, Baumann PC. Analysis of fish bile with HPLC fluorescence to determine environmental exposure to benzo(a)pyrene. Hydrobiologia.

[111] Penning TM, Burczynski ME, Hung CF, McCoull KD, Palackal NT, Tsuruda L. Dihydrodiol dehydrogenases and polycyclic aromatic hydrocarbon activation: Generation of reactive and redox active O-quinones. Chemical Research in Toxicology. 1999;**12**:1-18

[113] Moriarty C. Eels: A Natural and Unnatural History. Londres: David &

1993;**26**:273-286

1998;**36**:587-597

1994;**13**:707-715

[98] Zm T, Amb G, Hansen R, Andersen O. 1-Hydroxypyrene as a biomarker of PAH exposure in the marine polychaete Nereis diversicolor. Marine Environmental Research.

[99] McMaster ME, Van Der Kraak GJ, Munkittrick KR. An epidemiological evaluation of the biochemical basis for steroid hormonal depressions in fish exposed to industrial wastes. Journal of Great Lakes Research.

[100] Van der Oost R, Beyer J, Vermeulen

NPE. Fish bioaccumulation and biomarkers in environmental risk assessment: A review. Environmental Toxicology and Pharmacology.

[101] Pointet K, Milliet A. PAHs analysis of fish whole gall bladders and livers from the Natural Reserve of Camargue by GCy MS. Chemosphere.

[102] Eggens ML, Opperhuizen A, Boon JP. Temporal variation of

CYP1A indices, PCB and 1-OH pyrene concentration in flounder, *Platichthys flesus*, from the Dutch Wadden Sea. Chemosphere. 1996;**33**:1579-1596

[103] Hylland K, Sandvik M, Skasre JU, Beyer J, Egaas E, Goksøyr A. Biomarkers in flounder (*Platichthys flesus*): An evaluation of their use in pollution monitoring. Marine Environmental

[104] Ruddock PJ, Bird DJ, McCalley DV. Bile metabolites of PAHs in three species of fish from the Severn Estuary. Ecotoxicology and Environmental

Research. 1996;**42**:223-227

Safety. 2002;**51**:97-105

[105] Ariese F, Kok SJ, Verkaik M, Gooijer C, Velthorst NH, Hofstraat JW. Synchronous fluorescence spectrometry of fish bile: A rapid screening method for the biomonitoring

2009;**67**:38-46

1996;**22**:153-171

2003;**13**:57-149

2000;**40**:293-299

**216**

[115] Wheeler A. The eels. The Fishes of the British Isles and North-West Europe. London: Macmillan & Co.; 1969. pp. 223-230

[116] Sandvik M, Horsberg TE, Skaare JU, Ingebrigtsen K. Comparison of dietary and waterborne exposure to benzo[a]pyrene: Bioavailability, tissue disposition and CYP1A1 induction in rainbow trout (*Oncorhynchus mykiss*). Biomarkers. 1998;**3**:399-410

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[118] Kammann U, Brinkmann M, Freese M, Pohlmann JD, Stoffels S, Hollert H, et al. PAH metabolites, GST and EROD in European eel (*Anguilla anguilla*) as possible indicators for eel habitat quality in German rivers. Environmental Science and Pollution Research. 2014;**1**:2519-2530

[119] Ruddock PJ, Bird DJ, McEvoy J, Peters LD. Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in European eels *Anguilla anguilla* from United Kingdom estuaries. Science of the Total Environment. 2003;**301**:105-117

[120] Wariaghli F, Kammann U, Hanel R, Yahyaoui A. PAH metabolites in bile of European Eel (*Anguilla anguilla*) from Morocco. Bulletin of Environmental Contamination and Toxicology. 2015;**95**(6):740-744

### *Edited by Muharrem Ince, Olcay Kaplan Ince and Gabrijel Ondrasek*

*Biochemical Toxicology - Heavy Metals and Nanomaterials* provides an overview of biochemical contamination, nanomaterials and toxic metals, and measurement techniques. It explains and clarifies important studies and compares and develops new and groundbreaking measurement techniques in the fields of organic and inorganic pollution and nanoscience. It is highly recommended for professionals and readers interested in the environment and human health.

Published in London, UK © 2020 IntechOpen © e\_zebolov / iStock

Biochemical Toxicology - Heavy Metals and Nanomaterials

Biochemical Toxicology

Heavy Metals and Nanomaterials

*Edited by Muharrem Ince,* 

*Olcay Kaplan Ince and Gabrijel Ondrasek*