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## **Meet the editors**

Sonia Soloneski is PhD in Natural Sciences and Professor Assistant of Molecular Cell Biology at the Faculty of Natural Sciences and Museum of La Plata, National University of La Plata, Argentina. She became a member of the National Scientific and Technological Research Council (CONICET) of Argentina in Genetic Toxicology field. Presently, she is a member of the Latin American

Association of Environmental Mutagenesis, Teratogenesis and Carcinogenesis (ALAMCTA), the Argentinean Society of Toxicology (ATA) and the Argentinean Society of Genetics (SAG). She has authored more than 300 scientific publications in the field, including scientific publications in research papers, reviewed journals and conferences worldwide. She is a referent for issues related to Genetic Toxicology, Mutagenesis and Ecotoxicology field.

Marcelo L. Larramendy, PhD, serves as Professor of Molecular Cell Biology at the School of Natural Sciences and Museum (National University of La Plata, Argentina). He is appointed as Senior Researcher of the National Scientific and Technological Research Council of Argentina. He is also a former member of the Executive Committee of the Latin American Association of Envi-

ronmental Mutagenesis, Teratogenesis and Carcinogenesis. He is author of more than 470 contributions, including scientific publications, research communications, and conferences worldwide. He has received several national and international awards. Prof. Larramendy is a regular lecturer at the international A. Hollaender Courses organized by the International Association of Environmental Mutagenesis Societies and former guest scientist at National Institutes of Health (USA) and University of Helsinki (Finland). He is expert in Genetic Toxicology and has been referee for more than 30 international scientific journals. He was a member of the International Panel of Experts at the International Agency for Research on Cancer (IARC, WHO, Lyon, France) in 2015 for the evaluation of DDT, 2,4- D and Lindane. Presently, Prof. Dr. Larramendy is Head of the Laboratory of Molecular Cytogenetics and Genotoxicology at the UNLP.

## Contents

## **Preface XI**


Maite R. Arana, Guillermo N. Tocchetti, Juan P. Rigalli, Aldo D. Mottino, Fabiana García and Silvina S.M. Villanueva


Chapter 8 **A Review of Cyanogenic Glycosides in Edible Plants 179** Islamiyat Folashade Bolarinwa, Moruf Olanrewaju Oke, Sulaiman Adebisi Olaniyan and Adeladun Stephen Ajala

## Chapter 9 **Toxic Effects as a Result of Herbal Medicine Intake 193** Nudrat Fatima and Naira Nayeem

## Preface

Chapter 8 **A Review of Cyanogenic Glycosides in Edible Plants 179**

Adebisi Olaniyan and Adeladun Stephen Ajala

Chapter 9 **Toxic Effects as a Result of Herbal Medicine Intake 193**

Nudrat Fatima and Naira Nayeem

**VI** Contents

Islamiyat Folashade Bolarinwa, Moruf Olanrewaju Oke, Sulaiman

Throughout the ages, toxicological science has provided information that has shaped and guided human society. Toxicology, as a distinct scientific discipline, is fairly modern. Howev‐ er, knowledge of poisons and poisoning incidents date back to ancient times. Undertakings as diverse as the healing arts, the industrial military, the agriculture, and the regulation/policy making all have their groundings in the modern toxicology. Toxicology, the science of poisons or the study of the untoward effects of chemicals or physical agents and complex biotoxins on biological systems, has moved over the centuries from the art of food gathering and murder to a highly sophisticated science of mechanisms. The identification and use of toxic substances have occurred throughout our recorded history, and every day, new poisons and toxins are discovered. Humanity has always had the desire to assure its health and safety, but drawing conclusions about harmful chemicals has required both knowledge and learning. Initially, this was done by a trial-and-error system, where substances were tested to see which were safe and which were more hazardous. Then, toxicology advanced significantly throughout the middle ages with an understanding of the relationship between working diseases and expo‐ sure to many toxins and historically, this discipline formed the basis of therapeutics and ex‐ perimental medicine. Paracelsus, a Swiss German physician and alchemist who pioneered the use of chemicals and minerals in medicine and is best known for articulating the concept that *"All things are poison and nothing (is) without poison; only the dose makes that a thing is no poison,"* is today considered the "father" of toxicology. The field of toxicology has come a long way in the centuries since Paracelsus. More than time, research toxicologists have studied the mecha‐ nisms of toxicity of a vast range of both naturally occurring and synthetic xenobiotics. More recently, toxicologists have embraced "green chemistry" as the design of chemical and prod‐ ucts with properties that minimize or eliminate the negative impacts on humans and on the environment. Modern toxicology has tried to move away from the traditional approach of ani‐ mal testing and toward a harm-free route of experimentation. Methods have been refined, more multicenter protocols have been performed, and international analysis and exchange of information have increased considerably. The increasing collection and evaluation of biomark‐ ers of exposure and effects is also providing growing opportunities for toxicological science. Actually, the science of toxicology has embraced research in other areas, including genomics, bioinformatics, and more recently, nanotoxicology, which is emerging as a new research disci‐ pline. Although live organisms have been used to assess and evaluate potentially toxic prod‐ ucts since the eighteenth century, new methodologies continue to be explored, and for some toxic biomarkers, there are no alternatives to animal testing. The ability to detect potential con‐ taminants in water, air and soil can help us identify emerging health threats to living organ‐ isms and ecosystems generated by climate change, determine future food sufficiency, and assess the danger of weapons of mass destruction, among others. Rapid detection of potential‐ ly harmful physical or chemical xenobiotics is essential for protecting biota in general and hu‐ man beings in particular.

Although a vast literature is available on toxicology, this book contains solid and important investigations into the diverse chemical hazards encountered in both anthropogenic and natu‐ ral environments and provides valuable information about the toxicity of several xenobiotics that can negatively influence the health of humans and ecosystems.

This single volume comprises nine high-quality chapters describing several issues related to the field of toxicology. The first chapter comprises an excellent review, starting with a complete appraisal of the function and structure of the skin, outlining characteristics of cutaneous toxici‐ ty, including contact dermatitis, photosensitivity, contact urticaria, chemical-induced acne, pig‐ mentary disturbance, drug rash, hair disturbance, and nail disturbance. This is followed by a second chapter providing information from *in vitro* and *in vivo* studies on the role of hepatic and intestinal multidrug resistance–associated protein 2 in detoxification processes, as well as on their regulation by xenobiotics, at the transcriptional and post-transcriptional levels. The third chapter presents an update describing the chemical and physical properties of nanoparticles and their characterization for proper toxicological evaluation related to exposure, environmen‐ tal fate and transport, and ecological and genotoxic effects, with a focus on the risk of these in aquatic environment biota. The fourth chapter provides information about embryotoxic and teratogenic effects induced by maltodextrin-coated cadmium sulfide quantum dots in chicken embryos and fetuses. The fifth chapter provides an overview of applications of zinc oxide nano‐ particles followed by a brief outline of methods of synthesis and characterization and the cur‐ rent knowledge on zinc oxide nanoparticle interactions with fish and their potential implications for human health. The sixth chapter depicts the impacts of microplastics in aquatic environments and their toxicological implications for fish populations. The seventh chapter is an interesting overview of several types of ecotoxicological biomarkers, presenting a set of ex‐ amples about employed species, advantages and disadvantages of different types of toxicity testing, and the use of exposed natural ecosystems or man-made analogue systems as the more environmentally realistic approach for ecotoxicological testing. The eighth chapter presents a review about cyanogenic glycosides in specific plant foods, the health implications of consum‐ ing cyanogenic plants, and the effects of various processing methods on cyanogenic glycosides, with updated information gathered from the published reports on cyanogenic glycosides. The book ends with a chapter describing the possible toxicities of herbal medicines and their differ‐ ent alternatives, creating a sense of awareness in people to avoid the use of herbal medicines without prescription and to motivate different underdeveloped and developing countries to promptly make regulations for herbal medicines.

The editors of *Toxicology - New Aspects to This Scientific Conundrum* are enormously grateful to all the contributing authors for sharing their knowledge and insight into this book project. They have made an extensive effort to arrange the information included in every chapter. This book is designed to provide an overview on the different toxicants and their effects on living organisms, including humans. The publication of this book is of high importance for those re‐ searchers, scientists, engineers, geneticists, pharmacologists, physicians, and veterinarians, as well as teachers and advanced-level students, who make use of these different investigations to understand both basic and applied toxic aspects of known and new xenobiotics and to guide them in the future investigations.

#### **Sonia Soloneski, PhD, and Marcelo L. Larramendy, PhD**

School of Natural Sciences and Museum National University of La Plata La Plata, Argentina

#### **Chapter 1 Provisional chapter**

#### **Drug-Induced Cutaneous Toxicity Drug-Induced Cutaneous Toxicity**

Katsuhiko Yoshizawa, Michiko Yuki and Katsuhiko Yoshizawa, Michiko Yuki and

Airo Tsubura Airo Tsubura

ral environments and provides valuable information about the toxicity of several xenobiotics

This single volume comprises nine high-quality chapters describing several issues related to the field of toxicology. The first chapter comprises an excellent review, starting with a complete appraisal of the function and structure of the skin, outlining characteristics of cutaneous toxici‐ ty, including contact dermatitis, photosensitivity, contact urticaria, chemical-induced acne, pig‐ mentary disturbance, drug rash, hair disturbance, and nail disturbance. This is followed by a second chapter providing information from *in vitro* and *in vivo* studies on the role of hepatic and intestinal multidrug resistance–associated protein 2 in detoxification processes, as well as on their regulation by xenobiotics, at the transcriptional and post-transcriptional levels. The third chapter presents an update describing the chemical and physical properties of nanoparticles and their characterization for proper toxicological evaluation related to exposure, environmen‐ tal fate and transport, and ecological and genotoxic effects, with a focus on the risk of these in aquatic environment biota. The fourth chapter provides information about embryotoxic and teratogenic effects induced by maltodextrin-coated cadmium sulfide quantum dots in chicken embryos and fetuses. The fifth chapter provides an overview of applications of zinc oxide nano‐ particles followed by a brief outline of methods of synthesis and characterization and the cur‐ rent knowledge on zinc oxide nanoparticle interactions with fish and their potential implications for human health. The sixth chapter depicts the impacts of microplastics in aquatic environments and their toxicological implications for fish populations. The seventh chapter is an interesting overview of several types of ecotoxicological biomarkers, presenting a set of ex‐ amples about employed species, advantages and disadvantages of different types of toxicity testing, and the use of exposed natural ecosystems or man-made analogue systems as the more environmentally realistic approach for ecotoxicological testing. The eighth chapter presents a review about cyanogenic glycosides in specific plant foods, the health implications of consum‐ ing cyanogenic plants, and the effects of various processing methods on cyanogenic glycosides, with updated information gathered from the published reports on cyanogenic glycosides. The book ends with a chapter describing the possible toxicities of herbal medicines and their differ‐ ent alternatives, creating a sense of awareness in people to avoid the use of herbal medicines without prescription and to motivate different underdeveloped and developing countries to

The editors of *Toxicology - New Aspects to This Scientific Conundrum* are enormously grateful to all the contributing authors for sharing their knowledge and insight into this book project. They have made an extensive effort to arrange the information included in every chapter. This book is designed to provide an overview on the different toxicants and their effects on living organisms, including humans. The publication of this book is of high importance for those re‐ searchers, scientists, engineers, geneticists, pharmacologists, physicians, and veterinarians, as well as teachers and advanced-level students, who make use of these different investigations to understand both basic and applied toxic aspects of known and new xenobiotics and to

**Sonia Soloneski, PhD, and Marcelo L. Larramendy, PhD**

School of Natural Sciences and Museum

National University of La Plata

La Plata, Argentina

that can negatively influence the health of humans and ecosystems.

VIII Preface

promptly make regulations for herbal medicines.

guide them in the future investigations.

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

http://dx.doi.org/10.5772/64473

#### **Abstract**

The skin is the largest organ in the body and is continually exposed to external stimuli, such as chemical and environmental substances. Cutaneous toxicity can be broadly classified according to the mechanism of onset, namely: contact dermatitis, i.e., damage resulting from contact with a substance (irritant dermatitis, allergic contact dermatitis, chemical burns); photosensitivity, i.e., caused by combined effects of a substance and ultraviolet light (phototoxic dermatitis, photoallergic contact dermatitis); contact urticaria; chemical-induced acne; pigmentary disturbance; drug rash; hair disturbance; nail disturbance; or tumor-induced. This review outlines the function and structure of the skin, outlining characteristics of these types of cutaneous toxicity. In recent years, advances have been made in the development of pharmaceutical products targeting specific molecules or genes and nanotechnology-based pharmaceutical products, raising concerns about the onset of toxicity by novel mechanisms involving new pharmaceutical products. Therefore, it is important to understand the basic toxicity-related changes described herein.

**Keywords:** cutaneous toxicity, drugs, chemicals, toxicity studies

## **1. Introduction**

Cutaneous adverse drug or chemical reactions in patients are not common. Among hospitalized patients, the incidence of adverse drug reactions concerning the skin ranges from 1% to 3%; however, the actual prevalence is much higher, as many mild forms of cutaneous adverse reactions are not reported [1]. We are constantly exposed to external stimuli, such as chemical and environmental substances, resulting in various skin symptoms. This article focuses on (1)

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

the function and structure of the skin and (2) characteristics of cutaneous toxicity of pharmaceutical products and chemical substances in humans and animals.

## **2. Function and structure of the skin**

Skin is the largest organ of the body, covering the surface and accounting for approximately 15–20% of body mass. In addition to its constant barrierrole, protecting the living body against external stimuli, skin is important for maintaining the body health (e.g., through regulation of body temperature, storage of fluids and electrolytes, and the synthesis of vitamin D) and also acts as an important component of the immune system. As a sensory organ, the skin can sense pain, touch, pressure, and temperature [2]. Histologically, the skin consists of the epidermis, dermis, and subcutaneous tissue. The epidermis is formed by keratinized squamous epithelia, stratified from the surface into the cornified layer, clear layer (only on the palms and soles), granular layer, spinous layer, and basal layer [3]. The epidermis also contains antigenpresenting Langerhans cells (mainly in the spinous layer); melanocytes (mainly in the basal layer), which produce melanin to protect epidermal cells against damage induced by ultraviolet light; and Merkel cells (mainly on the palms and soles), which are neuroendocrine cells. The dermis is composed of fibrous connective tissue with elastic and reticular fibers intermingled with collagen bundles, containing mast cells that are involved in allergic reactions, sweat glands, sebaceous glands, hair follicles, blood vessels, lymphatic vessels, and nerve fibers. Subcutaneous tissue is composed of loose connective tissue and subcutaneous adipose tissue. Adipose tissue is especially prominent in the footpads where it functions as a "shock absorber" and as an insulating layer [4]. Skin appendages are skin-associated structures that serve a particular function, including sensation, contractility, lubrication, and heat loss. They contain hairs (sensation, heat loss, filter for breathing, protection), sebaceous glands (secrete sebum onto hair follicles, which oils the hair), sweat glands (can produce sweat secreted with strong odor(apocrine) or with a faint odor(eccrine), and nails (protection). Hair growth occurs in three stages: anagen (growth phase), catagen (involution period), and telogen (resting phase during which hair shedding occurs) [5, 6]. The rate of hair growth and duration of the growth cycle vary in different areas of the body and are influenced by sex hormones and growth factors. Sebaceous glands are most often associated with hair follicles and produce sebum by holocrine secretion. Zymbal's gland is a specialized sebaceous gland in rodents located at the base of the external ear canal; the gland cells contain cytochrome P450 isoenzyme and peroxidases and are capable of chemical metabolism [7, 8]. Apocrine glands are distributed throughoutthe skin of mostlaboratory animals, whereas in humans they are located in axillary, pubic, and perianal areas, while they are only present in the plantar areas in rodents. There are a number of specialized apocrine glands, such as the anal sac gland of dogs, the ceruminous glands of the external ear canal, and the glands of Moll in the eyelids. Eccrine glands are found throughout the body in humans; however, these glands are limited to the footpads of carnivores and rodents.

In preclinical studies, cutaneous toxicity is rarely encountered, except in cutaneous application, intradermal administration, and subcutaneous administration. Cutaneous toxicity primarily involves either a direct local inflammatory reaction to the drug without involvement of an immunological mechanism or an indirect inflammatory reaction associated with a systemic manifestation [6]. In cutaneous application studies, both epidermis and skin appendages are important factors in transdermal drug absorption [9]. Experimental animals such as guinea pigs, monkeys, and swine exhibit similar absorption characteristics to humans [5]. Of all laboratory animals, swine skin is most structurally comparable to human skin [4]. Swine and humans have comparable stratum corneum, epidermal thickness, and hair follicle density, as well as similar chemical composition of the stratum corneum. Rodents have much thinner skin (especially the epidermis) with greater permeation compared to humans [4]. In general, skin is thicker over the dorsal and lateral surfaces and thinnest on the ventral and medial surfaces. Areas of skin that contact the ground, such as footpad and heels, have the thickest epidermis (**Figure 1**). The extent of transdermal drug absorption differs according to skin location. Sites in order of favorable absorption, due to the skin thickness, are the abdomen, forehead, palms, and soles of feet [5]. It should be noted that skin thickness varies considerably during the hair cycle (**Figure 2**). Skin thickness during the anagen stage is thickest and is thinnest during the catagen stage in rodents and rabbits. If the skin is damaged, the biological protective barrier function decreases, leading to a significant increase in drug absorption, which results in intensified systemic toxicity [10]. Microsomal enzymes in keratinocytes are capable of metabolizing topically applied chemicals, thus rendering them inactive or active. Dimethylbenz(a)anthracene (DMBA) becomes a potent skin carcinogen after metabolic activation by keratinocytes [11].

the function and structure of the skin and (2) characteristics of cutaneous toxicity of pharma-

Skin is the largest organ of the body, covering the surface and accounting for approximately 15–20% of body mass. In addition to its constant barrierrole, protecting the living body against external stimuli, skin is important for maintaining the body health (e.g., through regulation of body temperature, storage of fluids and electrolytes, and the synthesis of vitamin D) and also acts as an important component of the immune system. As a sensory organ, the skin can sense pain, touch, pressure, and temperature [2]. Histologically, the skin consists of the epidermis, dermis, and subcutaneous tissue. The epidermis is formed by keratinized squamous epithelia, stratified from the surface into the cornified layer, clear layer (only on the palms and soles), granular layer, spinous layer, and basal layer [3]. The epidermis also contains antigenpresenting Langerhans cells (mainly in the spinous layer); melanocytes (mainly in the basal layer), which produce melanin to protect epidermal cells against damage induced by ultraviolet light; and Merkel cells (mainly on the palms and soles), which are neuroendocrine cells. The dermis is composed of fibrous connective tissue with elastic and reticular fibers intermingled with collagen bundles, containing mast cells that are involved in allergic reactions, sweat glands, sebaceous glands, hair follicles, blood vessels, lymphatic vessels, and nerve fibers. Subcutaneous tissue is composed of loose connective tissue and subcutaneous adipose tissue. Adipose tissue is especially prominent in the footpads where it functions as a "shock absorber" and as an insulating layer [4]. Skin appendages are skin-associated structures that serve a particular function, including sensation, contractility, lubrication, and heat loss. They contain hairs (sensation, heat loss, filter for breathing, protection), sebaceous glands (secrete sebum onto hair follicles, which oils the hair), sweat glands (can produce sweat secreted with strong odor(apocrine) or with a faint odor(eccrine), and nails (protection). Hair growth occurs in three stages: anagen (growth phase), catagen (involution period), and telogen (resting phase during which hair shedding occurs) [5, 6]. The rate of hair growth and duration of the growth cycle vary in different areas of the body and are influenced by sex hormones and growth factors. Sebaceous glands are most often associated with hair follicles and produce sebum by holocrine secretion. Zymbal's gland is a specialized sebaceous gland in rodents located at the base of the external ear canal; the gland cells contain cytochrome P450 isoenzyme and peroxidases and are capable of chemical metabolism [7, 8]. Apocrine glands are distributed throughoutthe skin of mostlaboratory animals, whereas in humans they are located in axillary, pubic, and perianal areas, while they are only present in the plantar areas in rodents. There are a number of specialized apocrine glands, such as the anal sac gland of dogs, the ceruminous glands of the external ear canal, and the glands of Moll in the eyelids. Eccrine glands are found throughout the body in humans; however, these glands are limited to the footpads of

In preclinical studies, cutaneous toxicity is rarely encountered, except in cutaneous application, intradermal administration, and subcutaneous administration. Cutaneous toxicity primarily

ceutical products and chemical substances in humans and animals.

**2. Function and structure of the skin**

2 Toxicology - New Aspects to This Scientific Conundrum

carnivores and rodents.

**Figure 1.** Comparative histology of different skin locations in rats. (a) Scalp region at the vertex. (b) Nose region. (c) Inguinal region. (d) Back region. (e) Abdominal region. (f) Footpad region. (g) Eccrine sweat gland in the footpad. Note that scalp, inguinal, and abdominal skin are thin. In contrast, back and footpad skin are thicker. Footpad skin is the thickest, especially the stratum corneum and epidermis. Eccrine sweat glands are located only in the footpad of rodents; however, these glands are found throughout the human body.

**Figure 2.** Comparative histology of back skin during different hair cycle stages. (a) Anagen stage in the mouse. (b) Catagen stage in the mouse. (c) Anagen stage in the rat. (d) Catagen stage in the rat. (e) Anagen stage in the rabbit. (f) Catagen stage in the rabbit. Note that skin thickness is thickest at the anagen stage and thinnest at the catagen stage in rodents.

## **3. Types of cutaneous toxicity (Table 1)**

Cutaneous toxicity can be classified according to the mechanism of onset into the following: (1) contactdermatitis,i.e.,damageresultingfromcontactoftheskinwithadrug(irritantdermatitis, allergic contact dermatitis, and chemical burns); (2) photosensitivity, caused by the combined effect of a chemical substance and ultraviolet light (phototoxic dermatitis and photoallergic contactdermatitis);(3) contacturticaria;(4) chemical-inducedacne;(5)pigmentarydisturbance; (6)drugrash;(7)hairdisturbance;(8)naildisturbance;and(9)tumor-induced.Cutaneoustoxicity canalsobe classifiedaccordingtothe routeof exposure,i.e., eitherdue tosystemic effectsorlocal irritation of the skin (local toxicity) [5, 6].

#### **3.1. Contact dermatitis**

Contact dermatitis is skin inflammation occurring as a result of direct contact of the skin with a drug that can be classified into the following three types, according to the mechanism of onset.

## *3.1.1. Irritant dermatitis*

Irritant dermatitis is an inflammatory change caused by direct irritation of the skin that can be either acute or cumulative. Activation of mast cells, complement or prostaglandin synthesis results in reversible damage to the skin, observed as irritation within 4 hours following topical applicationofthechemicals.Irritantdermatitisischaracterizedbyinflammatorycellinfiltration, acanthosis,epidermalhyperkeratosis,andhyperplasiaassociatedwithotherepidermalchanges such as erosion/ulcer, necrosis, or vesicle formation [11]. Irritant dermatitis depends on the severity of the irritants and duration of their exposures [5, 6] (**Figure 3**). If the damage to the skin is irreversible, the lesion is clinically referred to as corrosion, which is characterized by full thickness necrosis of the epidermis penetrating into the underlying dermis [11]. In preclinical studies of topical application agents, skin irritation testing is conducted using rabbits or guinea pigs to evaluate drug-induced irritation using the Draize method (with a 5-grade score based on macroscopicassessmentoftheseverityoferythema,crusting,andedema)(**Figure4**and**Table2**). The Draize test consists of application of the chemical to the test site on shaved dorsal skin. The test sites undergo gross evaluation at 6, 24, and 72 postapplication.


**Table 1.** Classification of drug-induced cutaneous toxicity.

**Figure 2.** Comparative histology of back skin during different hair cycle stages. (a) Anagen stage in the mouse. (b) Catagen stage in the mouse. (c) Anagen stage in the rat. (d) Catagen stage in the rat. (e) Anagen stage in the rabbit. (f) Catagen stage in the rabbit. Note that skin thickness is thickest at the anagen stage and thinnest at the catagen stage in

Cutaneous toxicity can be classified according to the mechanism of onset into the following: (1) contactdermatitis,i.e.,damageresultingfromcontactoftheskinwithadrug(irritantdermatitis, allergic contact dermatitis, and chemical burns); (2) photosensitivity, caused by the combined effect of a chemical substance and ultraviolet light (phototoxic dermatitis and photoallergic contactdermatitis);(3) contacturticaria;(4) chemical-inducedacne;(5)pigmentarydisturbance; (6)drugrash;(7)hairdisturbance;(8)naildisturbance;and(9)tumor-induced.Cutaneoustoxicity canalsobe classifiedaccordingtothe routeof exposure,i.e., eitherdue tosystemic effectsorlocal

Contact dermatitis is skin inflammation occurring as a result of direct contact of the skin with a drug that can be classified into the following three types, according to the mechanism of onset.

Irritant dermatitis is an inflammatory change caused by direct irritation of the skin that can be either acute or cumulative. Activation of mast cells, complement or prostaglandin synthesis results in reversible damage to the skin, observed as irritation within 4 hours following topical applicationofthechemicals.Irritantdermatitisischaracterizedbyinflammatorycellinfiltration, acanthosis,epidermalhyperkeratosis,andhyperplasiaassociatedwithotherepidermalchanges such as erosion/ulcer, necrosis, or vesicle formation [11]. Irritant dermatitis depends on the severity of the irritants and duration of their exposures [5, 6] (**Figure 3**). If the damage to the skin is irreversible, the lesion is clinically referred to as corrosion, which is characterized by full thickness necrosis of the epidermis penetrating into the underlying dermis [11]. In preclinical

rodents.

**3. Types of cutaneous toxicity (Table 1)**

4 Toxicology - New Aspects to This Scientific Conundrum

irritation of the skin (local toxicity) [5, 6].

**3.1. Contact dermatitis**

*3.1.1. Irritant dermatitis*

**Figure 3.** Sequential stages of inflammatory changes in irritant contact dermatitis following a single exposure to sodium lauryl sulfate (SLS) in the guinea pig. (a) Epidermal necrosis and slight infiltration of neutrophils in dermis are observed 24 hours after exposure. (b) Epidermal necrosis and severe infiltration of neutrophils in epidermis and dermis are observed 24 hours after exposure. (c) Epidermal abscess (pustule) and acanthosis (epidermal regeneration) are observed 48 hours after exposure. Owing to its emulsifying properties, SLS is an anionic surfactant used in many hygienic and cleaning products, including shampoos, toothpastes, and shaving foams.

**Figure 4.** Macroscopic photos from a cumulative dermal irritation study in animals. (a) After the hair on the back of the rat is shaved, the drug is continually applied to the same area. The rat wears the Elizabethan collar to prevent the animal from biting or licking the exposure site. (b) Cumulative dermal irritation study in a rabbit (left: vehicle application, right: drug application). The site of drug application is observed with erythema, redness, swelling, and moistness. The change spreads beyond the site of application, indicating a strong irritant property of the drug.


This table has been modified from [4].

**Table 2.** Skin irritation test (Draize scale).

#### *3.1.2. Allergic contact dermatitis*

**Figure 3.** Sequential stages of inflammatory changes in irritant contact dermatitis following a single exposure to sodium lauryl sulfate (SLS) in the guinea pig. (a) Epidermal necrosis and slight infiltration of neutrophils in dermis are observed 24 hours after exposure. (b) Epidermal necrosis and severe infiltration of neutrophils in epidermis and dermis are observed 24 hours after exposure. (c) Epidermal abscess (pustule) and acanthosis (epidermal regeneration) are observed 48 hours after exposure. Owing to its emulsifying properties, SLS is an anionic surfactant used in many hy-

**Figure 4.** Macroscopic photos from a cumulative dermal irritation study in animals. (a) After the hair on the back of the rat is shaved, the drug is continually applied to the same area. The rat wears the Elizabethan collar to prevent the animal from biting or licking the exposure site. (b) Cumulative dermal irritation study in a rabbit (left: vehicle application, right: drug application). The site of drug application is observed with erythema, redness, swelling, and moistness. The

change spreads beyond the site of application, indicating a strong irritant property of the drug.

gienic and cleaning products, including shampoos, toothpastes, and shaving foams.

6 Toxicology - New Aspects to This Scientific Conundrum

Allergic contact dermatitis is a condition caused by a delayed (type IV) allergic reaction. A low molecular weight drug binds as hapten to a protein in the body to act as a complete antigen. Characteristically, inflammation is induced approximately 12 hours following recontact of a sensitized animal with the drug. Known sensitizing substances include preservatives contained in topical application agents, nickel sulfate, potassium dichromate, neomycin, aroma chemicals, formaldehyde, rubber/latex medical supplies, and plants (e.g., rhus lacquer).

#### *3.1.3. Chemical burns*

Chemical burns are an injury caused by a chemical substance that is extremely corrosive or irritating (e.g., strong acid or strong alkali), often involving itching and/or ulceration due to local coagulative necrosis (**Figure 5**). No currently available pharmaceutical products cause this type of injury. Accidental exposure to skin or oral ingestion of these chemicals represents a pediatric emergency problem and these chemicals have a history of being common agents used for suicide [12, 13]. Cement burn is well known in the developed world. The majority of patients are either workers in the construction industry or do-it-yourself enthusiasts, commonly kneeling or standing in cement. The mechanism of injury is a combination of the effects of cement alkalinity and mechanical abrasion. Besides denaturing protein, alkalis saponify fatproducing liquefactive necrosis [14].

**Figure 5.** Sodium hydroxide-induced burn in the back skin of a rat. Severe coagulative necrosis is observed in all cutaneous layers. Insert is a higher magnification image of the same photo.

**Figure 6.** Macroscopic photo from a phototoxic study of 8-methoxypsoralen in guinea pigs. After the hair on the back of the guinea pig was shaved, the drug was applied to the same area and irradiated with ultraviolet light, and the reaction was subsequently evaluated. (a) The site on the left was irradiated with ultraviolet light (UVA) after drug application, while the site on the right was not irradiation after drug application. Erythema is observed at the site with ultraviolet light irradiation. This reaction to 8-methoxypsoralen with ultraviolet light has been utilized in ultraviolet light therapy (PUVA) for psoriasis in humans. (b) Apoptotic epidermal cells (sunburn cells) are observed at the site with ultraviolet light irradiation.

## **3.2. Photosensitive dermatitis**

## *3.2.1. Photosensitivity*

Photosensitive dermatitis (photosensitivity) is a general term that refers to skin inflammation caused by the combined effect of a drug and light. It can be classified into two types, either with or without involvement of an immunological mechanism. Numerous systemic and topical drugs, aroma chemicals, plants, and cosmetics have been reported to induce this condition. Some examples of photosensitizing drugs are phenothiazine, tetracyclines, sulfonamides, chlorpromazine, nalidixic acid, and fluorocoumarins (psoralens).

## *3.2.2. Phototoxic dermatitis*

Phototoxic dermatitis is skin damage caused by a drug that is sensitive to light (ultraviolet light), not by the drug alone, but after absorption of photon energy, without involvement of an immune mechanism. Free radicals and peroxidative injuries have been reported to be involved in this reaction. In preclinical studies of topical application agents, phototoxicity testing is conducted using guinea pigs for evaluation of drug phototoxicity (**Figure 6**).

## *3.2.3. Photoallergic contact dermatitis*

Photoallergic contact dermatitis is a condition caused by a delayed (type IV) allergic reaction. A drug sensitive to light (ultraviolet light) absorbs photon energy and is transformed into a substance (i.e., hapten) that combines with a protein in the body to act as a complete antigen. Characteristically, inflammation is induced approximately 12 hours following recontact of a sensitized animal with the drug.

## **3.3. Contact urticaria**

**Figure 5.** Sodium hydroxide-induced burn in the back skin of a rat. Severe coagulative necrosis is observed in all cuta-

**Figure 6.** Macroscopic photo from a phototoxic study of 8-methoxypsoralen in guinea pigs. After the hair on the back of the guinea pig was shaved, the drug was applied to the same area and irradiated with ultraviolet light, and the reaction was subsequently evaluated. (a) The site on the left was irradiated with ultraviolet light (UVA) after drug application, while the site on the right was not irradiation after drug application. Erythema is observed at the site with ultraviolet light irradiation. This reaction to 8-methoxypsoralen with ultraviolet light has been utilized in ultraviolet light therapy (PUVA) for psoriasis in humans. (b) Apoptotic epidermal cells (sunburn cells) are observed at the site

neous layers. Insert is a higher magnification image of the same photo.

8 Toxicology - New Aspects to This Scientific Conundrum

with ultraviolet light irradiation.

Contact urticaria is acute redness or rash that occurs within several minutes to one hour following exposure to a drug. It can be caused by a direct effect of the drug on vascular walls, by an indirect effect on vascular walls via histamine release from mast cells (without involvement of an immune mechanism), or by an IgE-mediated immediate (type I) allergic reaction with involvement of an immune mechanism. For immune contact urticaria, known conditions include systemic reactions to penicillin or food, as well as urticaria due to natural rubber products (latex allergy), but it is generally difficult to reproduce such conditions in preclinical studies using experimental animals.

## **3.4. Chemical-induced acne**

Chemical-induced acne is a disease of hair follicles caused by a chemical substance and is characterized by keratin plugs in hair follicles due to excessive proliferation of keratinocytes in hair follicles (comedo), sebum retention and inflammation [11]. Known examples of chemical-induced acne include occupational skin disorders of oil acne, caused by frequent exposure of the skin to cutting oils, as well as chloracne, induced by dioxins such as TCDD and PCB [15]. Clinically, the lesions are located around the eyes, ears, back, and genitalia; and other symptoms include hyperpigmentation, conjunctivitis, and ocular discharge. A notorious event occurred when Ukraine President Viktor Yushchenko was stricken with facial chloracne resulting from deliberate poisoning with TCDD during his presidential campaign [16].

#### **3.5. Pigmentary disturbance**

Pigmentary disturbance is only observed in animals with scarce hair or animals that have been shaved, thus is difficult to detect in preclinical studies. Altered pigmentation is a condition that sometimes follows skin inflammation and is characterized histopathologically by an increase or decrease in the number of melanocytes as well as melanin production. Hyperpigmentation can occur in association with increased melanin production due to drug-induced activation of melanocytes, hemosiderin deposition due to hemorrhage, or deposition of a heavy metal or drug itself (**Figures 7** and **8**). Melanin production is increased by busulfan, cyclophosphamide, long-term high-dose ACTH, and inorganic arsenic. In addition, chlorpromazine or minocycline can form a complex with melanin or hemosiderin with deposition in the skin, leading to blue-gray discoloration of the skin. In contrast, hypopigmentation results from loss of melanin due to damage to melanocytes. Depigmenting agents such as phenols, catechols, and hydroquinone have a similar structure to tyrosine, thus can inhibit melanin synthesis and induce hypopigmentation (**Figure 8**). Recently, an unexpected outbreak of patients with leukoderma occurred in Japan with use of brightening/lightening cosmetics containing rhododendrol, which is a competitive tyrosinase inhibitor, thereby inhibiting melanin synthesis [17]. This type of leukoderma is induced by not only apoptosis of melanocytes but also subsequent immune reactions with CD8-positive T cell infiltration toward melanocytes [18, 19].

**Figure 7.** Hyperpigmentation due to melanin deposition in dermis at a drug injection site in the monkey. (a) Crust and epidermal hyperplasia are observed in the epidermis, and black pigment is observed in the dermis. (b) High power field of (a). Note that the black pigment is scattered throughout the dermis.

**Figure 8.** Macroscopic photo from a dermal application study of two drugs in mini-pigs (dark Yucatan pigs). The normal skin of Yucatan pigs appears black, because the skin contains a large amount of melanin pigment. With one drug applied to the left regions of the image, darkening of the skin is shown compared to the normal portion of skin, indicating excessive pigmentation due to drug application. With another drug applied to the right side of the image, lightening of the skin is shown with decreased pigmentation.

#### **3.6. Drug rash (cutaneous reaction)**

other symptoms include hyperpigmentation, conjunctivitis, and ocular discharge. A notorious event occurred when Ukraine President Viktor Yushchenko was stricken with facial chloracne resulting from deliberate poisoning with TCDD during his presidential campaign [16].

Pigmentary disturbance is only observed in animals with scarce hair or animals that have been shaved, thus is difficult to detect in preclinical studies. Altered pigmentation is a condition that sometimes follows skin inflammation and is characterized histopathologically by an increase or decrease in the number of melanocytes as well as melanin production. Hyperpigmentation can occur in association with increased melanin production due to drug-induced activation of melanocytes, hemosiderin deposition due to hemorrhage, or deposition of a heavy metal or drug itself (**Figures 7** and **8**). Melanin production is increased by busulfan, cyclophosphamide, long-term high-dose ACTH, and inorganic arsenic. In addition, chlorpromazine or minocycline can form a complex with melanin or hemosiderin with deposition in the skin, leading to blue-gray discoloration of the skin. In contrast, hypopigmentation results from loss of melanin due to damage to melanocytes. Depigmenting agents such as phenols, catechols, and hydroquinone have a similar structure to tyrosine, thus can inhibit melanin synthesis and induce hypopigmentation (**Figure 8**). Recently, an unexpected outbreak of patients with leukoderma occurred in Japan with use of brightening/lightening cosmetics containing rhododendrol, which is a competitive tyrosinase inhibitor, thereby inhibiting melanin synthesis [17]. This type of leukoderma is induced by not only apoptosis of melanocytes but also subsequent immune reactions with CD8-positive T cell infiltration toward

**Figure 7.** Hyperpigmentation due to melanin deposition in dermis at a drug injection site in the monkey. (a) Crust and epidermal hyperplasia are observed in the epidermis, and black pigment is observed in the dermis. (b) High power

field of (a). Note that the black pigment is scattered throughout the dermis.

**3.5. Pigmentary disturbance**

10 Toxicology - New Aspects to This Scientific Conundrum

melanocytes [18, 19].

Drug rash (cutaneous reaction) is the most common adverse drug reaction reported to occur with antibiotics. The most serious forms of drug rash are toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome, which are known to occur with the use of various drugs, including penicillin derivative or cephem derivative antibiotics, antipyretic analgesics (particularly NSAIDs), allopurinol, amine antiepileptic drugs (phenytoin and carbamazepine), and sulfa drugs. The Ministry of Health, Labour and Welfare of Japan announced that of 110,023 cases of adverse drug reactions reported from 2005 to 2009, approximately 2.2% of the cases (2370) were toxic epidermal necrolysis or Steven-Johnson syndrome [20]. Although the mechanism of onset remains unknown in many instances, a type III allergic reaction is often speculated.

Many new antitumor drugs with specific molecular targets have been approved in recent years (the so-called "targeted therapies"), and their adverse effects are highly specific with respect to the skin. Cutaneous reactions to these therapies are among the most frequently observed and, when severe or protracted, can result in significant morbidity, requiring dose modification or drug discontinuation [21, 22]. Hyperplastic changes of the epidermis can be attributed to numerous causes, including response to stimulation from growth factors, such as epidermal growth factor (EGF). The repeated administration of EGF to cynomolgus monkeys results in cutaneous desquamation and epidermal hyperplasia [23]. Epidermal growth factor receptor (EGFR), multikinase, c-Kit, BRAF, or MEK inhibitors induce papulopustular rash, maculopapular rash, and hand-foot syndrome in humans [24, 25]. EGFR inhibitor-induced lesions are associated with the inhibition of EGFR in undifferentiated, proliferating keratinocytes in the basal and suprabasal layers of the epidermis [26]. Other inhibitor-related rashes appear to be associated with the inhibition of vascular endothelial growth factor (VEGF) receptors in the skin [24].

#### **3.7. Hair disturbance**

Many drugs induce hair disorders, such as hair loss, stimulated hair growth, or, more rarely, changes in the hair shape and color [27]. Hair loss (hypotrichosis or alopecia) is a common problem that affects approximately 60 million men, women, and children in the United States, with a total cost for medical consultation and treatment of US\$1.3 billion per year [28]. The onset of alopecia (toxic alopecia) often depends on the hair cycle at the time of drug administration. Drugs with an androgenic effect can cause alopecia by acting on the resting phase of the hair cycle to shorten the cycle. In addition, drugs with an antimitotic effect (e.g., anticancer drugs) or irradiation can cause alopecia by inducing apoptosis of hair follicles during the anagen phase of the hair follicle, thereby causing atrophy of hair follicles and prolongation of the resting phase of the hair cycle (chemotherapy or radiation-induced follicular dystrophy) [29, 30] (**Figure 9**). Hypertrichosis refers to drug-induced promotion of hair growth or induction of the anagen phase [31] **Figure 10**, and has been reported in organ transplant recipients and animal models treated with cyclosporine [32], as well as an antihypertensive (minoxidil) and a drug for treating benign prostatic hyperplasia (finasteride) [33]. Minoxidil and finasteride have been approved for clinical use as drugs to stimulate hair growth [34]. Hypertrichosis is observed as an increase in the length, thickness, and number of eyelashes in glaucoma patients treated with prostaglandin F2α agonists [35]. Bimatoprost has been used as a therapy for eyelash insufficiency or as eyelash restorer. As in the cases of the pigmentary disturbances described above, these changes can only be observed in animals with scarce hair or animals with shaved hair and are thus difficult to detect in typical preclinical studies.

**Figure 9.** *N*-Methyl-*N*-nitrosourea (MNU)-induced follicular dystrophy in C57BL mice. (a) Systemic hair loss and whitening skin color are observed in the mouse treated with MNU (right side), compared to the control mouse with abundant hair (left side). MNU is an alkylating agent used as an antimitotic chemical, and induces apoptosis of hair follicles during the anagen phase of the hair follicle causing atrophy of hair follicles and prolongation of the resting phase of the hair cycle. (b) Anagen stage of hair cycle in a control mouse. (c) Catagen stage of hair cycle (follicular dystrophy) in a MNU-treated mouse. (b) and (c) are at the same magnification.

**Figure 10.** Immunosuppressant drug-induced hypertrichosis in a mouse follicular dystrophy model. (a) Compared to *N*-methyl-*N*-nitrosourea (MNU)-induced follicular dystrophy in the treated mouse (left side), the mouse treated with the immunosuppressant drug after MNU exposure has abundant body hair (right side) (b) MNU-induced follicular dystrophy is characterized by a prolonged catagen stage of hair cycle and thinness of the back skin. (b) and (c) are at the same magnification. (c) The hair cycle in the mouse treated with the drug after MNU exposure was found to be in the anagen stage with thickening of the back skin. (b) and (c) are at the same magnification.

## **3.8. Nail disturbance**

**3.7. Hair disturbance**

12 Toxicology - New Aspects to This Scientific Conundrum

Many drugs induce hair disorders, such as hair loss, stimulated hair growth, or, more rarely, changes in the hair shape and color [27]. Hair loss (hypotrichosis or alopecia) is a common problem that affects approximately 60 million men, women, and children in the United States, with a total cost for medical consultation and treatment of US\$1.3 billion per year [28]. The onset of alopecia (toxic alopecia) often depends on the hair cycle at the time of drug administration. Drugs with an androgenic effect can cause alopecia by acting on the resting phase of the hair cycle to shorten the cycle. In addition, drugs with an antimitotic effect (e.g., anticancer drugs) or irradiation can cause alopecia by inducing apoptosis of hair follicles during the anagen phase of the hair follicle, thereby causing atrophy of hair follicles and prolongation of the resting phase of the hair cycle (chemotherapy or radiation-induced follicular dystrophy) [29, 30] (**Figure 9**). Hypertrichosis refers to drug-induced promotion of hair growth or induction of the anagen phase [31] **Figure 10**, and has been reported in organ transplant recipients and animal models treated with cyclosporine [32], as well as an antihypertensive (minoxidil) and a drug for treating benign prostatic hyperplasia (finasteride) [33]. Minoxidil and finasteride have been approved for clinical use as drugs to stimulate hair growth [34]. Hypertrichosis is observed as an increase in the length, thickness, and number of eyelashes in glaucoma patients treated with prostaglandin F2α agonists [35]. Bimatoprost has been used as a therapy for eyelash insufficiency or as eyelash restorer. As in the cases of the pigmentary disturbances described above, these changes can only be observed in animals with scarce hair or animals with shaved hair and are thus difficult to detect in typical preclinical studies.

**Figure 9.** *N*-Methyl-*N*-nitrosourea (MNU)-induced follicular dystrophy in C57BL mice. (a) Systemic hair loss and whitening skin color are observed in the mouse treated with MNU (right side), compared to the control mouse with abundant hair (left side). MNU is an alkylating agent used as an antimitotic chemical, and induces apoptosis of hair follicles during the anagen phase of the hair follicle causing atrophy of hair follicles and prolongation of the resting phase of the hair cycle. (b) Anagen stage of hair cycle in a control mouse. (c) Catagen stage of hair cycle (follicular

dystrophy) in a MNU-treated mouse. (b) and (c) are at the same magnification.

Nail changes that reflect a previous general condition are a barometer of health that can be used to predict the presence or absence of an abnormality several weeks before its presentation [6]. Transverse ridges (Beau's line), washboard nail plates, and onycholysis are known to occur with use of metoprolol, retinoids, anticancer drugs, or irradiation [5]. Adverse effects of targeted molecular therapies, such as EGFR inhibitors, are also highly specific with respect to nails in human patients [22, 36]. In addition, yellow nail discoloration is known to occur with penicillamine, and black nail discoloration with Futraful (tegafur), anticancer drugs, or gold drugs. Administration of nucleoside analogs to dogs results in nail loss and footpad erosions with associated radiomimetic defects in the stratum germinativum [4]. In general, onycholysis can be induced by anticancer drugs or irradiation in experimental animals, while other changes are difficult to detect in preclinical studies.

## **3.9. Skin carcinogenesis**

Some photoirritants, such as 8-methoxypsoralen, have been associated with UV-induced skin carcinogenesis. Treatment of psoriasis by photochemotherapy (PUVA) with oral methoxsalen, a psoralen, in conjunction with UVA radiation, is associated with an increased risk of irregular pigmented skin lesions, squamous cell carcinoma, and malignant melanoma [37, 38]. Chemically induced skin tumors have been associated with numerous topically applied and systemically administered compounds in rodents; however, there appear to be few clinically used drugs that are suspected of being involved in skin carcinogenesis in humans. Rodent models of skin carcinogenesis are widely used for studies of carcinogenic mechanisms and the evaluation of carcinogenesis associated with chemical substances. Huff et al. performed a retrospective investigation of carcinogenicity tests on 379 chemical compounds conducted by the US National Toxicology Program (NTP) and reported that increased skin carcinogenesis was observed with 19 chemical compounds [39] (**Table 3**). Currently used methods used to determine skin carcinogenesis of drugs/chemical substances or methods to clarify carcinogenic mechanisms include: 2-year dermal application carcinogenicity studies (**Figure 11**); DMBA/ TPA two-stage skin carcinogenesis models using 12-O-tetradecanoylphorbol-13-acetate (TPA) and 7,12-dimethylbenzanthracene (DMBA) (DMBA as an initiator, TPA as a promoter) [40]; studies in Tg.AC transgenic mice with expression of the v-Ha-ras gene in the epidermis [41]; and studies in SENCAR (SENsitivity to CARcinogen) mice [42]. Two-stage skin carcinogenesis models using metallothionein-I/II knockout mice have shown significant increases in skin carcinogenesis, thereby indicating an important role of metallothionein as an inhibitory factor of carcinogenesis in skin [43]. p53 is a protein that causes cell cycle arrest, apoptosis, or senescence that is crucial in the process of tumor suppression in several cell types [44]. In the

**Figure 11.** A 2-year dermal application study of a drug in CD1 mice. (a) Macroscopically, multiple reddish skin tumors are observed on the back. (b) Histological findings are consistent with squamous cell papilloma.

DMBA/TPA two-stage skin carcinogenesis model, the absence of p53 in stratified epithelia leads to the appearance of a higher number of tumors that grow faster and become malignant more frequently than tumors arising in mice with the wild type p53 genotype [45]. The carcinogenic risk of a chemical after topical application is traditionally investigated in rats; however, in recent years, Tg.AC mice have become a popular alternative. The skin of Tg.AC mice is genetically initiated, thus the induction of epidermal papilloma in response to dermal or oral exposure to a chemical agent acts as a reporter phenotype for the carcinogenicity of the test chemical [11, 46]. The SENCAR mouse is an outbred strain (not genetically engineered) that was selected specifically for increased skin tumor multiplicity and decreased tumor latency in response to known dermal carcinogens [41]. A recent report described a possible animal model for human keratoacanthoma involving a single intraperitoneal injection of 50 or 75 mg/kg *N*-methyl-*N*-nitrosourea in male Sprague-Dawley rats at 4 weeks of age [47] (**Figure 12**). Keratoacanthoma is a benign tumor believed to arise from the epithelium of hair follicles [48]. Peroxisome proliferator-activated receptor agonists have been associated with the development of hemangiosarcomas in mice and hamsters and liposarcomas and fibrosarcomas in rats [49].

**Figure 12.** Animal model for human keratoacanthoma following a single intraperitoneal injection of *N*-methyl-*N*-nitrosourea in a male rat.

#### **3.10. Other cutaneous toxicity due to systemic toxicity**

pigmented skin lesions, squamous cell carcinoma, and malignant melanoma [37, 38]. Chemically induced skin tumors have been associated with numerous topically applied and systemically administered compounds in rodents; however, there appear to be few clinically used drugs that are suspected of being involved in skin carcinogenesis in humans. Rodent models of skin carcinogenesis are widely used for studies of carcinogenic mechanisms and the evaluation of carcinogenesis associated with chemical substances. Huff et al. performed a retrospective investigation of carcinogenicity tests on 379 chemical compounds conducted by the US National Toxicology Program (NTP) and reported that increased skin carcinogenesis was observed with 19 chemical compounds [39] (**Table 3**). Currently used methods used to determine skin carcinogenesis of drugs/chemical substances or methods to clarify carcinogenic mechanisms include: 2-year dermal application carcinogenicity studies (**Figure 11**); DMBA/ TPA two-stage skin carcinogenesis models using 12-O-tetradecanoylphorbol-13-acetate (TPA) and 7,12-dimethylbenzanthracene (DMBA) (DMBA as an initiator, TPA as a promoter) [40]; studies in Tg.AC transgenic mice with expression of the v-Ha-ras gene in the epidermis [41]; and studies in SENCAR (SENsitivity to CARcinogen) mice [42]. Two-stage skin carcinogenesis models using metallothionein-I/II knockout mice have shown significant increases in skin carcinogenesis, thereby indicating an important role of metallothionein as an inhibitory factor of carcinogenesis in skin [43]. p53 is a protein that causes cell cycle arrest, apoptosis, or senescence that is crucial in the process of tumor suppression in several cell types [44]. In the

14 Toxicology - New Aspects to This Scientific Conundrum

**Figure 11.** A 2-year dermal application study of a drug in CD1 mice. (a) Macroscopically, multiple reddish skin tumors

are observed on the back. (b) Histological findings are consistent with squamous cell papilloma.

#### *3.10.1. Acne formation due to anti-inflammatory analgesics*

This is a common clinical adverse reaction to NSAIDs or steroids and involves proliferation of acne bacteria leading to worsening of inflammation (steroid acne). In preclinical studies, spontaneous interdigit inflammation may worsen in beagle dogs following NSAID administration, eventually leading to skin ulcers in all extremities in severe cases (**Figure 13**).


+: Positive, CE: apparent increase in incidence, ?: increased incidence but not significantly, −: negative, +/−: positive or negative, TR No.: National Toxicology Program (NTP) study number. This table has been modified from [39].

**Table 3.** Chemical compounds reported to produce skin carcinogenesis from systemic exposure (US NTP study).

**Figure 13.** Nonsteroidal anti-inflammatory analgesic drug (NSAID)-induced skin lesion in a subacute toxicity study using beagle dogs. (a) Macroscopic observation of an interdigit lesion in the foot pad. Severe swelling and ulceration due to inflammation with local bacterial infection are observed. (b) In severe cases, the local lesions progress to skin ulcers on all extremities (subcutaneous phlegmon).

#### *3.10.2. Drug-induced skin atrophy*

**Name of chemical compound Route Any skin carcinogenesis (2-year**

water

water

Drinking water

Drinking water

Dermal application

negative, TR No.: National Toxicology Program (NTP) study number.

C.I. acid red 114 Drinking

16 Toxicology - New Aspects to This Scientific Conundrum

C.I. Direct Blue 15 Drinking

C.I. basic red 9 monohydrochloride

3,3′-Dimethoxybenzidine

3,3′-Dimethoxybenzidine

Tris(aziridinyl)-phosphine

This table has been modified from [39].

4-Vinyl-1-cyclohexene

sulfide

diepoxide

3,3′-Dimethoxybenzidine-4,4′-

dihydrochloride

dihydrochloride

diisocyanate

**carcinogenesis study)**

3-Amino-9-ethylcarbazole Dietary + + + + + 093 Benzene Oral CE CE CE CE – 289 Chloroethane Inhalation ? ? ? CE + 346

2,4-Diaminoanisole sulfate Dietary + + + + + 084

2,4-Dinitrotoluene Dietary + + – – + 054 Fenthion Dietary – – ? – +/− 103 Glycidol Oral CE CE CE CE + 374 Nithiazide Dietary – + + ? + 146 5-Nitro-O-anisidine Dietary + + ? + + 127 Nitrofurazone Dietary ? CE – CE + 337 Rhodamine 6G Dietary ? ? – – – 364

+: Positive, CE: apparent increase in incidence, ?: increased incidence but not significantly, −: negative, +/−: positive or

**Table 3.** Chemical compounds reported to produce skin carcinogenesis from systemic exposure (US NTP study).

**F344 rats B6C3F1 mice Male Female Male Female**

CE CE + 405

CE CE – 397

CE CE + 372

CE CE + 390

Dietary CE CE CE CE + 285

Dietary + + – – + 128

Subcutaneous + + + + + 058

CE CE CE CE + 362

**Mutagenicity (Ames)**

**TR No.**

> Skin atrophy can be observed with long-term, repeated use of corticosteroids due to inhibitory effects on cell proliferation and/or fiber production, leading to decreases throughout the epidermis, skin appendages (hair follicles, sweat glands, and sebaceous glands) and subcutaneous adipose tissue [50] (**Figure 14**). Skin atrophy is also commonly observed with systemic exposure to anticancer drugs in preclinical studies.

**Figure 14.** Corticosteroid-induced cutaneous atrophy in the rat. (a) In normal rat skin, the skin is thick with large hair shafts, sebaceous glands, and subcutaneous adipose tissue. (b) In the drug-exposed skin, the skin is thin with a severe decrease in all cutaneous layers, skin appendages (hair follicles and sebaceous glands), and subcutaneous adipose tissue.

#### *3.10.3. Skin ulcer due to peripheral circulatory insufficiency*

A lesion similar to cutaneous gangrene seen in diabetic patients can be induced in monkeys with certain drugs, and is speculated as a consequence of peripheral circulatory insufficiency due to the involvement of a vascular disorder [5].

## *3.10.4. Drug-induced granulomatous reaction*

Hypodermic injections of certain drugs induce granulomatous inflammation located at the injection site, which is highly painful for the patients (**Figure 15**). Granulomas induced by luteinizing hormone-releasing hormone analogues have been reported in some patients for the treatment of prostatic cancers [51]. Histopathologically, epithelioid granulomatous inflammation with small vacuoles derived from the constituent ingredients of drug microcapsules has been observed [52]. In some patient cases, vaccinations induce granulomatous reactions at the injection site due to specific inflammation and irritation [53]. Recently, treatment with interferon has been associated with cutaneous granulomatous reactions and sarcoid reactions [54].

**Figure 15.** Drug-induced granuloma in subcutaneous tissue of the rat. (a) Granuloma is observed in the subcutaneous tissue. (b) Many foreign body giant cells that phagocytose lipoid materials are observed. Lipoid materials are derived from the contents of the drug. (c) Dystrophic calcification is observed in the obsolete lesions.

## **4. Closing remarks**

This review has outlined the types and characteristics of drug-induced cutaneous toxicity, as well as providing descriptions of the methods of cutaneous toxicity testing required for safety evaluation. It should be emphasized that cutaneous toxicity of drugs or chemical substances may appear in various forms. In recent years, advances have been made in the development of pharmaceutical products targeting specific molecules, genes, or nanotechnology-based pharmaceutical products. Due to the potential onset of cutaneous toxicity involving novel mechanisms with new pharmaceutical products, it will continue to be important to understand the basic toxic changes described here.

## **Acknowledgements**

*3.10.3. Skin ulcer due to peripheral circulatory insufficiency*

due to the involvement of a vascular disorder [5].

*3.10.4. Drug-induced granulomatous reaction*

18 Toxicology - New Aspects to This Scientific Conundrum

sarcoid reactions [54].

**4. Closing remarks**

A lesion similar to cutaneous gangrene seen in diabetic patients can be induced in monkeys with certain drugs, and is speculated as a consequence of peripheral circulatory insufficiency

Hypodermic injections of certain drugs induce granulomatous inflammation located at the injection site, which is highly painful for the patients (**Figure 15**). Granulomas induced by luteinizing hormone-releasing hormone analogues have been reported in some patients for the treatment of prostatic cancers [51]. Histopathologically, epithelioid granulomatous inflammation with small vacuoles derived from the constituent ingredients of drug microcapsules has been observed [52]. In some patient cases, vaccinations induce granulomatous reactions at the injection site due to specific inflammation and irritation [53]. Recently, treatment with interferon has been associated with cutaneous granulomatous reactions and

**Figure 15.** Drug-induced granuloma in subcutaneous tissue of the rat. (a) Granuloma is observed in the subcutaneous tissue. (b) Many foreign body giant cells that phagocytose lipoid materials are observed. Lipoid materials are derived

This review has outlined the types and characteristics of drug-induced cutaneous toxicity, as well as providing descriptions of the methods of cutaneous toxicity testing required for safety

from the contents of the drug. (c) Dystrophic calcification is observed in the obsolete lesions.

All authors have read and approved the final manuscript. The authors declare no competing financial interests. This project was supported by a grant from MEXT-Supported Programs for the Strategic Research Foundations at Private Universities (Kansai Medical University).

## **Author details**

Katsuhiko Yoshizawa\* , Michiko Yuki and Airo Tsubura

\*Address all correspondence to: yoshizak@hirakata.kmu.ac.jp

Pathology II, Kansai Medical University, Hirakata, Osaka, Japan

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[31] Yoshizawa K, Nambu H, Yamamoto D, Yang J, Kiyozuka Y, Shikata N, Tsubura A. Timespecific occurrence of alopecia in neonatal C57BL mice treated with *N*-methyl-*N*nitrosourea and the therapeutic efficacy of tacrolimus hydrate. Pathol Int 2000;50:175–

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[34] Piraccini B, Starace M, Alessandrini A, Guarrera M, Fiorucci MC, Lorenzi S. Efficacy and tolerability of 5% minoxidil solution (CarexidilR) in male and female androgenetic alopecia: a 6-month open multicentric study. G Ital Dermatol Venereol 2011;146:1–8

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[37] Archier E, Devaux S, Castela E, Gallini A, Aubin F, Le Maitre M, Aractingi S, Bachelez H, Cribier B, Joly P, Jullien D, Misery L, Paul C, Ortonne JP, Richard MA. Carcinogenic risks of psoralen UV-A therapy and narrowband UV-B therapy in chronic plaques psoriasis: a systemic literature review. J Eur Acad Dermatol Venereol 2012;26(Suppl 3):

[38] Stern RS, Nichols KT, Vakeva LH. Malignant melanoma in patients treated for psoriasis with methoxsalen (psoralen) and ultraviolet A radiation (PUVA). The PUVA follow-up study. N Engl J Med 1997;336:1041–1045. DOI: 10.1056/NEJM199704103361501. [39] Huff J, Cirvello J, Haseman J, Bucher J. Chemicals associated with site-specific neoplasia in 1394 long-term carcinogenesis experiments in laboratory rodents. Environ Health

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22 Toxicology - New Aspects to This Scientific Conundrum

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## **Hepatic and Intestinal Multidrug Resistance-Associated Protein 2: Transcriptional and Post-transcriptional Regulation by Xenobiotics Hepatic and Intestinal Multidrug Resistance-Associated Protein 2: Transcriptional and Post-transcriptional Regulation by Xenobiotics**

Maite R. Arana, Guillermo N. Tocchetti, Maite R. Arana, Guillermo N. Tocchetti, Juan P. Rigalli, Aldo D. Mottino, Fabiana García and

Juan P. Rigalli, Aldo D. Mottino, Fabiana García and Silvina S.M. Villanueva Silvina S.M. Villanueva

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

http://dx.doi.org/10.5772/64755

#### **Abstract**

[53] Rosenblatt AE, Stein SL. Cutaneous reactions to vaccinations. Clin Dermatol

[54] El-Khalawany M, Mohammad I, Aboeldahab S, Thabet A. Cutaneous granulomas associated with interferon therapy. Am J Dermatopathol 2016 (in press). DOI: 10.1097/

2015;33:327–332. DOI: 10.1016/j.clindermatol.2014.12.009.

DAD.0000000000000547.

24 Toxicology - New Aspects to This Scientific Conundrum

We are daily exposed to a large number of pharmacological drugs, environmental pollutants, and natural toxins, which represent a potential toxic insult. The organism possesses a sophisticated system of detoxification particularly expressed in the liver, intestine, and kidney. This system consists of intracellular biotransformation enzymes that convert the toxins into more hydrophilic derivatives followed by their elimination through membrane transporters. Multidrug resistance-associated protein 2 (MRP2, ABCC2) is an important member of the ATP-binding cassette (ABC) superfamily of transporters localized at the apical membrane of polarized cells, such as hepatocytes, enterocytes, and renal tubular cells. MRP2 is proposed as a major actor in the elimination of endo- and xenobiotics, mainly conjugated with glucuronic acid, glutathione, and sulfate. The small intestine and the liver constitute relevant detoxification organs expressing MRP2 and therefore preventing absorption and promoting the hepatobiliary clearance of xenobiotics. MRP2 expression and/or function can be modulated by therapeutic drugs, herbal products, dietary compounds, and environmental pollutants. Consequently, MRP2 modulation could cause changes in tissue exposure, with potential toxicological and pharmacological consequences. This chapter reviews the information available on the role of hepatic and intestinal MRP2 in detoxification processes, and their regulation by xenobiotics, considering in particular its toxicological relevance.

**Keywords:** MRP2, intestine, liver, detoxification, clearance, toxicity

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

## **1. Introduction**

#### **1.1. Detoxification of xenobiotics**

Most organisms are daily exposed to a large number of different xenobiotics, such as therapeutic drugs, environmental pollutants, and natural toxins. The major routes of exposure to these chemicals comprise incorporation with the diet, inhalation or absorption through the skin. Preservation of health depends largely on the body's ability to eliminate these harmful substances. In this regard, the organism possesses a sophisticated system of detoxification mainly expressed in organs such as the liver, intestine, and kidney. The detoxification process consists of intracellular biotransformation enzymes that neutralize the toxins and membrane transporters for their subsequent elimination from the cell. The biotransformation process is carried out by the same biochemical machinery that metabolizes endogenous compounds, often of similar chemical structure. Even though a compound can be excreted without undergoing any change, it is usually converted by biotransformation enzymes into a more hydrophilic derivative prior to elimination. The biotransformation reactions are carried out by phase I enzymes such as cytochrome P450 (CYP) members and by phase II conjugating enzymes such as glutathione S-transferase (GST; EC 2.5.1.18), UDP-glucuronosyltransferase (UGT; EC 2.4.1.17), and sulfotransferase (SULT; EC 2.8.2.) [1, 2]. Phase I enzymes usually act in tandem with phase II enzymes, which ultimately results in incorporation of anionic groups into the xenobiotic molecule. The hydrophilic derivatives can be excreted from the cells by phase III or membrane transport systems, with the anionic groups acting as *affinity markers* for a series of transporters of the multidrug resistance-associated protein (MRP) family. These proteins are members of the ATP-binding cassette (ABC) drug efflux transporters and mediate the active extrusion of biotransformation products for their subsequent elimination from the body [1, 2]. Direct elimination of xenobiotics without suffering any biotransformation is also possible and is denominated *"phase 0 metabolism"* [3].

What follows is a description of the role of the multidrug resistance-associated protein 2 (MRP2, ABCC2) as an important component of the ABC family involved in xenobiotic disposition by liver and intestine.

#### **1.2. Multidrug resistance-associated protein 2 and its role in the detoxification of xenobiotics**

MRP2 structure consists of a large core segment, containing two cytosolic nucleotide-binding domains (NBDs), two membrane-spanning domains (MSDs), and a linker segment L1, shared by other ABC members. Additionally, MRP2 contains a third NH2-terminal membranespanning domain MSD0, also called terminal transmembrane domain, with five transmembrane helices resulting in an extracellular NH2-terminus and an intracellular linker segment 0 (L0) (**Figure 1**) [4]. MRP2 is characteristically expressed at the apical membrane of polarized cells such as hepatocytes, enterocytes, and renal tubular cells [4–6], where it plays a primary role in the elimination of specific compounds. Particularly, the highest concentration of this transporter is found in the liver and intestine [7], two organs playing a prominent role in xenobiotic detoxification commonly known as "first-pass metabolism and clearance". Protein expression of MRP2 is highest in enterocytes of the proximal small intestine, decreasing in direction to the terminal ileum [8], gradient shared with the expression of phase II metabolizing enzymes such as GST and UGT [9, 10], thus suggesting that metabolism and transport processes may act coordinately. In the liver, the major biotransformation organ, MRP2 is abundantly expressed in the bile canalicular membranes of the hepatocyte [11], where it plays a role in bile formation through secretion of endogenous substrates such as glutathione (GSH) and glutathione conjugates. Canalicular MRP2 also constitutes the main route of elimination of xenobiotics conjugated with GSH, sulfate or glucuronic acid [12]. Similarly, immunohistochemical analysis in normal rat liver demonstrated that UGTs and rMrp2 are localized in the same regions [13, 14], also indicating that they may work in cooperation. MRP2 in either localization contributes significantly to disposition of potentially harmful compounds thus decreasing their toxicity.

**1. Introduction**

**1.1. Detoxification of xenobiotics**

26 Toxicology - New Aspects to This Scientific Conundrum

possible and is denominated *"phase 0 metabolism"* [3].

disposition by liver and intestine.

Most organisms are daily exposed to a large number of different xenobiotics, such as therapeutic drugs, environmental pollutants, and natural toxins. The major routes of exposure to these chemicals comprise incorporation with the diet, inhalation or absorption through the skin. Preservation of health depends largely on the body's ability to eliminate these harmful substances. In this regard, the organism possesses a sophisticated system of detoxification mainly expressed in organs such as the liver, intestine, and kidney. The detoxification process consists of intracellular biotransformation enzymes that neutralize the toxins and membrane transporters for their subsequent elimination from the cell. The biotransformation process is carried out by the same biochemical machinery that metabolizes endogenous compounds, often of similar chemical structure. Even though a compound can be excreted without undergoing any change, it is usually converted by biotransformation enzymes into a more hydrophilic derivative prior to elimination. The biotransformation reactions are carried out by phase I enzymes such as cytochrome P450 (CYP) members and by phase II conjugating enzymes such as glutathione S-transferase (GST; EC 2.5.1.18), UDP-glucuronosyltransferase (UGT; EC 2.4.1.17), and sulfotransferase (SULT; EC 2.8.2.) [1, 2]. Phase I enzymes usually act in tandem with phase II enzymes, which ultimately results in incorporation of anionic groups into the xenobiotic molecule. The hydrophilic derivatives can be excreted from the cells by phase III or membrane transport systems, with the anionic groups acting as *affinity markers* for a series of transporters of the multidrug resistance-associated protein (MRP) family. These proteins are members of the ATP-binding cassette (ABC) drug efflux transporters and mediate the active extrusion of biotransformation products for their subsequent elimination from the body [1, 2]. Direct elimination of xenobiotics without suffering any biotransformation is also

What follows is a description of the role of the multidrug resistance-associated protein 2 (MRP2, ABCC2) as an important component of the ABC family involved in xenobiotic

**1.2. Multidrug resistance-associated protein 2 and its role in the detoxification of xenobiotics** MRP2 structure consists of a large core segment, containing two cytosolic nucleotide-binding domains (NBDs), two membrane-spanning domains (MSDs), and a linker segment L1, shared by other ABC members. Additionally, MRP2 contains a third NH2-terminal membranespanning domain MSD0, also called terminal transmembrane domain, with five transmembrane helices resulting in an extracellular NH2-terminus and an intracellular linker segment 0 (L0) (**Figure 1**) [4]. MRP2 is characteristically expressed at the apical membrane of polarized cells such as hepatocytes, enterocytes, and renal tubular cells [4–6], where it plays a primary role in the elimination of specific compounds. Particularly, the highest concentration of this transporter is found in the liver and intestine [7], two organs playing a prominent role in xenobiotic detoxification commonly known as "first-pass metabolism and clearance". Protein expression of MRP2 is highest in enterocytes of the proximal small intestine, decreasing in

**Figure 1.** (A) Molecular structure of MRP2/ABCC2. MSDs: membrane-spanning domains; NBDs: nucleotide-binding domains; L0: linker segment 0. (B) Coordinated action between phase II metabolizing enzymes and MRP2 in the enterocyte. Hydrophobic xenobiotics (X) may enter the cell by diffusion through the apical membrane of the enterocyte. After that, they may suffer metabolic phase I reaction by cytochrome P450 and/or subsequent conjugation by phase II enzymes, such as UDP-glucuronosyltransferase (UGT) localized in the endoplasmic reticulum or glutathione S-transferase (GST) or sulfotransferase (SUL) localized in the cytosol. In the phase III, the more hydrophilic metabolites (Y) may be actively secreted into the intestinal lumen by MRP2. (C) Coordinated action between phase II metabolizing enzymes and MRP2 in the hepatocyte. Here, hydrophobic xenobiotics (X) enter the cell through the basolateral pole of the membrane in the hepatocyte. After conjugation by phase II metabolizing enzymes, the final product (**Y**) may be secreted into bile by MRP2.

Selected substrates of MRP2 are included in **Table 1**. Studies using Groningen Yellow (GY)/ TR- Wistar [15, 16] or Eisai hyperbilirubinemic rats (EHBR) [17] that are rMrp2 deficient as a result of mutations leading to premature stop codons have helped to identify transporter substrates initially. Interestingly, ingredients of our daily diet are substrates for MRP2. That is the case of the tea component epicatechin, the dietary supplement chrysin [18, 19], and the meat-derived dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) [20]. Finally, it is important to emphasize that there are MRP2 substrates which influence transporter expression thus influencing its own bioavailability. That is the case, for example, of tamoxifen [21].


**Table 1.** Selected MRP2 substrates.

## **2. Regulation of expression and activity of MRP2 by xenobiotics**

Expression of MRP2 can be regulated at different levels [22], which can be grouped in two main categories: (i) transcriptional level, implying changes in mRNA synthesis rate, or (ii) posttranscriptional level, comprising complex processes involving or not changes in MRP2 mRNA levels. Regarding the transcriptional regulation, the MRP2 promoter contains a number of binding sites recognizing a variety of transcription factors, which in turn can be activated by either endo- or xenobiotics [23, 24]. Thus, a wide variety of drugs, environmental pollutants, and natural toxins behave as ligands of transcription factors/nuclear receptors such as farnesoid X-activated receptor (FXR), pregnane X receptor (PXR), liver X receptor α (LXRα), and constitutive androstane receptor (CAR), to ultimately induce the synthesis of MRP2 mRNA [25]. They modulate MRP2 transcriptionally through binding to the response element ER-8, which extends from −401 to −376 bp of the MRP2 promoter [25]. Additionally, the study of MRP2 promoter identified binding sites for other transcription factors such as the nuclear factor-erythroid 2-related factor-2 (Nrf2), the peroxisome proliferator-activated receptor alpha (PPARα), CCAAT/enhancer-binding protein-β (C/EBPβ), and hepatic nuclear factors (HNFs), which can also influence the MRP2 expression at the transcriptional level. Finally, a particular transcriptional regulation involves the intracellular nucleotide cyclic adenosine monophosphate (cAMP) pathway, likely triggered by xenobiotics acting predominantly through binding to plasma membrane receptors. This was so far demonstrated in the intestine [26], where the treatment of Caco-2 human intestinal cells with the membrane-permeable analogue dibutyryl cAMP or the adenylyl cyclase activator forskolin led to a significant induction in hMRP2 protein and mRNA expression. Reporter gene and chromatin immunoprecipitation assays performed in this same study showed an increased binding of the transcription factors c-JUN and activating transcription factor-2 (ATF2) to a regulatory region containing activator protein-1 (AP-1) and cAMP response element (CRE) binding sites within the MRP2 promoter.

On the other hand, post-transcriptional MRP2 regulation can involve the dynamic endocytic retrieval and exocytic insertion of this transporter between the canalicular membrane and an intracellular pool of vesicles [27]. A variety of signal transduction pathways involving the activation of the mitogen-activated protein kinases (MAPK) A and C take part in these events [27–29]. Also, mRNA splicing may account for post-transcriptional regulation. As an example, alternative splicing of hMRP2 mRNA has been shown to be a cause underlying Dubin–Johnson syndrome as a consequence of synthesis of nonfunctional protein [30, 31]. Additionally, MRP2 can be translationally modulated. In this regard, Jones et al. [32] observed that under certain situations rMrp2 protein in rats is modified without changes in mRNA levels. This is not just attributed to a modified rate of protein degradation but to the presence of several rMrp2 transcription initiation sites in the 5' untranslated region [7, 33]. The alternative use of these sites leads to the production of different rMrp2 mRNAs with differential translational efficiency.

What follows is a description of the regulatory properties of MRP2 as an important component of the ABC family in liver and intestine. The effects of xenobiotics on MRP2 expression and activity were particularly considered. Transcriptional and post-transcriptional regulations were described separately.

## **2.1. Transcriptional regulation**

substrates initially. Interestingly, ingredients of our daily diet are substrates for MRP2. That is the case of the tea component epicatechin, the dietary supplement chrysin [18, 19], and the meat-derived dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) [20]. Finally, it is important to emphasize that there are MRP2 substrates which influence transporter expression thus influencing its own bioavailability. That is the case, for ex-

> *Anticancer drugs*: doxorubicin, epirubicin, etoposide, irinotecan, methotrexate, mitoxantrone, cisplatin, tamoxifen, vincristine, vinblastine, camptothecin *Antibiotics*: ampicillin, azithromycin, cefodizime, ceftriaxone, grepafloxacine,

*HIV drugs*: adenovir, cidofovir, indinavir, lopinavir, nelfinavir, ritonavir,

pravastatine, quercetin 4′-β-glucoside, temocaprilate, conjugates of acetaminophen, indomethacin, phenobarbital, sulfinpyrazone.

ochratoxin A, 2-amino-1-methyl-6-phenylimidazol[4,5-b]pyridin, 4- (methylnitrosamino)-1-(3-pyridyl)-1-buta-nol, α-naphtylisothio-cyanate,

heavy metal complexes (arsenic glutathione, Sb, Zn, Cu, Mn, Cd) *Dyes*: fluo-3, carboxydichloro fluorescein, sulfobromophthalein

*Flavonoids*: epicatechin, chrysin

Expression of MRP2 can be regulated at different levels [22], which can be grouped in two main categories: (i) transcriptional level, implying changes in mRNA synthesis rate, or (ii) posttranscriptional level, comprising complex processes involving or not changes in MRP2 mRNA levels. Regarding the transcriptional regulation, the MRP2 promoter contains a number of binding sites recognizing a variety of transcription factors, which in turn can be activated by either endo- or xenobiotics [23, 24]. Thus, a wide variety of drugs, environmental pollutants, and natural toxins behave as ligands of transcription factors/nuclear receptors such as farnesoid X-activated receptor (FXR), pregnane X receptor (PXR), liver X receptor α (LXRα), and constitutive androstane receptor (CAR), to ultimately induce the synthesis of MRP2 mRNA [25]. They modulate MRP2 transcriptionally through binding to the response element

**2. Regulation of expression and activity of MRP2 by xenobiotics**

*Other drugs*:genistein-7-glucoside, p-aminohippurate, olmesartan, phloridzin,

*Toxicants*: S-glutathionyl-2,4-dinitrobenzene, S-glutathionyl ethacrynic acid,

ample, of tamoxifen [21].

28 Toxicology - New Aspects to This Scientific Conundrum

Glutathione

Steroids

Bilirubin glucuronides Conjugated bile salts Leukotrienes C4, D4, E4

(17β-glucuronosyl estradiol)

ethinylestradiol-3-O-glucuronide

**Table 1.** Selected MRP2 substrates.

Triiodo-L-thyronine

**Endogenous compounds Exogenous compounds**

irinotecan

saquinavir

Several xenobiotics including therapeutic drugs, environmental pollutants, and natural toxins have shown to activate different transcription factors and nuclear receptors thus exhibiting the potential of increasing MRP2 expression [34]. However, not all xenobiotics induce the expression of MRP2. Indomethacin, a nonsteroidal anti-inflammatory drug, reduced the expression of rat rMrp2 at the mRNA and protein levels in the liver [35]. This reduction was associated with a diminished mRNA expression of the hepatic nuclear receptors CAR, FXR, PXR,retinoic acid receptor α (RARα), and retinoid X receptor α (RXRα). This down-regulation of nuclear receptors is consistent with observations in endotoxin-treated rats that also proved to cause rMrp2 down-regulation at the transcriptional level [36, 37]. Intestinal injury caused by indomethacin can increase endotoxin levels in portal blood [38], which in turn induces several immune responses and oxidative stress, as shown by the reduced levels of hepatic GSH and increased levels of nitric oxide (NO) and nitrosothiols in portal blood [35]. In this regard, El Kasmi et al. demonstrated that dextran sulfate sodium-induced intestinal injury also downregulates hepatic mMrp2 expression in mice liver and that the intestinal microbiota and TLR4 (Toll-like receptor 4) are involved in this effect [39]. In addition, it was shown that mice with intestinal injury that received soy oil-based parenteral nutrition containing phytosterols exhibited an exacerbated decrease in mMrp2 mRNA levels. The phytosterol stigmasterol was at least partially involved and associated with increased levels of interleukin 6 (IL-6) mRNA and reduced levels of FXR mRNA in liver [39].

In contrast to indomethacin, several drugs are able to induce the expression of MRP2. For example, spironolactone (SL), a drug used to treat patients with edema and ascites, has been shown to increase bile flow in rats due to the up-regulation of rMrp2 at the transcriptional level, probably in response to increased PXR levels [40]. This up-regulation in rMrp2 resulted in increased efflux activity of the model substrate of rMrp2 dinitrophenyl-S-glutathione (DNP-SG) *in vitro*. These findings were in accordance with observations in patients exhibiting increased clearance of drugs co-administered with SL [41, 42]. Benznidazole (BZL), a nitroimidazole administered for treatment of Chagas disease, was also shown to induce rMrp2 protein expression [43] in rats. This induction is presumably mediated by PXR, since knockdown of the nuclear receptor in the hepatic cell line HepG2 prevented the induction of hMRP2 by this drug [44]. The antituberculosis agents rifampicin and isoniazid that cause liver injury also up-regulate hepatic rMrp2 protein expression in rats [45]. Interestingly, monoammonium glycyrrhizin, commonly present in Chinese herbal formulas used for hepatic protection, prevented such increases in rMrp2 expression. In addition rifampicin and isoniazid increased lipid peroxidation and GSH levels in hepatic tissue, indicating the presence of oxidative stress, which was also prevented by monoammonium glycyrrhizin.

Acetaminophen (APAP) represents one of the most common over-the-counter drugs, used as an effective and safe analgesic and antipyretic. Nevertheless, APAP overdose is very frequent and associated with severe liver injury. Although the mechanism underlying APAP toxicity is not completely understood, the CYP-biotransformation product N-acetyl-p-benzoquinone imine (NAPQI) was described as a mediator of APAP toxic effects since it promotes GSH depletion and binds itself to cellular proteins [46]. APAP phase II metabolites resulting from conjugation with glucuronic acid and sulfate are known MRP2 substrates. Similarly, MRP2 transports the GSH conjugate of NAPQI, thus contributing to reduce the toxic burden exerted by APAP. In this regard, the administration of a single hepatotoxic dose of APAP to Wistar rats resulted in an increase in rMrp2 expression in liver plasma membranes [47]. In a similar study, an induction hepatic mMrp2 following Nrf2 activation was demonstrated in mice, clearly suggesting the presence of an adaptive mechanism to the APAP-triggered injury [48, 49]. In line with these observations, therapeutic activation of Nrf2 has been proposed as a possible strategy to ameliorate APAP-associated hepatotoxicity [50–52]. Nrf2 bears a special toxicological relevance due to its activation by pro-oxidant compounds and reactive metabolites usually associated with situations of drug overdose or exposure to environmental toxicants. Under homeostatic conditions, Nrf2 is sequestered in the cytosol by the Kelch-like ECH-associated protein 1 (Keap1) which promotes Nrf2 ubiquitination and proteosomal degradation. Upon an oxidative stimulus, it takes place a modification in the oxidation status of particular cysteine residues in the Keap1 molecule leading to Nrf2 dissociation and migration to the nucleus where it binds to antioxidant response elements (ARE) within the promoters and activates the transcription of target genes [53] including antioxidant and GSH synthesis enzymes and also drug transporters like MRP2 [54]. Management of Nrf2 activation could be applied to other cases of oxidative stress-associated hepatic injury. For instance, the Nrf2 activator N-acetylcysteine was described to ameliorate the rMrp2 down-regulation exerted by the pro-oxidant phytochemical timosaponin A3 [55]. Similarly, other Nrf2 activators are being tested in clinical trials for the treatment of hepatic and extrahepatic diseases [53].

by indomethacin can increase endotoxin levels in portal blood [38], which in turn induces several immune responses and oxidative stress, as shown by the reduced levels of hepatic GSH and increased levels of nitric oxide (NO) and nitrosothiols in portal blood [35]. In this regard, El Kasmi et al. demonstrated that dextran sulfate sodium-induced intestinal injury also downregulates hepatic mMrp2 expression in mice liver and that the intestinal microbiota and TLR4 (Toll-like receptor 4) are involved in this effect [39]. In addition, it was shown that mice with intestinal injury that received soy oil-based parenteral nutrition containing phytosterols exhibited an exacerbated decrease in mMrp2 mRNA levels. The phytosterol stigmasterol was at least partially involved and associated with increased levels of interleukin 6 (IL-6) mRNA

In contrast to indomethacin, several drugs are able to induce the expression of MRP2. For example, spironolactone (SL), a drug used to treat patients with edema and ascites, has been shown to increase bile flow in rats due to the up-regulation of rMrp2 at the transcriptional level, probably in response to increased PXR levels [40]. This up-regulation in rMrp2 resulted in increased efflux activity of the model substrate of rMrp2 dinitrophenyl-S-glutathione (DNP-SG) *in vitro*. These findings were in accordance with observations in patients exhibiting increased clearance of drugs co-administered with SL [41, 42]. Benznidazole (BZL), a nitroimidazole administered for treatment of Chagas disease, was also shown to induce rMrp2 protein expression [43] in rats. This induction is presumably mediated by PXR, since knockdown of the nuclear receptor in the hepatic cell line HepG2 prevented the induction of hMRP2 by this drug [44]. The antituberculosis agents rifampicin and isoniazid that cause liver injury also up-regulate hepatic rMrp2 protein expression in rats [45]. Interestingly, monoammonium glycyrrhizin, commonly present in Chinese herbal formulas used for hepatic protection, prevented such increases in rMrp2 expression. In addition rifampicin and isoniazid increased lipid peroxidation and GSH levels in hepatic tissue, indicating the presence of oxidative stress,

Acetaminophen (APAP) represents one of the most common over-the-counter drugs, used as an effective and safe analgesic and antipyretic. Nevertheless, APAP overdose is very frequent and associated with severe liver injury. Although the mechanism underlying APAP toxicity is not completely understood, the CYP-biotransformation product N-acetyl-p-benzoquinone imine (NAPQI) was described as a mediator of APAP toxic effects since it promotes GSH depletion and binds itself to cellular proteins [46]. APAP phase II metabolites resulting from conjugation with glucuronic acid and sulfate are known MRP2 substrates. Similarly, MRP2 transports the GSH conjugate of NAPQI, thus contributing to reduce the toxic burden exerted by APAP. In this regard, the administration of a single hepatotoxic dose of APAP to Wistar rats resulted in an increase in rMrp2 expression in liver plasma membranes [47]. In a similar study, an induction hepatic mMrp2 following Nrf2 activation was demonstrated in mice, clearly suggesting the presence of an adaptive mechanism to the APAP-triggered injury [48, 49]. In line with these observations, therapeutic activation of Nrf2 has been proposed as a possible strategy to ameliorate APAP-associated hepatotoxicity [50–52]. Nrf2 bears a special toxicological relevance due to its activation by pro-oxidant compounds and reactive metabolites usually associated with situations of drug overdose or exposure to environmental toxicants. Under

and reduced levels of FXR mRNA in liver [39].

30 Toxicology - New Aspects to This Scientific Conundrum

which was also prevented by monoammonium glycyrrhizin.

Environmental pollutants such as arsenite significantly increased rMrp2 protein expression in rat liver after 2 weeks of exposure. Longer exposure treatment (4 or 6 weeks) also increased rMrp2 expression but to a lesser extent [56]. Arsenite not only regulates Mrp2 but is also an MRP2 substrate, so transporter induction may help to counteract the toxic effects of arsenite in the liver. In agreement with this, arsenite content in bile decreased with the exposure time in the same manner as rMrp2 protein induction. Lipid peroxidation was increased and GSH peroxidase activity was reduced in the liver at 4 and 6 weeks of arsenite exposure, indicating a probable effect of oxidative stress in attenuating hepatic rMrp2 induction. This regulation of Mrp2 may explain the dual effects reported for arsenite exposure [57].

The T-2 mycotoxin is commonly found in different crops. Prolonged exposure (3 weeks) of poultry to T-2 reduced cMrp2 mRNA expression in the liver of broiler chickens [58]. Although the authors suggest that PXR may be involved in the down-regulation observed, no studies were conducted to prove that. The organochlorine pesticides 2,4'-dichlorodiphenyltrichloroethane (DDT), 4,4'-DDT, chlordane, heptachlor, dieldrin, and lindane, highly resistant to degradation, were shown to increase hMRP2 mRNA in HepaRG cells after a 48-h exposure [59]. This regulation could be mediated by PXR, since this nuclear receptor expression was increased in human hepatocytes after treatment with chlordane, dieldrin, and endosulfan [60].

Bisphenol A (BPA) is a typical contaminant of food, water, and air. A study evaluating the association between the expression of drug transporters in fetal liver and BPA exposure showed a positive correlation between BPA levels and hMRP2 expression. A similar correlation was described between BPA levels and Nrf2 expression, suggesting also a mediation of this transcription factor in the hMRP2 regulation by BPA and agreeing well with a previous report showing Nrf2 activation by BPA *in vitro* [61, 62].

Transcriptional regulation of MRP2 by xenobiotics was also demonstrated in extrahepatic tissues like the small intestine. An example of therapeutic drugs affecting MRP2 gene expression is rifampicin, a well-known PXR agonist which has been demonstrated to increase hMRP2 expression and activity in healthy volunteers. Fromm et al. reported an up-regulation of hMRP2 mRNA and protein in duodenal biopsies [63]. Later, Oswald et al. showed the impact of this regulation on oral availability and efficacy of the coadministered drug ezetimibe [64]. The same inducing effect was reported using the human-derived cell line LS180 [65]. Similarly, carbamazepine is another PXR agonist [66] shown to increase hMRP2 mRNA and protein levels in human healthy volunteers and also to reduce intestinal absorption of the co-administered MRP2 substrate talinolol [67].

Even though there are only few reports on drug–drug interactions associated with PXRdependent modulation of intestinal MRP2, many other agents have been shown to regulate its expression via PXR. Pregnenolone-16α-carbonitrile, a synthetic steroid, induced mMrp2 mRNA expression in jejunum of C57BL/6 mice (200 mg/kg, i.p., 4 days). A concomitant upregulation of PXR mRNA was observed and the involvement of this nuclear receptor was confirmed using PXR knockout mice [68]. The antiviral agents and PXR agonists efavirenz and saquinavir [69] also up-regulated hMRP2 expression in LS180 cells [65, 70]. Since these drugs are usually involved in long-term treatments, coadministration with MRP2 substrates could result in unwanted drug–drug interactions. In the same human intestinal cell line, the effect of the endothelin receptor antagonist bosentan [71] and the antineoplastic mitotane [72] was demonstrated. They both exerted a significant hMRP2 induction concomitantly with PXR activation at pharmacologically relevant concentrations.

In some studies, the increases in MRP2 expression were correlated experimentally with increased transport activity. Examples of these drugs are BZL and SL, both PXR agonists. Perdomo et al. reported an induction of rMrp2 protein in jejunum of BZL-treated rats (100 mg/ kg, i.p., 3 days) [43]. This change was accompanied with an increased efflux of DNP-SG to the intestinal lumen. These results not only demonstrate higher secretion of rMrp2 substrates but also strongly suggest a restriction in the absorption of xenobiotics incorporated luminally in BZL-treated rats. Similarly, SL was demonstrated to increase serosal to mucosal transport of DNP-SG well correlating with increased rMrp2 mRNA and protein expression in rat proximal jejunum. The PXR antagonist ketoconazole was able to prevent this induction, suggesting mediation by this nuclear receptor [73].

Cimetidine and quinidine have been shown to increase both hMRP2 and PXR expressions in T84 cells [74]. Similarly, the anticonvulsant phenobarbital induced PXR mRNA and hMRP2 mRNA and protein at the same time in Caco-2 cells [75]. On the contrary, MRP2 expression is down-regulated by drugs that decrease PXR expression. Haslam et al. reported such an effect in T84 cells for the cholesterol-lowering drug atorvastatin and the anticancer agents topotecan and irinotecan [74].

Xenobiotics can regulate expression of intestinal MRP2 interacting with nuclear receptors other than PXR. That is the case of the bcl-2 inhibitor obatoclax, which at nanomolar concentrations induced hMRP2 mRNA in LS180 cells through activation of the aryl hydrocarbon receptor [76]. Another example is the proteasome inhibitor bortezomib, which also increases hMRP2 mRNA expression in SW-480 human cells [77]. Even though the mediator has still to be identified, the transcription factor Nrf2 appears to be the main candidate considering its simultaneous upregulation and also the well-demonstrated relationship between MRP2 induction and Nrf2 activation in tissues like liver [49], kidney [78], and brain [79].

Among the natural compounds modulating intestinal MRP2, the isothiocyanates sulforaphane (SF) and erucin (ER) were well studied. Derived from cruciferous vegetables, they have drawn the attention of the researchers due to their anticancer properties [80]. After microarray studies, Traka et al. reported hMRP2 up-regulation in Caco-2 cells treated with SF (50 μM, 24 h) [81]. They confirmed this finding by RT-PCR analysis. Using the same model, Jakubíková et al. showed a similar hMRP2 mRNA increase after treatment with SF and with ER (20 μM, 24 h) [82]. This effect appears to be partly mediated by phosphoinositide 3-kinase (PI3K)/AKT, considering the inhibition exhibited by the PI3K/AKT inhibitor LY294002. Later, in analogous experimental conditions, Harris and Jeffery demonstrated an induction also at protein level [83]. Considering that Nrf2 is activated by SF [84], this transcription factor may play a role in MRP2 up-regulation.

Other naturally occurring xenobiotics found to modulate intestinal MRP2 are the polyphenols quercetin and resveratrol (RES). The first one was demonstrated to up-regulate hMRP2 at protein level in Caco-2 cells (50 and 100 μM, 72 h). A coordinated induction of the phase II enzyme UGT1A6 was also observed, suggesting reduced absorption and enhanced secretion of glucuronides at intestinal level [85]. Studies using RES have become of strong interest after the increasing evidence regarding its beneficial health effects. A short time ago, RES ability to down-regulate MRP2 at mRNA and protein levels has been proven in rat intestine and Caco-2 cells [86]. The functional impact was evaluated in the latter model by determination of intracellular retention of the MRP2 substrate methotrexate (MTX). Treatment with RES increased the accumulation of MTX, suggesting a suppressed efflux activity. Moreover, in an attempt to clarify the mechanism, the authors found a concomitant inhibition of the insulinlike growth factor receptor 1 (IGF-1R)/AKT/ERK signaling pathway.

## **2.2. Post-transcriptional regulation**

in human healthy volunteers and also to reduce intestinal absorption of the co-administered

Even though there are only few reports on drug–drug interactions associated with PXRdependent modulation of intestinal MRP2, many other agents have been shown to regulate its expression via PXR. Pregnenolone-16α-carbonitrile, a synthetic steroid, induced mMrp2 mRNA expression in jejunum of C57BL/6 mice (200 mg/kg, i.p., 4 days). A concomitant upregulation of PXR mRNA was observed and the involvement of this nuclear receptor was confirmed using PXR knockout mice [68]. The antiviral agents and PXR agonists efavirenz and saquinavir [69] also up-regulated hMRP2 expression in LS180 cells [65, 70]. Since these drugs are usually involved in long-term treatments, coadministration with MRP2 substrates could result in unwanted drug–drug interactions. In the same human intestinal cell line, the effect of the endothelin receptor antagonist bosentan [71] and the antineoplastic mitotane [72] was demonstrated. They both exerted a significant hMRP2 induction concomitantly with PXR

In some studies, the increases in MRP2 expression were correlated experimentally with increased transport activity. Examples of these drugs are BZL and SL, both PXR agonists. Perdomo et al. reported an induction of rMrp2 protein in jejunum of BZL-treated rats (100 mg/ kg, i.p., 3 days) [43]. This change was accompanied with an increased efflux of DNP-SG to the intestinal lumen. These results not only demonstrate higher secretion of rMrp2 substrates but also strongly suggest a restriction in the absorption of xenobiotics incorporated luminally in BZL-treated rats. Similarly, SL was demonstrated to increase serosal to mucosal transport of DNP-SG well correlating with increased rMrp2 mRNA and protein expression in rat proximal jejunum. The PXR antagonist ketoconazole was able to prevent this induction, suggesting

Cimetidine and quinidine have been shown to increase both hMRP2 and PXR expressions in T84 cells [74]. Similarly, the anticonvulsant phenobarbital induced PXR mRNA and hMRP2 mRNA and protein at the same time in Caco-2 cells [75]. On the contrary, MRP2 expression is down-regulated by drugs that decrease PXR expression. Haslam et al. reported such an effect in T84 cells for the cholesterol-lowering drug atorvastatin and the anticancer agents topotecan

Xenobiotics can regulate expression of intestinal MRP2 interacting with nuclear receptors other than PXR. That is the case of the bcl-2 inhibitor obatoclax, which at nanomolar concentrations induced hMRP2 mRNA in LS180 cells through activation of the aryl hydrocarbon receptor [76]. Another example is the proteasome inhibitor bortezomib, which also increases hMRP2 mRNA expression in SW-480 human cells [77]. Even though the mediator has still to be identified, the transcription factor Nrf2 appears to be the main candidate considering its simultaneous upregulation and also the well-demonstrated relationship between MRP2 induction and Nrf2

Among the natural compounds modulating intestinal MRP2, the isothiocyanates sulforaphane (SF) and erucin (ER) were well studied. Derived from cruciferous vegetables, they have drawn the attention of the researchers due to their anticancer properties [80]. After microarray studies,

MRP2 substrate talinolol [67].

32 Toxicology - New Aspects to This Scientific Conundrum

activation at pharmacologically relevant concentrations.

activation in tissues like liver [49], kidney [78], and brain [79].

mediation by this nuclear receptor [73].

and irinotecan [74].

As anticipated, post-transcriptional regulation of MRP2 activity may also occur in response to exposition to xenobiotics. Faldaprevir is a drug used to treat patients with hepatitis C in spite that it causes hyperbilirubinemia. Acute treatment of human and rat hepatocytes with faldaprevir inhibited hMRP2-/rMrp2-mediated efflux of bilirubin glucuronides into bile [87]. This may partially explain the impaired bilirubin disposition by faldaprevir. However, toxicology studies in monkeys and patients showed that the bilirubin accumulated during faldaprevir treatment was mainly unconjugated, suggesting that the main cause is probably inhibition of glucuronidation ather than excretion of the conjugated metabolites. Simeprevir, another drug used to treat hepatitis, causes hyperbilirubinemia composed by conjugated and unconjugated bilirubin, which supports an impaired hMRP2 efflux activity [88].

Ethynylestradiol (EE) and genistein (GNT) are estrogenic compounds of particular relevance considering their oral incorporation as components of contraceptives formulations and soyderived food, respectively. Interestingly, they were demonstrated to regulate intestinal MRP2 at post-transcriptional level, although showing a noticeable dose and model dependence. Thus, it was initially found that EE at a cholestatic dose (5 mg/kg b.w. day, for 5 consecutive days, s.c.) down-regulates rMrp2 expression and function in rat proximal jejunum, without changes in mRNA levels [89]. However, at pharmacological concentrations (0.5–5 pM) EE upregulates hMRP2 expression and activity in Caco-2 cells. This hMRP2 induction was estrogen receptor β (ERβ)-mediated but not associated with changes at the mRNA level, thus suggesting a post-transcriptional regulation [90]. The implication of ERβ in such kind of regulation is possible since it was reported to regulate miRNAs expression [91] which, in turn, can alter hMRP2 expression [92]. Similarly, Caco-2 cells exposed to GNT (1 μM, 48 h) exhibited an ERβ-mediated hMRP2 induction at protein level without changes in expression at the mRNA level. This was associated with increased transporter activity and enhanced protection against 1-chloro-2,4-dinitrobenzene, an MRP2 substrate precursor [90]. GNT can also modulate MRP2 activity in an acute fashion, i.e., without changes in transporter expression. Some years ago, it was reported that this isoflavone competitively inhibits hepatic rMrp2 in isolated and perfused rat liver model [93]. In line with this result, Yokooji et al. demonstrated that GNT administered intravenously reduced rMrp2-mediated secretion of irinotecan hydrochloride and its metabolites in rat liver and intestine [94].

The uricosuric drug probenecid represents another example of xenobiotics affecting intestinal MRP2 activity without modifying MRP2 expression. Probenecid is a classical competitive inhibitor of organic anions transport also used in the clinical practice to enhance plasma levels of antibiotics. Although nonspecific, its inhibitory effect on intestinal MRP2 was clearly demonstrated in rats [95] and in Caco-2 cells [90]. Nowadays, more potent and specific MRP inhibitors like MK571 are preferred for characterization of transport specificity. Finally, various members of the large family of nonsteroidal anti-inflammatory drugs are recognized MRP2 modulators [96]. In intestine, indomethacin was shown to inhibit MRP2 and to increase sulfasalazine transepithelial permeability, both in rat small intestine and in Caco-2 cell monolayers [97]. In agreement with these findings, Caco-2 cells coincubated with indomethacin exhibited an increased permeability to the hMRP2 substrates fluvastatin [98] and colchicine [99].

## **3. Conclusion**

Although the organisms are permanently exposed to a wide range of xenobiotics such as therapeutic drugs, environmental pollutants, and natural toxins, they possess a sophisticate system of detoxification in which metabolizing enzymes and transport proteins play an essential role. Evidence from *in vitro* and *in vivo* studies unambiguously demonstrates that MRP2 is a crucial actor in protecting specific tissues from xenobiotic toxicity. It is noteworthy that MRP2 also plays a crucial role in elimination of endogenous metabolites. A list of relevant and well-recognized substrates of MRP2 is presented in **Table 1**. Two relevant tissues expressing MRP2 are the liver and the small intestine. It should be noted that other participants not contemplated in this review, such as a bunch of metabolizing enzymes as well as members of the family of ABC transporters different from MRP2, are additionally involved in xenobiotic detoxification.

Expression and activity of MRP2 can be modulated at both transcriptional and post-transcriptional levels. The nuclear receptor PXR plays a major role in transcriptional regulations. PXR functions as sensor for many agents and its activation leads to a coordinated response on biotransformation enzymes and transport systems. Examples of these agents are rifampicin, carbamazepine, pregnenolone-16α-carbonitrile, efavirenz, saquinavir, BZL, SL, etc. Other xenobiotics such as bortezomib, BPA, and sulforaphane regulate MRP2 as a result of the interaction with Nrf2. Alternatively, regulation of MRP2 activity by xenobiotics can occur without changes in transporter expression, as it was demonstrated to GNT. Finally, it is important to note that regulation of MRP2 activity by therapeutic agents can result in changes in their therapeutic efficacy or safety, or alternative in drug–drug interactions if other drugs, substrates of MRP2, are simultaneously administered.

**Abbreviations**: ABC, ATP-binding cassette; AP-1, activator protein-1; APAP, acetaminophen; ARE, antioxidant response elements; ATF2, activating transcription factor-2; BPA, bisphenol A; BZL, benznidazole; C/EBPβ, CCAAT/enhancer-binding protein-β; cAMP, cyclic adenosine monophosphate; CAR, constitutive androstane receptor; CRE, cAMP response element; CYP, cytochrome P450; DDT, dichlorodiphenyltrichloroethane; DNP-SG, dinitrophenyl-S-glutathione; EE, ethynylestradiol; EHBR, Eisai hyperbilirubinemic rats; ER, erucin; ERβ, estrogen receptor β; FXR, farnesoid X-activated receptor; GNT, genistein; GY, Groningen Yellow; GSH, glutathione; GST, glutathione S-transferase; HNFs, hepatic nuclear factors; IGF-1R, insulinlike growth factor receptor 1; IL-6, interleukin 6; Keap1, Kelch-like ECH-associated protein 1; L0, linker segment 0; LXRα, liverXreceptorα; MAPK, mitogen-activated protein kinases; MRP, multidrug resistance-associated protein; MRP2, multidrug resistance-associated protein 2; hMRP2, human MRP2; rMrp2, rat Mrp2; mMrp2, mouse Mrp2; cMrp2, chickenMrp2; MSDs, membrane-spanning domains; MTX, methotrexate; NAPQI, N-acetyl-p-benzoquinone imine; NBDs, nucleotide-binding domains; NO, nitric oxide; Nrf2, nuclear factor-erythroid 2- related factor-2; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PI3K, phosphoinositide 3 kinase; PPARα, peroxisome proliferator activated receptor alpha; PXR, pregnane X receptor; RARα,retinoic acid receptor α;RES,resveratrol;RXRα,retinoidXreceptor α; SF, sulforaphane; SL, spironolactone; SULT, sulphotransferase; TLR4, tool-like receptor 4; UGT, UDP-glucuronosyltransferase.

## **Author details**

possible since it was reported to regulate miRNAs expression [91] which, in turn, can alter hMRP2 expression [92]. Similarly, Caco-2 cells exposed to GNT (1 μM, 48 h) exhibited an ERβ-mediated hMRP2 induction at protein level without changes in expression at the mRNA level. This was associated with increased transporter activity and enhanced protection against 1-chloro-2,4-dinitrobenzene, an MRP2 substrate precursor [90]. GNT can also modulate MRP2 activity in an acute fashion, i.e., without changes in transporter expression. Some years ago, it was reported that this isoflavone competitively inhibits hepatic rMrp2 in isolated and perfused rat liver model [93]. In line with this result, Yokooji et al. demonstrated that GNT administered intravenously reduced rMrp2-mediated secretion of irinotecan hydrochloride and its metab-

The uricosuric drug probenecid represents another example of xenobiotics affecting intestinal MRP2 activity without modifying MRP2 expression. Probenecid is a classical competitive inhibitor of organic anions transport also used in the clinical practice to enhance plasma levels of antibiotics. Although nonspecific, its inhibitory effect on intestinal MRP2 was clearly demonstrated in rats [95] and in Caco-2 cells [90]. Nowadays, more potent and specific MRP inhibitors like MK571 are preferred for characterization of transport specificity. Finally, various members of the large family of nonsteroidal anti-inflammatory drugs are recognized MRP2 modulators [96]. In intestine, indomethacin was shown to inhibit MRP2 and to increase sulfasalazine transepithelial permeability, both in rat small intestine and in Caco-2 cell monolayers [97]. In agreement with these findings, Caco-2 cells coincubated with indomethacin exhibited an increased permeability to the hMRP2 substrates fluvastatin [98] and

Although the organisms are permanently exposed to a wide range of xenobiotics such as therapeutic drugs, environmental pollutants, and natural toxins, they possess a sophisticate system of detoxification in which metabolizing enzymes and transport proteins play an essential role. Evidence from *in vitro* and *in vivo* studies unambiguously demonstrates that MRP2 is a crucial actor in protecting specific tissues from xenobiotic toxicity. It is noteworthy that MRP2 also plays a crucial role in elimination of endogenous metabolites. A list of relevant and well-recognized substrates of MRP2 is presented in **Table 1**. Two relevant tissues expressing MRP2 are the liver and the small intestine. It should be noted that other participants not contemplated in this review, such as a bunch of metabolizing enzymes as well as members of the family of ABC transporters different from MRP2, are additionally involved in xenobiotic

Expression and activity of MRP2 can be modulated at both transcriptional and post-transcriptional levels. The nuclear receptor PXR plays a major role in transcriptional regulations. PXR functions as sensor for many agents and its activation leads to a coordinated response on biotransformation enzymes and transport systems. Examples of these agents are rifampicin, carbamazepine, pregnenolone-16α-carbonitrile, efavirenz, saquinavir, BZL, SL, etc. Other

olites in rat liver and intestine [94].

34 Toxicology - New Aspects to This Scientific Conundrum

colchicine [99].

**3. Conclusion**

detoxification.

Maite R. Arana1 , Guillermo N. Tocchetti1 , Juan P. Rigalli1,2, Aldo D. Mottino1 , Fabiana García3 and Silvina S.M. Villanueva1\*

\*Address all correspondence to: villanueva@ifise-conicet.gov.ar

1 Instituto de Fisiología Experimental (IFISE-CONICET)-Facultad de Ciencias Bioquímicas y Farmacéuticas-Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina

2 Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University, Heidelberg, Germany

3 Facultad de Ciencias Médicas-Universidad Nacional de Rosario-CONICET-Cátedra Fisiología Humana, Rosario, Santa Fe, Argentina

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#### **Chapter 3 Provisional chapter**

#### **Nanotoxicology: A Review Nanotoxicology: A Review**

Chavon Walters, Edmund Pool and Chavon Walters, Edmund Pool and

Vernon Somerset Vernon Somerset

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44 Toxicology - New Aspects to This Scientific Conundrum

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

http://dx.doi.org/10.5772/64754

#### **Abstract**

Nanotoxicology represents a new and growing research area in toxicology. It deals with the assessment of the toxicological properties of nanoparticles (NPs) with the intention of determining whether (and to what extent) they pose an environmental or societal threat. Inherent properties of NPs (including size, shape, surface area, surface charge, crystal structure, coating, and solubility/dissolution) as well as environmental factors (such as temperature, pH, ionic strength, salinity, and organic matter) collectively influence NP behavior, fate and transport, and ultimately toxicity. The mechanisms underlying the toxicity of nanomaterials (NMs) have recently been studied extensively. Reactive oxygen species (ROS) toxicity represents one such mechanism. An overproduction of ROS induces oxidative stress, resulting in inability of the cells to maintain normal physiological redoxregulated functions. In the context of this book, this chapter includes topics pertaining to chemical and physical properties of NMs and characterization for proper toxicological evaluation, exposure, and environmental fate and transport, and ecological and genotoxic effects. This chapter reviews the available research pertaining specifically to NMs in the aquatic environment (in plants, aquatic invertebrates, and fish) and their use in biomarker studies.

**Keywords:** nanomaterials, characterization techniques, nanotoxicology, biomarkers, analytical methods, in vitro studies, in vivo studies

## **1. Introduction**

Nanotoxicology is regarded as the assessment of the toxicological properties of nanoparticles (NPs) with the intention of determining whether (and to what extent) they may pose an environmental or societal threat. Nanotechnology has advanced exponentially over the past

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

decade, with nanoscale materials being exploited in several applications and in several disciplines (including industry, science, pharmacy, medicine, electronics, and communication products). Vance et al. [1] reported a 30-fold increase in nano-based products between 2011 and 2015 (**Figure 1**) and an estimated global market of over \$1 trillion in 2015 [2]. Metal NPs (specifically, carbon and silver NPs) represent the largest and fastest growing group of NPs (**Figure 2**). Hence, human and environmental exposure is already occurring and is predicted to increase dramatically. This growth in nanotechnology has not advanced without concerns regarding their potential adverse environmental impacts. Several reviews have reported on the toxicity of various NPs [3, 4]. However, much is still unknown.

**Figure 1.** Nanomaterial growth trend 2010–2015 [1].

**Figure 2.** Composition of nanomaterials (adapted from Vance et al. [1]).

Nanomaterials (NMs) are generally defined as a substance having particles with at least one dimension of 1–100 nm in length. Their novel physical and chemical characteristics have made them useful in several applications; however, these very properties can be potentially toxic. Once introduce into aquatic ecosystems, the fate and toxicity of NPs and its uptake by aquatic organisms depend on several factors. Both the properties of NP (such as size, shape, and coatings) and water chemistry (such as dissolved organic carbon, ionic strength, pH, temperature) will largely influence the extent to which NPs will either remain in suspension, partition to dissolved organic carbon in the water column, form aggregates, or adsorb to suspended particles. In aquatic organisms, the accumulation of NPs is dependent on both the uptake and the elimination of the NP out of the organism. These processes also regulate the bioaccumulation (and bioavailability) of NPs. The availability of appropriate methodologies is needed to address key issues in nanotoxicology and to gain a better understanding of nanoparticle toxicity mechanisms (including oxidative stress, cytotoxicity, genotoxicity, and inflammatory responses). Exposure to NMs is largely through ingestion and adsorption to surface epithelia such as the gills.

decade, with nanoscale materials being exploited in several applications and in several disciplines (including industry, science, pharmacy, medicine, electronics, and communication products). Vance et al. [1] reported a 30-fold increase in nano-based products between 2011 and 2015 (**Figure 1**) and an estimated global market of over \$1 trillion in 2015 [2]. Metal NPs (specifically, carbon and silver NPs) represent the largest and fastest growing group of NPs (**Figure 2**). Hence, human and environmental exposure is already occurring and is predicted to increase dramatically. This growth in nanotechnology has not advanced without concerns regarding their potential adverse environmental impacts. Several reviews have reported on the

toxicity of various NPs [3, 4]. However, much is still unknown.

**Figure 1.** Nanomaterial growth trend 2010–2015 [1].

46 Toxicology - New Aspects to This Scientific Conundrum

**Figure 2.** Composition of nanomaterials (adapted from Vance et al. [1]).

Nanomaterials (NMs) are generally defined as a substance having particles with at least one dimension of 1–100 nm in length. Their novel physical and chemical characteristics have made them useful in several applications; however, these very properties can be potentially toxic. Aquatic ecosystems are progressively coming under pressure due to the presence of emerging anthropogenic contaminants, including NMs, posing health hazards to inhabitant organisms. In recent years, increasing data demonstrated that NPs could induce toxicity and genotoxicity under a variety of exposure scenarios. An accepted mechanism by which NPs may induce cytotoxicity is considered to be through the induction of reactive oxygen species (ROS) which can induce oxidative stress which in turn may lead to cytotoxicity, DNA damage, and other effects.

Although research on the environmental impacts of NMs has grown dramatically over the past decade, studies investigating the environmental fate, transport, and toxicity of a variety of nanomaterials are still lacking. This chapter will review the available research pertaining specifically to NMs in the aquatic environment (in plants, aquatic invertebrates, and fish) and their use in biomarkers studies.

## **2. Physical and chemical properties of NMs influencing their toxicity**

The behavior of NPs in various environmental matrices is complex and involves several processes. Properties of NMs are unique and different from conventional materials. Properties such as (1) particle size, (2) surface area and charge, (3) shape/structure, (4) solubility, and (5) surface coatings are known to affect NP toxicity.

Small owing to their small size, NMs have unique physical and chemical characteristics such as magnetic, optical, thermal, mechanical, electrical properties which make them suitable in several applications including in medicine, electronics, and energy production, and in several consumer products. However, these very properties have the potential to affect humans and the environment adversely. NPs can easily penetrate cell membranes and other biological barriers into living organisms causing cell damage. Studies reporting increased toxicity of NPs when compared to their larger bulk particles have led to a generally assumed hypothesis that NPs are more potent in causing damage. Lankvel et al. [5] reported the significance of particle size of AgNPs, reporting size-specific tissue distribution and size-specific toxicity. Scown et al. [6] reported the lowest aggregation potential for the smallest AgNPs (i.e., 10 nm vs. 35 and 600–1600 nm) and was most highly concentrated in the gills and liver. Gaiser et al. [7] studied the acute and chronic toxicities of nano- and bulk Ag and CeO2. Reported mortality rates for Ag and AgNP were as follows: micro-Ag at 0.1 mg/L was 13% and at 1 mg/L was 80%, while for AgNP at 0.1 mg/L was 57% and at 1 mg/L was 100%.

Although NPs size can be the most distinguishing characteristic when compared to conventional particles, shape and morphology also represent important factors when considering NP toxicity. Morphology (particles, spheres, rods, cubes, truncated triangles, wires, films, and coatings) affects NP kinetics and their transport in the environment. NP shape is also of importance as triangular nanoplates was reported to have greater inhibition of *Escherichia coli* than spherical- or rod-shaped AgNPs or Ag+ which can be attributed to the increased reactive, high atom density of the triangular nanoplates [8, 9].

When NPs are discarded, they can enter the aquatic environment as aggregates and soluble ions, which can be highly toxic to aquatic organisms. Aggregation and dissolution are key processes governing NP behavior and toxicity in the aquatic environment. These processes are largely driven by size and surface properties of NMs, as well as by the stability of natural colloids (such as dissolved organic matter). Colloidal stability is affected by several factors including the type environmental conditions such pH, temperature, and ionic strength. Romer et al. [10], investigating the stability of AgNPs, reported rapid aggregation in media with high ionic strength. Similarly, Walters et al. [11] reported higher toxicity due to the formation of smaller aggregates at elevated temperatures. These and other studies reported changes in organism exposure levels and consequent toxicity due to levels of aggregation [12]. Dissolution of NPs is also a significant process determining NPs effects in the aquatic environment. Most NPs do not dissolve in solution, but form colloid dispersions which will either remain dispersed or aggregate. As such, interactions with other colloid materials will affect the rate at which particles aggregate in an aqueous environment.

In the natural environment, NPs are not present in isolation. As such, it is important to consider the presence of other environmental stressors. For example, Walters et al. [13] reported that higher temperatures resulted in higher toxicity due to the formation of smaller aggregates at elevated temperatures and that AgNP dissolution and sedimentation contributed to a higher availability and toxicity of AgNP (and Ag+ ) to *Potamonautes perlatus*. Similarly, Liu and Hurt [14] reported higher dissolution rates of AgNPs with increased temperature. In the presence of dissolved oxygen (DO), AgNPs tend to aggregate and release Agions which in turns induce aggregation and oxidation [12].

Surface charge is a major factor in determining the particle dispersion characteristics and also will influence the adsorption of ions and biomolecules [15]. Baalousha [16] reported disaggregation of FeO NPs due to enhanced surface charge. Similarly, El Badawy et al. [17] reported surface charge-dependent toxicity of AgNPs. In addition, surface coating is indirectly related to aggregation and dissolution, as it is reported to increase the surface charge.

These unique physical and chemical properties of NPs raise concern as the conventional assumptions of chemical reactivity and behavior may not necessarily apply with regard to NPs.

## **3. Characterization**

600–1600 nm) and was most highly concentrated in the gills and liver. Gaiser et al. [7] studied the acute and chronic toxicities of nano- and bulk Ag and CeO2. Reported mortality rates for Ag and AgNP were as follows: micro-Ag at 0.1 mg/L was 13% and at 1 mg/L was 80%, while

Although NPs size can be the most distinguishing characteristic when compared to conventional particles, shape and morphology also represent important factors when considering NP toxicity. Morphology (particles, spheres, rods, cubes, truncated triangles, wires, films, and coatings) affects NP kinetics and their transport in the environment. NP shape is also of importance as triangular nanoplates was reported to have greater inhibition of *Escherichia coli*

When NPs are discarded, they can enter the aquatic environment as aggregates and soluble ions, which can be highly toxic to aquatic organisms. Aggregation and dissolution are key processes governing NP behavior and toxicity in the aquatic environment. These processes are largely driven by size and surface properties of NMs, as well as by the stability of natural colloids (such as dissolved organic matter). Colloidal stability is affected by several factors including the type environmental conditions such pH, temperature, and ionic strength. Romer et al. [10], investigating the stability of AgNPs, reported rapid aggregation in media with high ionic strength. Similarly, Walters et al. [11] reported higher toxicity due to the formation of smaller aggregates at elevated temperatures. These and other studies reported changes in organism exposure levels and consequent toxicity due to levels of aggregation [12]. Dissolution of NPs is also a significant process determining NPs effects in the aquatic environment. Most NPs do not dissolve in solution, but form colloid dispersions which will either remain dispersed or aggregate. As such, interactions with other colloid materials will affect the rate

In the natural environment, NPs are not present in isolation. As such, it is important to consider the presence of other environmental stressors. For example, Walters et al. [13] reported that higher temperatures resulted in higher toxicity due to the formation of smaller aggregates at elevated temperatures and that AgNP dissolution and sedimentation contributed to a higher

[14] reported higher dissolution rates of AgNPs with increased temperature. In the presence of dissolved oxygen (DO), AgNPs tend to aggregate and release Agions which in turns induce

Surface charge is a major factor in determining the particle dispersion characteristics and also will influence the adsorption of ions and biomolecules [15]. Baalousha [16] reported disaggregation of FeO NPs due to enhanced surface charge. Similarly, El Badawy et al. [17] reported surface charge-dependent toxicity of AgNPs. In addition, surface coating is indirectly related

These unique physical and chemical properties of NPs raise concern as the conventional assumptions of chemical reactivity and behavior may not necessarily apply with regard to

to aggregation and dissolution, as it is reported to increase the surface charge.

which can be attributed to the increased reactive,

) to *Potamonautes perlatus*. Similarly, Liu and Hurt

for AgNP at 0.1 mg/L was 57% and at 1 mg/L was 100%.

than spherical- or rod-shaped AgNPs or Ag+

48 Toxicology - New Aspects to This Scientific Conundrum

high atom density of the triangular nanoplates [8, 9].

at which particles aggregate in an aqueous environment.

availability and toxicity of AgNP (and Ag+

aggregation and oxidation [12].

NPs.

Nanomaterial behavior and toxicity are influenced by their physical and chemical properties. As such, characterization of NMs is essential in order to understand how their physical and chemical properties correlate with chemical, ecological, or biological responses. Full characterization of NPs includes determining the bulk (shape, size, phase, electronic structure, and crystallinity) and surface (surface area, arrangement of surface atoms, surface electronic structure, surface composition, and functionality) properties of the NM. In addition, environmental factors (such as temperature, pH, ionic strength, salinity, organic matter) may also affect NP behavior and toxicity.

Standardized tests established by the Organization for Economic Co-operation and Development (OECD) summarized some of the analytical methods commonly used in the characterization of NMs (OECD ENV/JM/MONO (2016)2. The morphology of NMs is frequently characterized using scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Energy-dispersive X-ray (EDX) spectrometry coupled to SEM is a common method to characterize elemental analysis of NMs. Particle size in aqueous phase may also be determined indirectly by dynamic light scattering (DLS) which measures the Brownian movement of the NPs, or by electrophoretic light scattering spectroscopy (ELS) which uses oscillating electric field. X-ray powder diffraction (XRD) is employed to measure particle size in the dry state and which applies the Scherrer method. Murdock et al. [18] investigated the use of DLS to characterize NM dispersion.

Brunauer-Emmet-Teller (BET) is used to measure surface area. Zeta potential measures surface charge in particles in the aqueous phase and is a fundamental parameters known to affect stability. Atomic force microscopy (AFM) and scanning tunneling microscopy (STM) allow three-dimensional imaging of nanometer scale surfaces and the measurement of forces between surfaces at the pico newton scale. UV-vis and Fourier transform infrared spectroscopy (FTIR) are spectroscopic techniques used in the characterization of fullerenes in solution [19, 20]. Surface enhanced Raman spectroscopy (SERS) is a surface-sensitive technique that enhances Raman scattering by nanostructures allowing the detection of single molecules. Chemical characterization techniques include Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), and Energy-dispersive X-ray spectroscopy (EDS) used to investigate the elemental composition of NMs.

## **4. Routes of exposure in the aquatic environment**

Due to the surge in nanotechnology, there have been significant increases in the number of various NPs released into the aquatic environment. **Figure 3** provides a summary of the possible routes in a typical aquatic environment to nanoparticles, potential interactions, and the possible clearance routes. Aquatic ecosystems are susceptible to environmental contamination since they are at the receiving end of contaminants, particularly from runoff sources. Identified sources of NPs in the aquatic environment include production facilities, production processes, wastewater treatment plants, and accidents during the transport. In addition, aquatic ecosystems are known to sequester and transport contaminants, including NMs. Baun et al. showed that NPs may adhere to algae which may then be consumed by filter-feeders and transfer to higher trophic levels. In the aquatic environment, NPs may aggregate thus reducing the NPs available for direct uptake in the aqueous phase by aquatic organisms. However, aggregated NPs may settle into sediment thereby posing a threat to benthic organisms. In the aquatic environment, NMs are generally associated with sediments [21]. Sediments and soil represent porous environmental matrices which typically have large specific surface areas.

**Figure 3.** Possible pathways of nanoparticles in the aquatic environment.

Uptake of nanoparticles into the aquatic biota is a major concern. Nanomaterials are able to cross biological barriers, gaining entering due to their small size. In aquatic organisms, the major routes of entry are via ingestion or direct passage across the gill and other external surface epithelia. In invertebrates, the cellular immune system, gut epithelium, and hepatopancreas are likely to be targeted [22]. Recent studies with Mozambique tilapia (*Oreochromis mossambicus*) have indicated that nickel NPs may be internalized via these routes [23]. In addition, NPs that are taken up via ingestion through the digestive tract may accumulate in the hepatopancreas [24]. The hepatopancreas is responsible for metabolism and detoxification [25] and is known for intracellular lysosomal digestion of food via internalization by endocytosis [22]. Nanosize particles have also been demonstrated to enter the liver of fish.

Endocytosis (<100 nm) and phagocytosis (100–100,000 nm) represent the two processes by which NPs might be absorbed into eukaryotic cells. At the cellular level, internalizations of NPs occur via endocytosis. Iron oxide NP internalization via endocytosis has been reported by Auffan et al. [26].

NPs released into the environment are affected by environmental factors such as pH, temperature, and presence of organic matter. The pH affects NP surface charge and consequently also aggregation. This has been reported by Gilbert et al. [27] who reported a pH-driven aggregation and disaggregation with larger aggregate radius at higher pH. Furthermore, Adams and Kramer [28] reported increased mobility under increased acidification. Temperature is also known to affect aggregation. Walters et al. [11] reported that forma-tion of smaller aggregates at higher temperatures suggests higher toxicity. Liu and Hurt [14] reported higher dissolution rates of AgNPs with increased temperature. NPs can be immobilized as a result of sorption or binding to particles such as organic matter. These effects have been reported. For example, Chen and Elimelech [29] reported that, in the presence of humic acid, the adsorbed humic acid on the fullerene NPs led to steric repulsion, stabilization of the NP suspension, and reduced aggregation.

## **5. Mechanisms of NP toxicity**

Identified sources of NPs in the aquatic environment include production facilities, production processes, wastewater treatment plants, and accidents during the transport. In addition, aquatic ecosystems are known to sequester and transport contaminants, including NMs. Baun et al. showed that NPs may adhere to algae which may then be consumed by filter-feeders and transfer to higher trophic levels. In the aquatic environment, NPs may aggregate thus reducing the NPs available for direct uptake in the aqueous phase by aquatic organisms. However, aggregated NPs may settle into sediment thereby posing a threat to benthic organisms. In the aquatic environment, NMs are generally associated with sediments [21]. Sediments and soil represent porous environmental matrices which typically have large specific surface areas.

50 Toxicology - New Aspects to This Scientific Conundrum

**Figure 3.** Possible pathways of nanoparticles in the aquatic environment.

Uptake of nanoparticles into the aquatic biota is a major concern. Nanomaterials are able to cross biological barriers, gaining entering due to their small size. In aquatic organisms, the major routes of entry are via ingestion or direct passage across the gill and other external surface epithelia. In invertebrates, the cellular immune system, gut epithelium, and hepatopancreas are likely to be targeted [22]. Recent studies with Mozambique tilapia (*Oreochromis mossambicus*) have indicated that nickel NPs may be internalized via these routes [23]. In addition, NPs that are taken up via ingestion through the digestive tract may accumulate in the hepatopancreas [24]. The hepatopancreas is responsible for metabolism and detoxification Many studies have attempted to elucidate the mechanisms of NP toxicity and distinguish between their bulk counterparts. Nanomaterials differ from their bulk counterparts in several ways, including high surface/volume ratio. Other factors such as dissolution, size, shape, aggregation state, surface coatings, and solution chemistry also influence the toxicity of NPs.

The toxicity of various NMs AgNP [5–7, 30–32]), CuO NP [19, 33], TiO2 NP [34], and Ni NP [23] has been studied in various aquatic species, such as *Daphnia magna* [32, 33], fish [6, 35], algae [36], and marine [37] and freshwater [13] crabs. Silver, carbon, and titanium NMs are among the most widely used types NMs used as additives in cosmetics and pharmaceuticals. Also, different NMs exhibit different properties had hence have different toxicity potencies. For example, Heinlaan et al. [38] compared the toxicities of three nanometal oxides: ZnO NPs, CuO NPs, and TiO2 NPs. ZnO NPs was determined to be the most toxic; whereas Zhu et al. [39] reported CuO NP the most potent to cytotoxicity and genotoxicity.

The assessment of NP toxicity has largely been assessed *in vitro*, reporting inducing various negative effects at different levels of cellular organization. Typical end points measured include end points examined which include mortality, as well as sublethal effects such as development, growth, respiration, malformation, oxidative stress, and gene expression. Generation of reactive oxygen species (ROS) and free radicals causes oxidative stress (activation or inhibition of the antioxidant defense system), lipid peroxidation, and DNA damage. Toxicity of NPs will be discussed further in the following sections.

#### **5.1. Oxidative stress**

Oxidative stress is referred to as an imbalance between the production of reactive oxygen species (ROS) and the cells' ability to reduce ROS, which may be as a result an increased ROS production, a decrease in the cell's defense mechanisms, or a combination of both [40]. An overproduction of ROS may induce oxidative stress, resulting in cells failing to maintain normal physiological redox-regulated functions further resulting in oxidative modification of proteins to generate protein radicals [41], initiation of lipid peroxidation [42], DNA strand breaks and modification to nucleic acids [43], modulation of gene expression [44], thereby leading to cell death and genotoxic effects [45]. To minimize the effects of ROS-oxidative damage to cellular components, biological systems have developed a complex antioxidant system, comprised of both enzymatic and non-enzymatic defense mechanisms. **Figure 4** summarizes the redox cycle including ROS generation by NPs and the antioxidant defense system.

**Figure 4.** ROS production and defense mechanisms (adapted from Unfired et al. [46]).

The antioxidant defense system has evolved to provide a balance between the production and removal of ROS. These are catalyzed by a number of different enzymes including Phase I and Phase II enzymes. Phase I enzymes, such as cytochrome P450, initiate the detoxification process by introducing a polar moiety which renders a lipophilic contaminant more hydrophilic. Activity of Phase I enzymes typically leads to an increase in ROS production. Phase II enzymes are involved in conjugating metabolized xenobiotics to endogenous molecules. Phase III involves further modification and excretion.

The potential role of oxidative stress as a mechanism of toxicity of AgNPs was evaluated by several authors. Walters et al. [13] studied oxidative stress, viz. antioxidant enzyme activity following a 7-day exposure to AgNPs (100 nm) at 10 and 100 μg/mL. The levels of ROS and oxidative stress were concentration-dependant, with an x-fold increase compared to control levels. Federici et al. [47] measured ROS generation following 14 days of exposure to TiO2 NPs (average particle size = 21 nm at 0.1, 0.5, or 1.0 mg/mL). The level of ROS (i.e., thiobarbituric acid-reactive substances (TBARS)) in the gills, intestine, and brain of the rainbow trout (*Oncorhynchus mykiss*) increased in a concentration-dependent manner. Statistically significant increases (twofold or more) in the gills, intestine, and brain vs. the control group were also reported. In line with these findings, Oberholster et al. [48] investigated the generation of superoxide (O2 − ·) and consequent stimulation of antioxidant defense mechanisms following a 10-day exposure to spiked sediment with various concentrations of NMs. These authors also reported concentration-dependant increases in enzyme activities. In another study with AgNPs, Gomes et al. [49] reported oxidative damage for higher doses (0, 100, 300, 600, 1000, 1500 mg/kg) and exposure times (4 days vs. 28 days) in *Eisenia fetida*. Induction of oxidative stress and antioxidant enzymes by titanium oxide NP (TiO2 NP) in *Daphnia magna* was studied by Kim et al. [34]. The authors' results indicated that the levels in activities of catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) increase with increasing TiO2 NP concentration. The levels of activities of superoxide dismutase (SOD) were reduced in the tissues with increasing TiO2 NP concentration. In another study, Tedesco et al. [50] found signs of oxidative stress in the form of lysosomal instability. In line with these findings, Moore [4] reported decreased lysosomal stability resulting from apoptosis induction by AgNP.

## **5.2. Ecotoxicity**

**5.1. Oxidative stress**

52 Toxicology - New Aspects to This Scientific Conundrum

system.

Oxidative stress is referred to as an imbalance between the production of reactive oxygen species (ROS) and the cells' ability to reduce ROS, which may be as a result an increased ROS production, a decrease in the cell's defense mechanisms, or a combination of both [40]. An overproduction of ROS may induce oxidative stress, resulting in cells failing to maintain normal physiological redox-regulated functions further resulting in oxidative modification of proteins to generate protein radicals [41], initiation of lipid peroxidation [42], DNA strand breaks and modification to nucleic acids [43], modulation of gene expression [44], thereby leading to cell death and genotoxic effects [45]. To minimize the effects of ROS-oxidative damage to cellular components, biological systems have developed a complex antioxidant system, comprised of both enzymatic and non-enzymatic defense mechanisms. **Figure 4** summarizes the redox cycle including ROS generation by NPs and the antioxidant defense

**Figure 4.** ROS production and defense mechanisms (adapted from Unfired et al. [46]).

involves further modification and excretion.

The antioxidant defense system has evolved to provide a balance between the production and removal of ROS. These are catalyzed by a number of different enzymes including Phase I and Phase II enzymes. Phase I enzymes, such as cytochrome P450, initiate the detoxification process by introducing a polar moiety which renders a lipophilic contaminant more hydrophilic. Activity of Phase I enzymes typically leads to an increase in ROS production. Phase II enzymes are involved in conjugating metabolized xenobiotics to endogenous molecules. Phase III The potential ecotoxicity of NPs has currently provoked public and scientific dialogues due to debates around the risks and benefits of these materials. As such, studies on the ecotoxicological fate and effects of NMs have increased in recent years. There has been extensive research investigating the toxicity of NPs to aquatic organisms with several recent reviews reporting on ecotoxicology of NPs [3, 4]. Data on the biological effects of NPs indicate that NPs can be toxic to bacteria, algae, invertebrates, fish, and mammals. Nonetheless, nano-ecotoxicology studies remain poorly and unevenly distributed as most research undertaken has largely been restricted to a narrow range of test species. Most of the current ecotoxicological data pertaining to NMs have been done on *Daphnia magna*. These crustaceans represent the food and energy link between algae and fish [51]; therefore, these studies are particularly relevant. Park and Choi [32] studied the ecotoxicity effects of AgNPs to *D. magna* and reported increased mortality. Asghari et al. reported abnormal swimming in *D. magna* following exposure to AgNPs, while Heinlaan et al. [33] reported ultrastructural changes in the midgut of *D. magna* upon exposure to CuO NPs.

Nanoparticles are able to penetrate the semipermeable membranes of some aquatic organisms, thereby forming aggregates around the exoskeleton of aquatic organisms [52]. Uptake of various NPs by aquatic organisms crustacean *Daphnia magna* [33, 53], the polychaete *Nereis diversicolor* [30], and the freshwater algae Ochromonas *danica* [31] has been reported (**Figure 5**).

**Figure 5.** Nanoparticle internalization in some aquatic organisms.

Uptake of NPs generally occurs across the gills and other epithelial surfaces [6]. Scown et al. [6] reported size-dependant uptake of AgNPs (10–35 nm) and associated oxidative stress in the gills of *Danio rerio,* while Maria et al. [54] reported reduced LPO levels in gills and hepatopancreas of female *Carcinus maenas*. In crustaceans, toxicants are largely sequestered in the hepatopancreas and gills [55]. Walters et al. [13] reported higher levels on enzymatic activities in the hepatopancreas when compared to the gills suggesting that the hepatopancreas might be a more sensitive organ [56] to AgNP exposure and also implies a lower ability to scavenge O2 − [37].

Studies showing enhanced ecotoxicity of NPs when compared to their bulk counterparts have led to the assumption that NPs generally represent a more potent threat. Once such study investigated the toxicity of various NPs with bulk counterparts [57], the authors reported significantly differences in toxicity (24-h LC50) between Al2O3 NPs (82 mg/L) and bulk Al2O3 (153 mg/L) and between TiO2 NPs (80 mg/L) and bulk TiO2(136 mg/L).

Studies reporting on reproduction and developmental end points are common. Wu et al. [58] recently showed that AgNPs induced a variety of morphological malformations such as edema, spinal abnormalities, fin fold abnormalities, heart malformations, and eye defects in Japanese medaka (*Oryzias latipes*). Wiench et al. [59] performed a 21-day chronic Daphnia reproduction study using coated TiO2 NPs and reported that the NOEC for adult mortality was 30 mg/L, while the NOEC for offspring production was 3 mg/L. The 21-day EC10 and EC50 values for reproductive effects were 5 mg/L and 26.6 mg/L, respectively.

## **5.3. Genotoxicity**

**Figure 5.** Nanoparticle internalization in some aquatic organisms.

54 Toxicology - New Aspects to This Scientific Conundrum

O2 − [37].

Uptake of NPs generally occurs across the gills and other epithelial surfaces [6]. Scown et al. [6] reported size-dependant uptake of AgNPs (10–35 nm) and associated oxidative stress in the gills of *Danio rerio,* while Maria et al. [54] reported reduced LPO levels in gills and hepatopancreas of female *Carcinus maenas*. In crustaceans, toxicants are largely sequestered in the hepatopancreas and gills [55]. Walters et al. [13] reported higher levels on enzymatic activities in the hepatopancreas when compared to the gills suggesting that the hepatopancreas might be a more sensitive organ [56] to AgNP exposure and also implies a lower ability to scavenge

Studies showing enhanced ecotoxicity of NPs when compared to their bulk counterparts have led to the assumption that NPs generally represent a more potent threat. Once such study investigated the toxicity of various NPs with bulk counterparts [57], the authors reported significantly differences in toxicity (24-h LC50) between Al2O3 NPs (82 mg/L) and bulk Al2O3

Studies reporting on reproduction and developmental end points are common. Wu et al. [58] recently showed that AgNPs induced a variety of morphological malformations such as edema, spinal abnormalities, fin fold abnormalities, heart malformations, and eye defects in Japanese

(153 mg/L) and between TiO2 NPs (80 mg/L) and bulk TiO2(136 mg/L).

An important issue relating to the toxicity of NPs in biological media is the ability to cause damage to the genetic material, particularly since NPs have the capacity to cross cell membranes. In the section below, evidence of NP-induced genotoxicity is reviewed. DNA is a significant cellular component highly susceptible to oxidative damage. As such, there has been increasing interest in the analysis of the potential nanoparticle genotoxicity to aquatic organisms.

Genotoxic assessments of various NPs have largely been reported on in *vitro* studies. Reported abilities of NPs include chromosomal fragmentation, DNA strand breakages, point mutations, oxidative DNA adducts, and alterations in gene expression profiles and consequently may initiate and promote mutagenesis and carcinogenesis. Primary genotoxicity stemming from the direct interactions of NP with DNA following NP internalization has been reported [60]. Genotoxicity mediated by the generation of excess ROS, referred to as secondary genotoxicity, has been reported. Oberholster et al. [48], using DNA strand breakage as an indicator of genotoxicity, reported concentration-dependent effects to several NPs (α-alumina, β-alumina, precipitated silica; silica fume, calcined silica fume, colloidal antimony pentoxide, and superfine amorphous ferric oxide). DNA cleavage, an indicator of irreversible completion of apoptosis, occurred in organisms exposed to 5000 μg/kg of precipitated silica, amorphous ferric oxide, and colloidal antimony pentoxide NMs. The inter-nucleosomal DNA ladderbands occurred at 500 μg/kg of γ-alumina and α-alumina.

As with NP toxicity, NPs are also known to have more adverse genotoxic effects than their bulk counterparts. For example, Park and Choi [32] studied the genotoxicity of AgNPs on the freshwater crustacean *Daphnia magna*. Their results reported a higher degree of DNA damage in the form of DNA strand breaks in AgNPs when compared to Ag ions. Similarly, NP size is also known to affect its genotoxicity potential, inducing significant DNA and chromosomal damages compared to the larger NPs. This size effect was confirmed: the authors showed that smaller sized TiO2 NPs (10 nm) have significant chromosomal damage when compared to the larger TiO2NP (>200 nm) [61].

As such, there is a general consensus that smaller sized NPs produce higher reactivity and thus higher genotoxicity [62, 63]. However, particle size is not the only factor that determines particle (geno-)toxicity. Nanoparticle surface coating has also been reported to promote genotoxicity. Surface coating modifies the particle surface, and therefore, they may also alter the particle's genotoxicity. For instance, Hong et al. [64] reported positively charged coatings of iron oxide NPs which consequently resulted in increased DNA strand breaks, while the impact of genotoxicity of negatively charged coatings was insignificant. Similarly, Lui et al. [65] reported various genotoxic responses of iron oxide NPs depending on the type of coating. polyethylene glycol (PEG) coating exhibited mutagenic activity, while solid electrolyte interphase (SEI) exhibited no genotoxicity.

As with ecotoxicological effects, genotoxicity effects of coated NPs have also been investigated. Ahamed et al. [66] reported that coated AgNPs resulted in more upregulation of these proteins, suggesting that coated Ag NPs causes greater genotoxicity than uncoated AgNP. In similar study, AshaRani et al. [35] investigated the genotoxicity of coated silver NPs vs. uncoated silver NPs in the zebrafish embryos (*Danio rerio*). The authors reported that NPs which penetrated the nucleus cells able to take alterations and breakings to DNA.

## **6. Conclusions**

The use of NMs in consumer products and their potential environmental and human health risks is of increasing concern. As nanotechnologies and products increase, nanoproducts entering the aquatic ecosystems and other water sources too will increase, thereby increasing the potential threat to aquatic organisms. This chapter provides a review of nanotoxicology an emerging multidisciplinary field of science—with special focus on the effects of metal-NMs on aquatic invertebrates. NMs, depending on the size, shape, elemental materials, and the surface functional groups, induce oxidative stress thus leading to (nano)toxicity and genotoxicity. The risks associated with NMs (i.e., its fate, behavior, and toxicity in the environment) are largely unknown and difficult to predict. As the ultimate sink for conventional contaminants, the aquatic ecosystem is therefore predisposed to the potential effects of NPs.

Although our knowledge on the toxicity of various NMs in the aquatic environment has increased over the past few years, there is still a lack of knowledge regarding exposure concentrations, bioaccumulation in tissues, as well as environmental factors which could potentially affect its toxicity or bioaccumulation. Exposure to NPs is inevitable since NPs become more widely used, but there remains much more to be understood regarding their safety.

Although current toxicity testing protocols is generally applicable to identify deleterious effects associated with NPs, the mechanisms of action that govern toxicity of NMs are the subject of ongoing research. Research into new analytical methods is also required to address the special properties of NMs. The outcomes will thus enable researchers to predict the toxicological effects of AgNPs with the intent of guiding its development, application, and regulation. This will be important when considering measures for exposure control and environmental remediation of AgNPs.

## **Acknowledgements**

This research was supported by the Council for Scientific and Industrial Research (CSIR).

## **Author details**

polyethylene glycol (PEG) coating exhibited mutagenic activity, while solid electrolyte

As with ecotoxicological effects, genotoxicity effects of coated NPs have also been investigated. Ahamed et al. [66] reported that coated AgNPs resulted in more upregulation of these proteins, suggesting that coated Ag NPs causes greater genotoxicity than uncoated AgNP. In similar study, AshaRani et al. [35] investigated the genotoxicity of coated silver NPs vs. uncoated silver NPs in the zebrafish embryos (*Danio rerio*). The authors reported that NPs which penetrated

The use of NMs in consumer products and their potential environmental and human health risks is of increasing concern. As nanotechnologies and products increase, nanoproducts entering the aquatic ecosystems and other water sources too will increase, thereby increasing the potential threat to aquatic organisms. This chapter provides a review of nanotoxicology an emerging multidisciplinary field of science—with special focus on the effects of metal-NMs on aquatic invertebrates. NMs, depending on the size, shape, elemental materials, and the surface functional groups, induce oxidative stress thus leading to (nano)toxicity and genotoxicity. The risks associated with NMs (i.e., its fate, behavior, and toxicity in the environment) are largely unknown and difficult to predict. As the ultimate sink for conventional contami-

nants, the aquatic ecosystem is therefore predisposed to the potential effects of NPs.

Although our knowledge on the toxicity of various NMs in the aquatic environment has increased over the past few years, there is still a lack of knowledge regarding exposure concentrations, bioaccumulation in tissues, as well as environmental factors which could potentially affect its toxicity or bioaccumulation. Exposure to NPs is inevitable since NPs become more widely used, but there remains much more to be understood regarding their

Although current toxicity testing protocols is generally applicable to identify deleterious effects associated with NPs, the mechanisms of action that govern toxicity of NMs are the subject of ongoing research. Research into new analytical methods is also required to address the special properties of NMs. The outcomes will thus enable researchers to predict the toxicological effects of AgNPs with the intent of guiding its development, application, and regulation. This will be important when considering measures for exposure control and

This research was supported by the Council for Scientific and Industrial Research (CSIR).

interphase (SEI) exhibited no genotoxicity.

56 Toxicology - New Aspects to This Scientific Conundrum

**6. Conclusions**

safety.

environmental remediation of AgNPs.

**Acknowledgements**

the nucleus cells able to take alterations and breakings to DNA.

Chavon Walters1\*, Edmund Pool2 and Vernon Somerset1


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#### **Potential Harm of Maltodextrin‐Coated Cadmium Sulfide Quantum Dots in Embryos and Fetuses Potential Harm of Maltodextrin**‐**Coated Cadmium Sulfide Quantum Dots in Embryos and Fetuses**

Jorge Reyes‐Esparza, Janet Sánchez‐Quevedo, Antonieta Gómez‐Solís, Patricia Rodríguez‐Fragoso, Gerardo González De la Cruz and Lourdes Rodríguez‐Fragoso Jorge Reyes‐Esparza, Janet Sánchez‐Quevedo, Antonieta Gómez‐Solís, Patricia Rodríguez‐Fragoso, Gerardo González De la Cruz and Lourdes Rodríguez‐Fragoso

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

http://dx.doi.org/10.5772/64653

#### **Abstract**

Over the past years, there has been significant interest in the study of nanoparticles for clinical applications, particularly quantum dots (QDs). However, previous studies have also shown that QDs can reach the embryo through the placenta, a natural barrier for a large variety of organic substances with diverse molecular structures, and may cause developmental deformities. Due to its essential role in a toxicological profile and its relevance to human safety, knowledge regarding embryotoxicity is of great importance. Previous studies by this research group have shown that CdS‐maltodextrin QDs are biocompatible and nontoxic to cells and animals; however, QDs are able to induce embryotoxic effects. Therefore, as an effort to further address the issue, we studied the effects of CdS‐maltodextrin QDs on embryo and fetus development using an embryo‐ toxicity and teratogenicity assay on chicken embryos. Chicken embryos exposed to CdS‐maltodextrin QDs (0.001, 0.01, 0.1 and 1 µg/kg) *in ovo* for 72 h showed growth and developmental alterations during the early stage and at the end of their development in a dose‐dependent manner. Decreased development was observed during early stages (Stages 9/10 on the Hamburger‐Hamilton scale) when compared with untreated eggs (Stage 13). Chicken embryos exposed to lower CdS‐maltodextrin QDs doses (0.01, 0.1 and 1 ng/kg) and incubated *in ovo* for 21 h also showed growth and development alterations during the early stages and at the end of their development in a dose‐ dependent manner. However, reduced development was observed at the end of the development period (21 days), and this was associated with death of the chick. Current studies have also shown that CdS‐dextrin induces embryotoxicity and teratogenicity, affecting mainly the CNS, the neural tube and somites in chicken embryos. The nature of the observed abnormalities suggests that these effects could be directly associated with nanoparticle concentrations affecting somitogenesis. Therefore, according to the

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

results, there is a high probability that the prolonged accumulation of QDs in the maternal organism may be potentially harmful on embryo and fetus development. This study is limited to the analysis of embryotoxic and teratogenic effects induced by CdS‐ maltodextrin QDs.

**Keywords:** embryotoxicity, teratogenicity, nanoparticles, quantum dots, embryos

## **1. Introduction**

Countless publications about of the *in vitro* and *in vivo* effects of nanomaterials have appeared over the past years [1]. It is now well known that the same properties that make nanoparticles so attractive to biomedicine may contribute to their toxicological profile in biological systems [2, 3]. Nevertheless, there are still many unanswered questions about their toxicological aspects, such as embryotoxicity and teratogenicity.

It is well known that embryos and fetuses are most sensitive to harmful factors during critical periods [4]. Not only is differentiation during organogenesis a highly susceptible period to the induction of malformations, but also the fetal/neonatal developmental phases are just as prone to certain developmental deficits [5]. Therefore, embryotoxicity and teratogenicity assays are of great importance given their indispensable role in the toxicological profile that must be established for any new active substance relevant to human safety, including nanomaterials. In recent years, different studies have explored the passage of nanomaterials through the placenta, a natural barrier for a large variety of organic substances with diverse molecular structures [6–8]. It is now known that the passage of nanomaterials through the placental barrier may affect fetal cell proliferation, embryonic growth, and organ formation [9]. De‐ pending on the doses and time of exposure—from fertilization through the fetal period and eventually during lactation—the consequences can range from embryotoxicity to gross malformations, and a large variety of more subtle morphological, biochemical, and functional abnormalities have been detected [10–12].

However, knowledge concerning nanomaterial embryotoxicity and teratogenicity is yet limited because the toxicity of each nanoparticle depends on size, shape, and even surface cover, and so different nanomaterials may yield contradictory effects [13]. Because of this, in‐ depth knowledge of nanotoxicity and increased efforts devoted to the study of the toxic effects of nanomaterials on embryos and fetuses should be considered mandatory, as is with other investigational new drugs.

Our group is interested in the biomedical application of maltodextrin‐coated cadmium sulfide QDs, semiconductor nanoparticles of about 3.5 nm in size with superior optical properties when compared to conventional organic dyes [14]. Although the *in vitro* studies revealed that these CdS‐maltodextrin QDs produced distinct dose‐dependent toxic effects, *in vivo* studies demonstrated that, when administered to rodents, CdS‐maltodextrin QDs were biocompatible and nontoxic after 5 and 15 days [15]. Moreover, the pharmacokinetic data clearly showed that CdS‐maltodextrin QDs were not completely cleared from *in vivo* systems after 360 h. Then,

although CdS‐maltodextrin QDs appear to be nanomaterials with favorable pharmacokinetic properties to develop novel therapeutic and diagnostic modalities, according to previous results, CdS‐maltodextrin QDs seem to be allowed into the body through several barriers and then pass into the blood stream, from where they can reach organs and tissues and interact with biological structures [16].

Early results by this group demonstrated that CdS‐maltodextrin QDs were embryotoxic in a chicken embryo model [14]. The nature of the observed abnormalities suggests that these effects could be directly associated with nanoparticle concentrations. The observed effects indicate that prolonged accumulation of QDs in the maternal organism may increase the risk of adverse effects on embryo development. Since nanotoxicity studies targeting the reproduc‐ tive and developmental aspects are rather scant, and considering that QD mechanisms of action during embryogenesis are not fully understood, this study aims at further addressing the issue. We studied the effects of CdS‐maltodextrin QDs on embryo and fetus development using an embryotoxicity and teratogenicity assay on chicken embryos.

## **2. Materials and methods**

## **2.1. Embryotoxicity study**

results, there is a high probability that the prolonged accumulation of QDs in the maternal organism may be potentially harmful on embryo and fetus development. This study is limited to the analysis of embryotoxic and teratogenic effects induced by CdS‐

**Keywords:** embryotoxicity, teratogenicity, nanoparticles, quantum dots, embryos

Countless publications about of the *in vitro* and *in vivo* effects of nanomaterials have appeared over the past years [1]. It is now well known that the same properties that make nanoparticles so attractive to biomedicine may contribute to their toxicological profile in biological systems [2, 3]. Nevertheless, there are still many unanswered questions about their toxicological aspects,

It is well known that embryos and fetuses are most sensitive to harmful factors during critical periods [4]. Not only is differentiation during organogenesis a highly susceptible period to the induction of malformations, but also the fetal/neonatal developmental phases are just as prone to certain developmental deficits [5]. Therefore, embryotoxicity and teratogenicity assays are of great importance given their indispensable role in the toxicological profile that must be established for any new active substance relevant to human safety, including nanomaterials. In recent years, different studies have explored the passage of nanomaterials through the placenta, a natural barrier for a large variety of organic substances with diverse molecular structures [6–8]. It is now known that the passage of nanomaterials through the placental barrier may affect fetal cell proliferation, embryonic growth, and organ formation [9]. De‐ pending on the doses and time of exposure—from fertilization through the fetal period and eventually during lactation—the consequences can range from embryotoxicity to gross malformations, and a large variety of more subtle morphological, biochemical, and functional

However, knowledge concerning nanomaterial embryotoxicity and teratogenicity is yet limited because the toxicity of each nanoparticle depends on size, shape, and even surface cover, and so different nanomaterials may yield contradictory effects [13]. Because of this, in‐ depth knowledge of nanotoxicity and increased efforts devoted to the study of the toxic effects of nanomaterials on embryos and fetuses should be considered mandatory, as is with other

Our group is interested in the biomedical application of maltodextrin‐coated cadmium sulfide QDs, semiconductor nanoparticles of about 3.5 nm in size with superior optical properties when compared to conventional organic dyes [14]. Although the *in vitro* studies revealed that these CdS‐maltodextrin QDs produced distinct dose‐dependent toxic effects, *in vivo* studies demonstrated that, when administered to rodents, CdS‐maltodextrin QDs were biocompatible and nontoxic after 5 and 15 days [15]. Moreover, the pharmacokinetic data clearly showed that CdS‐maltodextrin QDs were not completely cleared from *in vivo* systems after 360 h. Then,

maltodextrin QDs.

66 Toxicology - New Aspects to This Scientific Conundrum

such as embryotoxicity and teratogenicity.

abnormalities have been detected [10–12].

investigational new drugs.

**1. Introduction**

Fertile White Leghorn chicken eggs were obtained from A.L.P.E. S.A. (Puebla, Mexico) and stored at 6°C. One hundred fertilized eggs were weighed, sterilized, and divided into nine groups. The first group served as a nontreated control and was considered the negative control. The second group was treated with 1 mL of Ringer solution. The third group was treated with caffeine (10 mg/mL) and was considered a positive control. The remaining six groups received CdS‐maltodextrin QDs in different concentrations (0.001, 0.01, 0.1, 1 µg/kg). One of these groups received caffeine (10 mg/mL) and was considered the positive control. An embryotox‐ icity assay was carried out as described by Jelinek and Marthan [17]. Test solutions (1 mL) were added to the air sac under sterile conditions. Each solution was injected after drilling into the shell at the blunt end of the egg; after injection, the holes were immediately sealed with melted paraffin wax. The eggs were then transferred to and maintained in a forced draft incubator at 37.5°C with a relative humidity of 65% until the desired stage of development was reached (72 h).

Embryos in each group were fixed in buffered formal saline (pH 7.4), dehydrated, and embedded in paraffin blocks. Paraffin tissue sections of 6 µm were stained with acetocarmine for routine histological examination. The embryo was examined and staged according to morphological criteria previously outlined by Hamburger and Hamilton [18]. Malformations were considered for the following specific structures: central nervous system (CNS), lens placode, otic placode, cardiovascular system (CVS), neural tube, as well as number of somites. Embryonic stages at the time of the CdS‐maltodextrin QD application varied from 14 to 16, which corresponds approximately to developed somites numbered 22–28. In order to monitor their uptake and distribution, we observed the embryos under a confocal microscope (Zeiss LSM510, USA). CdS‐Dx QDs were excited with 488 nm laser, and their signals were collected from 515 nm.

#### **2.2. Teratogenicity assay**

Fertile White Leghorn chicken eggs were obtained from A.L.P.E. S.A. (Puebla, Mexico) and stored at 6°C. One hundred fertilized eggs were weighed, sterilized, and divided into four groups. The first group served as a nontreated control and was considered the negative control. The other groups received CdS‐maltodextrin QDs in different concentrations (0.001, 0.01, 0.1, 1 µg/kg). A teratogenicity assay was carried out as described by Jelinek and Marthan [17]. Test solutions (1 mL) were added to the air sac under sterile conditions. Each solution was injected after drilling into the shell at the blunt end of the egg; after injection, the holes were immedi‐ ately sealed with melted paraffin wax. The eggs were then transferred to and maintained in a forced draft incubator at 37.5°C with a relative humidity of 65%. To determine the teratogenic effect of CdS‐maltodextrin QDs, we allowed the chicks to fully develop until they were able to hatch by themselves). Developmental stages and the presence of malformations were measured according to the Hamburger‐Hamilton scale.

#### **2.3. Statistical analysis**

Results are expressed as means + standard error of the mean (SEM). Significant differences were detected by one‐way analysis of variance using GraphPad Instat V2.03 (GraphPad Software Inc., San Diego, CA). The Tukey–Kramer multiple comparisons test was used when significant variations were found. Differences were considered significant at p < 0.05.

## **3. Results**

A chicken embryo model was used to assess CdS‐maltodextrin QDs embryotoxicity. **Figure 1** shows a representative chicken embryo treated with different doses of CdS‐maltodextrin QDs. As we can see, QD‐treated chicken embryos showed developmental delays in a dose‐depend‐ ent manner. Nonmalformations were found with doses of 0.001 µg/kg QDs. However, embryos treated with 0.01 µg/kg of CdS‐maltodextrin QDs showed significant changes in the CNS; 38% of embryos showed malformations that included morphological alterations and anencephaly (**Figure 2**). Embryos treated with 0.1 µg/kg of CdS‐maltodextrin QDs showed a significant developmental arrest at Stage 13 of the Hamburger‐Hamilton scale, and 50% of embryos showed various malformations. Those embryos showed several alterations in structures including lens placodes (10%), otic placodes (10%), CNS (50%) and neural tube (29%) (**Figure 2** and **Table 1**). These embryos had a smaller number of somites than nontreated embryos. The dose that produced the most critical alterations was 1 µg/kg; it produced 50% of embryo mortality at Stage 12 of the Hamburger‐Hamilton scale. Besides that, 71% of embryos had malformations, including lens placode 55%, otic placode 75%, CNS 71% (anencephaly and morphological alterations), lower number of somites, and 50% presented alterations in the neural tube (see **Figure 3** and **Table 1**).

LSM510, USA). CdS‐Dx QDs were excited with 488 nm laser, and their signals were collected

Fertile White Leghorn chicken eggs were obtained from A.L.P.E. S.A. (Puebla, Mexico) and stored at 6°C. One hundred fertilized eggs were weighed, sterilized, and divided into four groups. The first group served as a nontreated control and was considered the negative control. The other groups received CdS‐maltodextrin QDs in different concentrations (0.001, 0.01, 0.1, 1 µg/kg). A teratogenicity assay was carried out as described by Jelinek and Marthan [17]. Test solutions (1 mL) were added to the air sac under sterile conditions. Each solution was injected after drilling into the shell at the blunt end of the egg; after injection, the holes were immedi‐ ately sealed with melted paraffin wax. The eggs were then transferred to and maintained in a forced draft incubator at 37.5°C with a relative humidity of 65%. To determine the teratogenic effect of CdS‐maltodextrin QDs, we allowed the chicks to fully develop until they were able to hatch by themselves). Developmental stages and the presence of malformations were

Results are expressed as means + standard error of the mean (SEM). Significant differences were detected by one‐way analysis of variance using GraphPad Instat V2.03 (GraphPad Software Inc., San Diego, CA). The Tukey–Kramer multiple comparisons test was used when

A chicken embryo model was used to assess CdS‐maltodextrin QDs embryotoxicity. **Figure 1** shows a representative chicken embryo treated with different doses of CdS‐maltodextrin QDs. As we can see, QD‐treated chicken embryos showed developmental delays in a dose‐depend‐ ent manner. Nonmalformations were found with doses of 0.001 µg/kg QDs. However, embryos treated with 0.01 µg/kg of CdS‐maltodextrin QDs showed significant changes in the CNS; 38% of embryos showed malformations that included morphological alterations and anencephaly (**Figure 2**). Embryos treated with 0.1 µg/kg of CdS‐maltodextrin QDs showed a significant developmental arrest at Stage 13 of the Hamburger‐Hamilton scale, and 50% of embryos showed various malformations. Those embryos showed several alterations in structures including lens placodes (10%), otic placodes (10%), CNS (50%) and neural tube (29%) (**Figure 2** and **Table 1**). These embryos had a smaller number of somites than nontreated embryos. The dose that produced the most critical alterations was 1 µg/kg; it produced 50% of embryo mortality at Stage 12 of the Hamburger‐Hamilton scale. Besides that, 71% of embryos had malformations, including lens placode 55%, otic placode 75%, CNS 71% (anencephaly and morphological alterations), lower number of somites, and 50% presented alterations in the

significant variations were found. Differences were considered significant at p < 0.05.

from 515 nm.

**2.2. Teratogenicity assay**

68 Toxicology - New Aspects to This Scientific Conundrum

**2.3. Statistical analysis**

neural tube (see **Figure 3** and **Table 1**).

**3. Results**

measured according to the Hamburger‐Hamilton scale.

**Figure 1.** Effect of CdS‐maltodextrin QDs on embryogenesis. Embryos were staged in accordance with the Hamburger‐ Hamilton scale (S), and a dose‐dependent delay in embryo development was observed. Several deformities are evi‐ dent. Ot. p.: otic placode; Op. p.: lens placode; S: somites; NT: neural tube (Magnification 4×).

**Figure 2.** Gross abnormalities associated with CdS‐maltodextrin QDs. Application of 0.01, 0.1 and 1 µg/kg QDs in ster‐ ile chicken ringer solution to chicken embryo *in ovo* induced overall growth delay, as well as defects in the brain, neural tube and somites, as opposed to embryos treated with sterile chicken ringer alone (arrows). Embryos treated with 0.01 µg/kg QDs showed evident brain defects. 0.1 µg/kg QDs produced brain and neural tube defects; besides those defects, 1 µg/kg QD treatments resulted in malformations. The somites formed after QD treatment lack uniform rectangular alignment and border an abnormally formed neural tube. The localization of the asterisks represents neural tube de‐ formities. Applications of QDs caused multiple abnormalities in the otic and lens placodes. (Magnification 4×).

**Figure 3.** Fluorescence microscopic images showing the distribution and localization of CdS‐maltodextrin QDs in em‐ bryos. These images correspond to embryos treated with 0.1 and 1 µg/kg. The distribution and localization of CdS‐ MDx QDs were identified by a bright green imaging in the analyzed embryos. There is an evident presence of abnormalities in the brain, placodes, neural tube and somites. (Magnification 4×).


N = 14; CNS = central nervous system.

1 Nontreated embryos.

2 Hamburger‐Hamilton scale.

\* p < 0.05 as compared to control group.

**Table 1.** Effect of CdS‐maltodextrin QDs on embryos viability and development.

The fluorescent properties of CdS‐maltodextrin QDs allowed us to monitor their uptake and distribution directly by observing the bright green light they emit. CdS‐maltodextrin QDs uptake and bioimaging experiments were performed with a fluorescent microscope. **Figure 3** shows CdS‐maltodextrin QD distribution in chicken embryos. The uniform distribution of QDs in embryonic tissues was evident. The presence of malformations on the CNS, placodes and somites with doses of 0.1 µg/kg was evident too. However, more severe malformations were witnessed with doses of 1 µg/kg, such as absence of head, brain, otic placode and somites. Somites, mainly the ventral ones, were affected in size, morphology and number in a dose‐ dependent manner (**Figures 2** and **3**).

The teratogenic effect of CdS‐maltodextrin QDs was also evaluated using a chicken embryo model. Thus, we observed several malformations in embryos with QD doses of 0.01–1 µg/kg; for the teratogenicity study, we employed lower doses (0.01, 0.1 and 1 ng/kg) in order to allow for chick development. However, although lower doses were used as compared with the embryotoxicity study, the lack of development and birth defects was clear, and even with the same doses there were variations in embryo development. For example, embryos treated with 0.1 ng/kg QDs were at Stages 23 and 41, and embryos treated with 1 ng/kg were at Stages 20 and 32.

**Figure 3.** Fluorescence microscopic images showing the distribution and localization of CdS‐maltodextrin QDs in em‐ bryos. These images correspond to embryos treated with 0.1 and 1 µg/kg. The distribution and localization of CdS‐ MDx QDs were identified by a bright green imaging in the analyzed embryos. There is an evident presence of

**Control1 Ringer Caffeine 0.001 0.01 0.1 1**

abnormalities in the brain, placodes, neural tube and somites. (Magnification 4×).

70 Toxicology - New Aspects to This Scientific Conundrum

**Table 1.** Effect of CdS‐maltodextrin QDs on embryos viability and development.

**Type of malformations**

N = 14; CNS = central nervous system.

p < 0.05 as compared to control group.

Nontreated embryos.

Hamburger‐Hamilton scale.

1

2

\*

**Parameter Controls CdS‐maltodextrin QDs (µg/kg)**

**Viability (%)** 100 100 100 100 100 100 50 **Stage2** 16 ± 0.9 16 ± 0.5 12 ± 0.8\* 15 ± 1.0 14 ± 1.2 13 ± 1.1\* 12 ± 2.6\* **Malformations (%)** 0 0 100 0 38 50 71

**Optical placode (%)** 0 0 100 0 0 10 55 **Otic placode (%)** 0 0 100 0 0 10 75 **CNS (%)** 0 0 100 0 100 100 100 **Somites (#)** 26 ± 1.1 25 ± 1.5 20 ± 2.1 25 ± 1.8 24 ± 1.9 20 ± 4.3 15± 2.1 **Heart (%)** 0 0 100 0 0 0 0 **Neural tube (%)** 0 0 100 0 13 29 50

**Figure 4.** Morphological aspects of chicken after CdS‐maltodextrin QD treatment. Chicken treated with QDs showed different deformities. Full development was only observed with the lowest dose (0.01 ng/kg). Severe malformations were observed with 0.1 and 1 ng/kg. Effects on fetus development at Stages 20 and 23 were also observed.

These results showed that the treatment of chicken embryos with CdS‐maltodextrin QDs for 21 days caused reduced chicken viability (**Figure 4** and **Table 2**), and a dose‐dependent developmental delay was also observed. Only 35% of the chicks were alive and able to hatch when they were treated with 0.01 ng/kg of CdS‐maltodextrin QDs. The chicks were able to hatch by themselves and did not show apparent malformations, but these chicks did not attain full development (32–36 on the Hamburger‐Hamilton scale) and had a lack of lower limb motor coordination. Mortality was as high as 70% among chicks treated with 0.1 ng/kg of QDS. Of the chicks that were alive, only 20% were able to hatch by themselves, and 25% showed malformations consisting of lack of abdominal wall closure. The group of chickens treated with the highest doses (1 ng/kg) resulted in 100% mortality. These chicks showed a very early developmental stage (26–32 of the Hamburger‐Hamilton scale), and it was not even possible to detect specific organs. Chicks in advanced stages presented anencephaly and a lack of abdominal wall closure (**Figure 4** and **Table 2**).


1 Nontreated embryos.

2 Hamburger‐Hamilton scale.

\* p < 0.05 as compared to control group.

**Table 2.** Effect of CdS‐maltodextrin QDs on chicken viability and development.

## **4. Discussion**

Over the past years, there has been significant interest in the study of nanoparticles for clinical applications, particularly quantum dots (QDs). One of the most valuable QD properties is their fluorescence spectrum, which renders them optimal fluorophores for bioimaging applica‐ tions [19]. Due to their fluorescent features, QDs can be conjugated with bioactive moieties (e.g., antibodies or receptor ligands) to target specific biological events and cellular structures [20]; however, due to their small size and physical resemblance to physiological molecules, particular attention has being focused to potential risks of human beings. Understanding quantum dot potential toxicity should require a fundamental grasp of QDs properties. However, the most important problem is that each individual type of QD possesses its own unique physicochemical properties, which in turn determine its potential toxicity or lack thereof [21, 22].

developmental delay was also observed. Only 35% of the chicks were alive and able to hatch when they were treated with 0.01 ng/kg of CdS‐maltodextrin QDs. The chicks were able to hatch by themselves and did not show apparent malformations, but these chicks did not attain full development (32–36 on the Hamburger‐Hamilton scale) and had a lack of lower limb motor coordination. Mortality was as high as 70% among chicks treated with 0.1 ng/kg of QDS. Of the chicks that were alive, only 20% were able to hatch by themselves, and 25% showed malformations consisting of lack of abdominal wall closure. The group of chickens treated with the highest doses (1 ng/kg) resulted in 100% mortality. These chicks showed a very early developmental stage (26–32 of the Hamburger‐Hamilton scale), and it was not even possible to detect specific organs. Chicks in advanced stages presented anencephaly and a lack of

**Control1 Ringer 0.01 0.1 1**

0 0 0 25 44.4

Over the past years, there has been significant interest in the study of nanoparticles for clinical applications, particularly quantum dots (QDs). One of the most valuable QD properties is their

Lack of closure of abdominal wall with exposed viscera Lack of closure of abdominal wall with exposed viscera Anencephaly

Viability (%) 100 100 35 18 0 Hatch (%) 100 100 67 20 0 Stage (S)2 46 45 42 ± 3\* 30 ± 7\* 26 ± 6\*

> Lack of development Lack of motor coordination of lower limbs

abdominal wall closure (**Figure 4** and **Table 2**).

72 Toxicology - New Aspects to This Scientific Conundrum

Malformations None None Nonmalformations

**Table 2.** Effect of CdS‐maltodextrin QDs on chicken viability and development.

Malformations

Type of malformations

Nontreated embryos.

**4. Discussion**

Hamburger‐Hamilton scale.

p < 0.05 as compared to control group.

(%)

N = 20. 1

2

\*

**Parameters Controls CdS‐maltodextrin QDs (ng/kg)**

Many research groups have contributed with evidence that supports the application of a precautionary approach when creating products containing nanoparticles, such as QDs [23– 25]. According to their reports, there are several aspects to be considered. Firstly, QD size down to the nanoscale (between 1 and 100 nanometers); a small size allows nanoparticles to enter the body through several cellular barriers and pass into the bloodstream, from where they can reach organs and tissues and fully interact with biological structures, thus damaging normal functions in different ways [26]. Secondly, a potential source of confusion in assessing QD toxicity is that QD toxicity depends on multiple factors derived from both individual QD physicochemical properties and environmental conditions: QD charge, concentration, outer coating bioactivity (coating material, functional groups), as well as oxidative, photolytic, and mechanical stability have each been shown to be determining factors in QD toxicity [27]. The third aspect is that most nanomaterials enter the market without a toxicity analysis, and currently available testing is not suitable for a thorough assessment of its potential risks. The fourth aspect derives from several *in vitro* studies conducted on animals, which have shown that certain nanomaterials are toxic to subjects, and most likely are so to humans [28, 29]. The fifth and last aspect is that there are no regulations for nanomaterial synthesis, handling, use, and proper disposal in all countries.

We recently synthesized maltodextrin‐coated cadmium sulfide QDs (CdS‐maltodextrin) [14]. Although the *in vitro* studies revealed that these CdS‐maltodextrin QDs produced distinct dose‐dependent toxic effects, *in vivo* studies demonstrated that, when administered to rodents, CdS‐maltodextrin QDs were biocompatible and nontoxic after 15 days of exposure. Never‐ theless, the CdS‐maltodextrin QD pattern of biodistribution and accumulation in tissue was different after repeated doses [15]. The pharmacokinetic study of those QDs clearly showed that CdS‐maltodextrin QDs were not completely cleared from *in vivo* systems after 360 h after a single dose [16], suggesting that QDs may remain for long periods of time in some organs.

It is well known that biological barriers play an important role to determine QD biodistribu‐ tion [30, 31]. This group has demonstrated that CdS‐maltodextrin QDs are able to cross the blood–brain barrier (BBB) and spread into the brain after repeated doses in rodents without inducing morphological and functional changes [15]. However, the pharmacokinetic analysis showed that CdS‐maltodextrin QDs could remain in the brain for a very long period of time. On the other hand, we have also demonstrated the presence of CdS‐maltodextrin QDs in the blood–testis barrier (BTB) adjacent seminiferous tubules. The BTB was found to be intact and functional after a single dose as well as after repeated doses in rodents [15, 16]. Those results suggest that CdS‐maltodextrin QDs might also be able to cross the placental barrier, affecting embryos or fetuses. Therefore, concerned about the safety of CdS‐maltodextrin QDs and as an effort to further address the issue, the present work studied the effects of CdS‐maltodextrin QDs on the development of embryos and fetuses by using a chick embryo model. We used this model because it has long been appreciated that studying the embryonic chick *in ovo* provides a variety of advantages, including the potential to control the embryo's environment and its movement independently of maternal influences.

Exposure to chemicals during different stages of development—such as the preconceptional, periconceptional, embryonic, fetal and perinatal periods—has a varying impact on health. However, the embryonic period is the most critical period for embryogenesis, when mortality or different congenital anomalies are highly possible. NP direct embryotoxicity firstly depends on the ability of the compound to cross the maternal–fetal barrier, how much nanomaterial is accumulated in embryonic tissues, and its ability to induce damage [32]. Several reports have demonstrated contradictory effects of nanomaterials on embryos or their development. A decrease in embryonic weight after QD injection on the sixth day of embryogenesis (CdSe/ZnS QDs, 9.6%; CdT QDs, 6.2%) has been reported [33], whereas others have found that nanoma‐ terials did not produce embryotoxic or teratogenic effects during embryogenesis [34]. It has been shown that NPs (nSP and TiO2s 70 and 35 nm in diameter, respectively) can cross the placental barrier in pregnant mice and cause neurotoxicity in their offspring, and then remain in the placenta, fetal liver, and fetal brain [35]. Some authors argue that some NPs (CdSe and CdTe/CdS) in different sizes, at different dosages, and with different outer capping materials can increase the rate of early‐stage blastocyst death in mice and can be potentially transferred across the placenta to the fetus [32, 36]. On the other hand, some researchers have reported that the embryotoxic effects of some NPs can be modified if they are coated with silanes or by using a gold shell [37].

In the present study, we used different doses of 3.5‐nm‐sized CdS‐maltodextrin QDs, and the dose‐dependent embryotoxic effect was evident. Observation by fluorescence microscopy revealed the homogeneous distribution of QDs into the embryos. A significant concentration‐ related decline in embryonic growth was observed, as well as an increase in developmental defects, including various neuronal abnormalities. It has been reported that some nanomate‐ rials induce neuronal alteration, which suggests an NP capacity to interfere with normal neurotransmission pathways [35, 38]. Recent studies have demonstrated QD‐related neuro‐ toxicity in the CNS, as well as synaptic transmission and plasticity impairments, and deterio‐ rated brain functions in tested animals [39–41]. Another important finding from the present study was the presence of marked anomalies on somites. Somites are transient embryonic structures of the paraxial mesoderm that give rise to all off the striated muscular tissue in the adult body, the axial skeleton, and dermis during later embryogenesis. We found a significant reduction in the size of the somites n embryos treated with CdS‐maltodextrin QDs, which correlates with the overall body shortening observed. When we analyzed the size of each somite separately, we observed that the somites in treated embryos tended to have a different size, morphology or absence, contrary to controls.

Birth defects result from errors during embryonic development. Normal development involves careful orchestration of multiple events, including changes in gene transcription, cell shape, cell proliferation and tissue morphogenesis. Alterations in the developmental program may have dramatic effects on the organism. Sometimes, these effects are so drastic that lethality occurs early in the development, while others may cause birth defects. We found several defects in chicken after normal periods of development: chicks with poor development; CNS altera‐ tions or even anencephaly; lack of limb motor coordination; spasticity; and a lack of abdominal wall closure. Some limited data from animal reproductive studies and nanomaterials suggest a potentially increased risk of early miscarriages, impaired growth and birth defects [42, 43]. A particular birth defect may be caused by several mechanisms, including folate antagonism, neural crest cell disruption, endocrine disruption, oxidative stress, vascular disruption and specific receptor or enzyme‐mediated teratogenesis. In addition, some drugs may be involved in multiple mechanisms leading to birth defects [44].

suggest that CdS‐maltodextrin QDs might also be able to cross the placental barrier, affecting embryos or fetuses. Therefore, concerned about the safety of CdS‐maltodextrin QDs and as an effort to further address the issue, the present work studied the effects of CdS‐maltodextrin QDs on the development of embryos and fetuses by using a chick embryo model. We used this model because it has long been appreciated that studying the embryonic chick *in ovo* provides a variety of advantages, including the potential to control the embryo's environment and its

Exposure to chemicals during different stages of development—such as the preconceptional, periconceptional, embryonic, fetal and perinatal periods—has a varying impact on health. However, the embryonic period is the most critical period for embryogenesis, when mortality or different congenital anomalies are highly possible. NP direct embryotoxicity firstly depends on the ability of the compound to cross the maternal–fetal barrier, how much nanomaterial is accumulated in embryonic tissues, and its ability to induce damage [32]. Several reports have demonstrated contradictory effects of nanomaterials on embryos or their development. A decrease in embryonic weight after QD injection on the sixth day of embryogenesis (CdSe/ZnS QDs, 9.6%; CdT QDs, 6.2%) has been reported [33], whereas others have found that nanoma‐ terials did not produce embryotoxic or teratogenic effects during embryogenesis [34]. It has been shown that NPs (nSP and TiO2s 70 and 35 nm in diameter, respectively) can cross the placental barrier in pregnant mice and cause neurotoxicity in their offspring, and then remain in the placenta, fetal liver, and fetal brain [35]. Some authors argue that some NPs (CdSe and CdTe/CdS) in different sizes, at different dosages, and with different outer capping materials can increase the rate of early‐stage blastocyst death in mice and can be potentially transferred across the placenta to the fetus [32, 36]. On the other hand, some researchers have reported that the embryotoxic effects of some NPs can be modified if they are coated with silanes or by

In the present study, we used different doses of 3.5‐nm‐sized CdS‐maltodextrin QDs, and the dose‐dependent embryotoxic effect was evident. Observation by fluorescence microscopy revealed the homogeneous distribution of QDs into the embryos. A significant concentration‐ related decline in embryonic growth was observed, as well as an increase in developmental defects, including various neuronal abnormalities. It has been reported that some nanomate‐ rials induce neuronal alteration, which suggests an NP capacity to interfere with normal neurotransmission pathways [35, 38]. Recent studies have demonstrated QD‐related neuro‐ toxicity in the CNS, as well as synaptic transmission and plasticity impairments, and deterio‐ rated brain functions in tested animals [39–41]. Another important finding from the present study was the presence of marked anomalies on somites. Somites are transient embryonic structures of the paraxial mesoderm that give rise to all off the striated muscular tissue in the adult body, the axial skeleton, and dermis during later embryogenesis. We found a significant reduction in the size of the somites n embryos treated with CdS‐maltodextrin QDs, which correlates with the overall body shortening observed. When we analyzed the size of each somite separately, we observed that the somites in treated embryos tended to have a different

movement independently of maternal influences.

74 Toxicology - New Aspects to This Scientific Conundrum

size, morphology or absence, contrary to controls.

using a gold shell [37].

Somitogenesis, in particular, is a reiterated process occurring over time with strict periodicity. A pair of somites is formed every 90 min in the trunk of the chicken embryo from the anterior tip of the presomitic mesoderm (PSM). As development proceeds, ventral somitic cells migrate around the axial organs, giving rise to segmented structures such as vertebrae, intervertebral disks, and trunk and limb developments, whereas more dorsal somitic cells give rise to the dermis and all of the striated muscles of the adult body [45]. Somitogenesis can be achieved only by the integration of multiple signaling pathways involving intricate molecular machi‐ nery, cell proliferation and the genetic clock [46]. It has been reported that the use of transcrip‐ tion factor chemical inhibitors or genes induces severe defects in somite formation due to segmentation clock desynchronization [47]. Recent reports have shown evidence that the presence of reactive oxygen species (ROS) may affect the metabolic regulation of the ultradian biological oscillator with important pathophysiological implications for somitogenesis [48].

In our study, although higher doses were used in the embryotoxicity study, the lack of development and birth defects was clear, and even with the same doses there were variations in embryo development, which could be explained by individual susceptibility. Mortality was high, and the malformations and stages of development were not compatible with life. However, the presence of defects in limb neuromotor coordination, the lack of abdominal wall closure and neuronal alterations suggest that CdS‐maltodextrin QDs might be affecting the early stages of somitogenesis. We suggest that CdS‐maltodextrin QDs primarily affect the number of cells that segment together to form individual somites in chicken embryos and that this is responsible for the mortality and all the observed effects on the fetuses. It has been shown that CdS‐maltodextrin QDs are able to induce cytotoxicity and cell death, alter cell proliferation and induce the production of radical oxygen species (ROS) in a dose‐dependent manner [14]. QD‐induced perturbations of cellular mechanisms may cause different pathophysiological processes depending on concentration and duration of exposure [49–51].

In conclusion, our data indicate that CdS‐maltodextrin QDs induce embryotoxic and terato‐ genic effects with all doses used. QDs induced abnormalities associated with structures derived from somites in embryos and fetuses. Therefore, according to the results, there is a high probability that the prolonged accumulation of CdS‐maltodextrin QDs in the maternal organism may be potentially harmful to embryo and fetus development. However, further studies using mammalian species are needed in order to discard more toxic effects.

## **Author details**

Jorge Reyes‐Esparza1 , Janet Sánchez‐Quevedo1 , Antonieta Gómez‐Solís1 , Patricia Rodríguez‐Fragoso2\*, Gerardo González De la Cruz2 and Lourdes Rodríguez‐Fragoso1

\*Address all correspondence to: mrodriguezf@uaem.mx

1 Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, México

2 Departamento de Física, CINVESTAV – I.P.N., Mexico, D.F., Mexico

## **References**


[8] Wick P, Malek A, Manser P, Meili D, Maeder‐Althaus X, Diener L, Diener PA, Zisch A, Krug HF, von Mandach U. Barrier capacity of human placenta for nanosized materials. Environ Health Perspect. 2010; 118(3):432–6. doi:10.1289/ehp.0901200.

organism may be potentially harmful to embryo and fetus development. However, further

1 Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, México

[1] Ruenraroengsak P, Cook JM, Florence AT. Nanosystem drug targeting: facing up to complex realities. J Control Release. 2010; 141(3):265–76. doi:10.1016/j.jconrel.

[2] Tang Y, Han S, Liu H, Chen X, Huang L, Li X, Zhang J. The role of surface chemistry in determining *in vivo* biodistribution and toxicity of CdSe/ZnS core‐shell quantum dots. Biomaterials. 2013; 34(34):8741–55. doi:10.1016/j.biomaterials.2013.07.087.

[3] Hassan S, Singh AV. Biophysicochemical perspective of nanoparticle compatibility: a critically ignored parameter in nanomedicine. J Nanosci Nanotechnol. 2014; 14(1):402–

[4] Brendel K, Duhamel RC, Shepard TH. Embryotoxic drugs. Biol Res Pregnancy Perina‐

[5] Thompson BL, Levitt P, Stanwood GD. Prenatal exposure to drugs: effects on brain development and implications for policy and education. Nat Rev Neurosci. 2009; 10(4):

[6] Barua S, Mitragotri S. Challenges associated with penetration of nanoparticles across cell and tissue barriers: a review of current status and future prospects. Nano Today.

[7] Chu M, Wu Q, Yang H, Yuan R, Hou S, Yang Y, Zou Y, Xu S, Xu K, Ji A, Sheng L. Transfer of quantum dots from pregnant mice to pups across the placental barrier. Small. 2010;

, Antonieta Gómez‐Solís1

and

,

studies using mammalian species are needed in order to discard more toxic effects.

, Janet Sánchez‐Quevedo1

2 Departamento de Física, CINVESTAV – I.P.N., Mexico, D.F., Mexico

Patricia Rodríguez‐Fragoso2\*, Gerardo González De la Cruz2

\*Address all correspondence to: mrodriguezf@uaem.mx

**Author details**

Jorge Reyes‐Esparza1

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## **Environmental Fate of Zinc Oxide Nanoparticles: Risks and Benefits Environmental Fate of Zinc Oxide Nanoparticles: Risks and Benefits**

Asfina Beegam, Parvathy Prasad, Jiya Jose, Miguel Oliveira, Fernando G. Costa, Amadeu M.V.M. Soares, Paula P. Gonçalves, Tito Trindade, Nandakumar Kalarikkal, Sabu Thomas and Maria de Lourdes Pereira Asfina Beegam, Parvathy Prasad, Jiya Jose, Miguel Oliveira, Fernando G. Costa, Amadeu M.V.M. Soares, Paula P. Gonçalves, Tito Trindade, Nandakumar Kalarikkal, Sabu Thomas and Maria de Lourdes Pereira

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65266

#### **Abstract**

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Zinc oxide nanoparticles (ZnO-NPs) are among nanoscale materials displaying exponentially growing production due to their applications in the field of cosmetology, medicine, as antibacterial agent and catalyst. The ZnO nanomaterials release into the aquatic ecosystems through domestic and industrial wastewaters has the potential to induce pernicious effects on fish and other organisms. Increasing concerns on the environmental hazard to aquatic biota have been highlighted by the toxic potential of some metal-based nanomaterials. Several characteristics of ZnO-NPs (e.g. size, shape, surface charge and agglomeration state) play a central role in biological effects such as genotoxic, mutagenic or cytotoxic effects. Overall, Zn bioaccumulation, histopathological, and hematological changes with oxidative and cellular stress have been reported in ZnO-NPs exposed animals.

This chapter provides an overview on applications of ZnO-NPs followed by a brief outline on methods of synthesis and characterization, and the current knowledge on the ZnO-NPs interaction with fish as they are valuable models in ecotoxicology, sensitive to many contaminants, representing a potential source of food for humans. This chapter intends to provide information for a critical overview of the pros and cons of using these particles, factors influencing their effects, and potential human health implications.

**Keywords:** zinc oxide nanoparticles (ZnO-NPs), nano-ecotoxicology, fish, human health

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

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution

## **1. Introduction**

#### **1.1. Applications of zinc oxide nanoparticles**

Zinc oxide nanoparticles (ZnO-NPs) are commonly used in several domains of human activity such as cosmetics, paints, optoelectronics, and pharmaceuticals, due to their low cost and interesting properties (e.g., conductivity, chemical stability, catalytic properties, photonics and optoelectronics, antibacterial, antifungal, and UV filtering properties). ZnO-NPs are highly used in the cosmetic industry, typically in sunscreens and facial creams [1]. In the biomedical field, ZnO-NPs have been applied in cell imaging [2, 3], drug delivery, and have demonstrated promising results in cancer research (for review see [4, 5]).

ZnO-NPs have shown to decrease the viability of cultured cell lines derived from human cancers. ZnO-NPs induced a 50% reduction in cell viability in MCF7 (breast cancer) and A549 (lung cancer) cell cultures, at a very low concentration (31.2 μg ml−1), with size-dependent effectiveness [6]. A high toxicity on T98G (brain cancer) cells, moderate toxicity on KB (skin cancer) cells, and low toxicity on normal human HEK cells have also been reported [7]. ZnO-NPs have been proposed as genotoxic since they induced micronucleus in those cells. Apoptosis and intracellular production of reactive oxygen species (ROS) have been reported on melanoma cancer cells after exposure to different doses of ZnO-NPs [8]. These nanomaterials also exhibited activity against HepG2 (liver cells) cells depending on the dose [9]. A timedependent reduction in the viability of murine cancer cells after exposure of ZnO-NPs was recently documented by [10]. ZnO-NPs strong protein adsorption properties may also lead to its use in other biomedical applications. ZnO-NPs may be used to modulate metabolism and cellular responses, and have been proven useful for the detection of low levels of biomarkers (e.g., proteins/peptides [11]). ZnO-NPs have shown promising results as cholesterol sensors, controlling diabetes and hyperglycemia, modulation of some allergic reactions, via inhibition of mast cell degranulation [12] as well in tissue engineering scaffolds to enhance angiogenesis [13].

As for other nanoparticles, ZnO-NPs may also be toxic for some microorganisms, making them potential antibacterial, antifungal, and antiviral agents. This is an important feature of these nanomaterials considering the increasing concerns related to the proliferation of pathogenic microorganisms that are multiresistant to conventional antibiotics. ZnO-NPs may interact with the bacterial surface and/or with the bacterial core, exhibiting different bactericidal mechanisms. Antimicrobial properties of ZnO-NPs have been demonstrated on bacteria such as *Bacillus subtilis, Escherichia coli* and *Pseudomonas fluorescens* [14], *Staphylococcus aureus* and *Salmonella typhimurium*, as well as on the fungi *Aspergillus flavus* and *Aspergillus fumigatus* [15]. In a study aiming to evaluate the immunological and antibacterial mechanisms of ZnO-NPs against human pathogens, Rashmirekha et al. [16] reported a higher effect of ZnO-NPs against *Staphylococcus aureus* when compared to *Mycobacterium bovis-BCG*. ZnO-NPs were able to disrupt bacterial cell membrane integrity, decrease cell surface hydrophobicity, and downregulate the transcription of oxidative stress-resistance genes in bacteria. The intradermal administration of ZnO-NPs reduced the skin infection, bacterial load, and inflammation in mice. ZnO-NPs treatment also increased the bacterial killing by inducing ROS. Virostatic potential of micro-/nanofilopodia-like ZnO structures against herpes simplex virus-1 was reported by Mishra and colleagues [17]. Antoine and colleagues [18] synthesized 200 nm to 1 μm ZnO tetrapod-like structures, by flame transport method, to check the antiviral properties of ZnO micro- and nanostructures against HSV-2. ZnO tetrapods blocked the HSV-2 entry into the target cells and stopped the virus dispersal among already infected cells. The prophylactic treatment showed a decrease in HSV-2 internalization in both UV-treated and in nontreated conditions. The decreased internalization supports the preventive function of ZnO tetrapod nanoparticles against the ability of viruses to enter into the susceptible cells. ZnO tetrapods treatment decreased the cell fusion and syncytial formation of CHO-K1 cells.

ZnO-NPs synthesized by Nohynek et al. (2007), using wet chemical methods, revealed a high antibacterial activity, due to its inherent ability to absorb UV irradiation and optical transparency. This makes ZnO-NPs an important compound for the cosmetic industry, namely in formulations for sunscreens and facial creams [1]. The antibacterial activities of ZnO-NPs, as mentioned above, significantly contribute to its value in food processing industry, as a potent sanitizing agent for disinfecting and sterilizing food industry equipment and containers against foodborne pathogenic bacteria. ZnO-NPs are able to disrupt *E. coli* and *S. aureus* cell membrane causing cytoplasmic leakage and able to inhibit and kill the foodborne pathogens [19].

At industrial level, ZnO-NPs have various applications in catalysis and electronics [20]. ZnO-NPs can be used in infrared and chemical sensors, in the manufacture of rubber and cigarettes (used as filter) and preparation of creams and ointments used to treat skin diseases. The range of possible applications of ZnO-NPs also includes agriculture. Studies have shown potential beneficial effects of ZnO-NPs on seed germination, water purification, and soil remediation. Peanut seeds treated with 25 nm ZnO-NPs (1000 ppm) displayed high germination, seeding vigor, and plant growth [21], while decreased ryegrass germination has been reported after ZnO-NPs exposure [22]. Furthermore, the potential of ZnO-NPs to reduce microbial biomass and diversity [23] must also be taken into account.

## **1.2. Synthesis and characterization of ZnO nanoparticles**

**1. Introduction**

[13].

**1.1. Applications of zinc oxide nanoparticles**

82 Toxicology - New Aspects to This Scientific Conundrum

promising results in cancer research (for review see [4, 5]).

Zinc oxide nanoparticles (ZnO-NPs) are commonly used in several domains of human activity such as cosmetics, paints, optoelectronics, and pharmaceuticals, due to their low cost and interesting properties (e.g., conductivity, chemical stability, catalytic properties, photonics and optoelectronics, antibacterial, antifungal, and UV filtering properties). ZnO-NPs are highly used in the cosmetic industry, typically in sunscreens and facial creams [1]. In the biomedical field, ZnO-NPs have been applied in cell imaging [2, 3], drug delivery, and have demonstrated

ZnO-NPs have shown to decrease the viability of cultured cell lines derived from human cancers. ZnO-NPs induced a 50% reduction in cell viability in MCF7 (breast cancer) and A549 (lung cancer) cell cultures, at a very low concentration (31.2 μg ml−1), with size-dependent effectiveness [6]. A high toxicity on T98G (brain cancer) cells, moderate toxicity on KB (skin cancer) cells, and low toxicity on normal human HEK cells have also been reported [7]. ZnO-NPs have been proposed as genotoxic since they induced micronucleus in those cells. Apoptosis and intracellular production of reactive oxygen species (ROS) have been reported on melanoma cancer cells after exposure to different doses of ZnO-NPs [8]. These nanomaterials also exhibited activity against HepG2 (liver cells) cells depending on the dose [9]. A timedependent reduction in the viability of murine cancer cells after exposure of ZnO-NPs was recently documented by [10]. ZnO-NPs strong protein adsorption properties may also lead to its use in other biomedical applications. ZnO-NPs may be used to modulate metabolism and cellular responses, and have been proven useful for the detection of low levels of biomarkers (e.g., proteins/peptides [11]). ZnO-NPs have shown promising results as cholesterol sensors, controlling diabetes and hyperglycemia, modulation of some allergic reactions, via inhibition of mast cell degranulation [12] as well in tissue engineering scaffolds to enhance angiogenesis

As for other nanoparticles, ZnO-NPs may also be toxic for some microorganisms, making them potential antibacterial, antifungal, and antiviral agents. This is an important feature of these nanomaterials considering the increasing concerns related to the proliferation of pathogenic microorganisms that are multiresistant to conventional antibiotics. ZnO-NPs may interact with the bacterial surface and/or with the bacterial core, exhibiting different bactericidal mechanisms. Antimicrobial properties of ZnO-NPs have been demonstrated on bacteria such as *Bacillus subtilis, Escherichia coli* and *Pseudomonas fluorescens* [14], *Staphylococcus aureus* and *Salmonella typhimurium*, as well as on the fungi *Aspergillus flavus* and *Aspergillus fumigatus* [15]. In a study aiming to evaluate the immunological and antibacterial mechanisms of ZnO-NPs against human pathogens, Rashmirekha et al. [16] reported a higher effect of ZnO-NPs against *Staphylococcus aureus* when compared to *Mycobacterium bovis-BCG*. ZnO-NPs were able to disrupt bacterial cell membrane integrity, decrease cell surface hydrophobicity, and downregulate the transcription of oxidative stress-resistance genes in bacteria. The intradermal administration of ZnO-NPs reduced the skin infection, bacterial load, and inflammation in mice. ZnO-NPs treatment also increased the bacterial killing by inducing ROS. Virostatic

ZnO-based materials have been the subject of several reviews in the past years. A detailed survey on the literature concerning the synthesis and properties of nanosized ZnO can be found elsewhere [24]. ZnO is an inorganic crystalline compound with a band-gap energy located in the UV region that widens as the size of the particles decreases below a threshold of a few nanometers. Both in the bulk form and as a nanoscale material, ZnO is an important material for several applications including in electronics and optical devices [24]. ZnO-NPs may be synthesized by a variety of methods, selected based on the desired application, morphology, and size. Chemical and physical parameters (e.g., solvent type, precursors, pH, and temperature) are of high relevance in the synthesis protocols. A variety of shapes (nanorods, nanosphere, nanotubes, nanowires, nanoneedles, nanorings, spirals, drums, polyhedrons, disks, flowers, stars, boxes, and plates) may be produced, each displaying morphological-dependent physicochemical properties [25] that allow the exploitation of a variety of applications.

Some preparative methods include chemical vapor deposition, precipitation in aqueous solution, hydrothermal synthesis, sol-gel method, and synthesis using microemulsions and mechanochemical processes. These methods allow the production of particles differing in shape and size. Some available reviews present a thorough explanation of the principles and techniques involved in the different procedures [26]. Briefly, the mechanochemical method is based on high-energy dry milling; the controlled precipitation method involves hydrolysis of a Zn(II) solution, in conditions that limit uncontrolled growth of particles, eventually followed by a thermal treatment to improve crystallinity; the hydrothermal method is a simple and environmentally friendly technique that involves the thermal treatment of Zn(II) aqueous solutions under auto-generated pressure, by using an autoclave as the reaction vessel. ZnO-NPs have also been prepared using a Zn(II) precursor in the presence of plant extracts [27, 28] as cost-effective approaches, with promising results in terms of bioapplications. Selected examples of synthesis methods for ZnO-NPs are presented below.

ZnO-NPs were synthesized by Aneesh et al. [29] by hydrothermal treatment of Zn(CH3COO2) 2H2O (0.1 M) solutions. ZnO-NPs were prepared by varying the growth temperature and concentration of the Zn(II) precursor. X-ray diffraction (XRD) performed on powdered samples revealed nanoparticles of *wurtzite*-type structure. This synthesis yielded particles of sizes between 7 and 24 nm. Overall, particle size increased with growth temperature and decreased with concentration of precursor. Ramimoghadam et al. [30] synthesized ZnO-NPs also by a hydrothermal method, using palm olein as biotemplate. Different morphologies including flake-flower and 3D star-like structure were obtained. The concentration of palm olein has an effective role on observed morphological changes of the synthesized nanoparticles. These changes are possibly due to the reaction between carboxylic groups of palm olein and hydroxyl groups at the surface of ZnO. The biotemplates could be also used to modify the surface properties of ZnO-NPs.

Soni and Koser [31] used a hydrolysis method for the synthesis of ZnO-NPs, with different concentrations of a surface-protecting agent (thioglycerol). UV-VIS spectroscopy revealed blueshifts in the absorption bands of the samples, as compared to the spectrum of typical bulk ZnO, as an indication for the presence of nanosized ZnO. The absorption band edge was observed in the UV region at wavelength 365, 362, and 364 nm for ZnO-NPs synthesized using 0.12, 0.3, and 0.5 ml of capping agent, respectively. The samples were composed of particles with average sizes between 3.5 and 3.9 nm, depending on the amount of molar concentration of the capping agent. Increasing the concentration of capping agent, the average particle size decreased and the respective band gap widens due to quantum size effects [24]. Also using a hydrolysis method, Wang et al. [32] have synthesized nanometric ZnO using cetyltrimethylammonium bromide (CTAB) as surfactant. These authors reported highcrystalline nanoparticles of 50 nm average diameter. CTAB affected the process of nucleation and growth of crystallites during the synthesis also preventing the formation of ZnO agglomerates.

Giri et al. [33] synthesized hexagonal ZnO-NPs and nanorods by low-temperature oxidation of metallic Zn powder in the presence of acetic acid and trifluoroacetic acid. The final colorless powders were a first indication for the presence of ZnO. In this method, acetic acid and trifluoroacetic acid induced the growth of hexagonal-type ZnO-NPs and ZnO nanorods, respectively, whose crystalline nature was confirmed by XRD. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images displayed hexagonal cross section of the nanoparticles and nanorods. The samples showed strong UV absorption peaked at 378 nm. A green synthesis method was used by Oudhia et al. [34] for ZnO nanotubes aiming biomedical application. For this purpose, neem leaf extract as biotemplate was used. The XRD pattern indicated the *wurtzite*-type structure of ZnO-NPs. The average crystalline size of the synthesized nanotubes was estimated as 25 nm by using Debye-Scherrer equation applied to the XRD patterns of the samples.

Some preparative methods include chemical vapor deposition, precipitation in aqueous solution, hydrothermal synthesis, sol-gel method, and synthesis using microemulsions and mechanochemical processes. These methods allow the production of particles differing in shape and size. Some available reviews present a thorough explanation of the principles and techniques involved in the different procedures [26]. Briefly, the mechanochemical method is based on high-energy dry milling; the controlled precipitation method involves hydrolysis of a Zn(II) solution, in conditions that limit uncontrolled growth of particles, eventually followed by a thermal treatment to improve crystallinity; the hydrothermal method is a simple and environmentally friendly technique that involves the thermal treatment of Zn(II) aqueous solutions under auto-generated pressure, by using an autoclave as the reaction vessel. ZnO-NPs have also been prepared using a Zn(II) precursor in the presence of plant extracts [27, 28] as cost-effective approaches, with promising results in terms of bioapplications. Selected

ZnO-NPs were synthesized by Aneesh et al. [29] by hydrothermal treatment of Zn(CH3COO2) 2H2O (0.1 M) solutions. ZnO-NPs were prepared by varying the growth temperature and concentration of the Zn(II) precursor. X-ray diffraction (XRD) performed on powdered samples revealed nanoparticles of *wurtzite*-type structure. This synthesis yielded particles of sizes between 7 and 24 nm. Overall, particle size increased with growth temperature and decreased with concentration of precursor. Ramimoghadam et al. [30] synthesized ZnO-NPs also by a hydrothermal method, using palm olein as biotemplate. Different morphologies including flake-flower and 3D star-like structure were obtained. The concentration of palm olein has an effective role on observed morphological changes of the synthesized nanoparticles. These changes are possibly due to the reaction between carboxylic groups of palm olein and hydroxyl groups at the surface of ZnO. The biotemplates could be also used to modify the sur-

Soni and Koser [31] used a hydrolysis method for the synthesis of ZnO-NPs, with different concentrations of a surface-protecting agent (thioglycerol). UV-VIS spectroscopy revealed blueshifts in the absorption bands of the samples, as compared to the spectrum of typical bulk ZnO, as an indication for the presence of nanosized ZnO. The absorption band edge was observed in the UV region at wavelength 365, 362, and 364 nm for ZnO-NPs synthesized using 0.12, 0.3, and 0.5 ml of capping agent, respectively. The samples were composed of particles with average sizes between 3.5 and 3.9 nm, depending on the amount of molar concentration of the capping agent. Increasing the concentration of capping agent, the average particle size decreased and the respective band gap widens due to quantum size effects [24]. Also using a hydrolysis method, Wang et al. [32] have synthesized nanometric ZnO using cetyltrimethylammonium bromide (CTAB) as surfactant. These authors reported highcrystalline nanoparticles of 50 nm average diameter. CTAB affected the process of nucleation and growth of crystallites during the synthesis also preventing the formation of ZnO

Giri et al. [33] synthesized hexagonal ZnO-NPs and nanorods by low-temperature oxidation of metallic Zn powder in the presence of acetic acid and trifluoroacetic acid. The final colorless powders were a first indication for the presence of ZnO. In this method, acetic acid and

examples of synthesis methods for ZnO-NPs are presented below.

face properties of ZnO-NPs.

84 Toxicology - New Aspects to This Scientific Conundrum

agglomerates.

Barreto et al. [35] synthesized ZnO-NPs by microwave-assisted method and checked the effect of precursor reagent, temperature, irradiation time, and additives on the morphology of synthesized nanoparticles by using (Zn(NO3)26H2O, Zn(CH3COO2)2H2O, or ZnCl2) as precursor. Radiation temperature of 80–140°C and increase in the irradiation time give high purity and homogenous size and shape of nanoparticles. The final pH is another important variable which causes significant changes in the morphology of the final particles. The addition of the anionic surfactant (AOT, sodium di-2-ethylhexyl sulfosuccinate) to the reaction medium allowed the synthesis of smaller particles. Kumar and Rani [36] synthesized ZnO-NPs by using microemulsions as nanoreactor for the synthesis of ZnO using ZnSO4 salt. The stable reverse micelle microemulsion was prepared by mixing a nonionic surfactant, Triton X-100, PVP (used as co-stabilizing agent), cyclohexane, and distilled water. XRD diffraction analysis shows the typical hexagonal *wurtzite*-type structure of ZnO. TEM revealed nanoparticles of 10–12 nm in average size and rod shaped, and UV-VIS spectroscopy was use to estimate the optical band gap of the samples.

Tsuzuki and Cormick [37] synthesized nanocrystallites of ZnO of 26 nm size by a mechanochemical method using ZnCO3 as the precursor. It was observed that a milling time of 4 h was enough for the synthesis of ZnO-NPs. Song et al. [2] synthesized ellipsoidal ZnO-NPs with high crystal quality by another mechanochemical method. It was observed that depending on the solvent used, the ZnO-NPs remained dispersed with a mean diameter of 21 nm (nonpolar solvents), whereas in more polar solvents the nanoparticles gradually aggregated to a diameter of about 200 nm. Photoluminescence spectra of ZnO-NPs have been reported.

The routine methodologies used to characterize ZnO-NPs (colloids and powders) are those commonly applied to characterize other types of nanoparticles and include dynamic light scattering (DLS) techniques, UV-VIS absorption spectroscopy, selected area electron diffraction (SAED), and powder X-ray diffraction (XRD). The size and shape of the nanoparticles are directly analyzed by using microscopy (transmission electron microscopy—TEM, atomic force microscopy—AFM, or scanning electron—SEM). The crystalline phase (typically *wurtzite* type) can be identified by using XRD and the surface charge of the colloidal NPs through zeta potential measurements. A number of processes involving NPs are mediated by the surface. Although its characterization is not straightforward, important information can be acquired by using Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS) among other techniques.

## **2. Nanoparticles in aquatic systems**

Increase in production and applications of ZnO-NPs is expected to result in its increased release into the environment. Ultimately, the aquatic ecosystems will probably be the main recipients, mainly as a result of industrial and domestic wastewaters [38]. As one of the most produced nanoparticles in the European Union, with an estimated production of around 1600 t [39], ZnO-NPs environmental release may occur as early as their production, production of products containing nanoparticles, during their use and end of life of those products. Despite the knowledge that nanoparticles are increasingly being used in different fields of human activity, the quantification of their release in the environment, at any given time, is quite challenging due to the limited data on their current and expected prevalence in commercial products [40–42]. The technical difficulties associated with quantification of ZnO-NPs levels in the environment led to the need to predict environmental concentrations based on market penetration of nanomaterials, known usage of the products as well as fate/behavior. According to the available data, the theoretically predicted average environmental concentration of ZnO-NPs in European surface waters is 0.09 μg L−1 (with 85% confidence intervals: 0.05–0.29) [39]. No standards have yet been established for permissible levels of nanoparticles in the environment. Nonetheless, in addition to dose, physicochemical properties of nanoparticles (e.g., size, shape, chemical composition, aggregation) as well as ionic strength and pH of receiving media play determinant roles on their behavior, bioavailability, and biological effects of nanoparticles [43]. Once released into the environment, ZnO-NPs may display different behaviors. Nanoparticles in the environment may stay in suspensions as individual particles; dissolve; aggregate; form larger particles and ultimately sediment; adsorb onto water constituents (e.g., dissolved organic matter); transform chemically (e.g., due to redox reactions) or biologically (e.g., in the presence of microorganisms) in the marine environment [44]. Once in the environment, most ZnO-NPs are likely to precipitate due to its poor colloidal stability [45]. The available studies indicate that aggregation of ZnO-NPs, as for other nanoparticles, increases with ionic strength. In high ionic strength environments, reduction in electrostatic repulsion forces between the nanoparticles occurs, promoting aggregation and sedimentation. However, the presence of natural substances such as humic acids may help to steric and electrostatic stabilization of ZnO-NPs, aiding in their transport, mobility, and dispersion [46]. Other highly relevant alterations that may occur in the environment and lead to toxic effects of ZnO-NPs are the dissolution and redox transformations. ZnO-NPs may dissolve, releasing Zn ions which may induce toxic effects [47], with reported faster dissolutions at smaller sizes [48]. Redox reactions on ZnO-NPs' surface may lead to the production of ROS, which are able to oxidize organic compounds and lead to oxidative stress. Thus, for risk assessments of nanoparticles such as ZnO-NPs, different factors have to be taken into account based on the wide variety of reactivities and properties of a particular type of nanoparticle [49].

The available studies on the behavior of ZnO-NPs in water systems have been performed under laboratory conditions, focusing mostly on freshwater. Available data indicate that dissolution is dependent on concentration, with the lowest dissolution percentage at the highest ZnO concentrations [50].

## **3. Interaction of nanoparticles with aquatic organisms**

**2. Nanoparticles in aquatic systems**

86 Toxicology - New Aspects to This Scientific Conundrum

reactivities and properties of a particular type of nanoparticle [49].

concentrations [50].

The available studies on the behavior of ZnO-NPs in water systems have been performed under laboratory conditions, focusing mostly on freshwater. Available data indicate that dissolution is dependent on concentration, with the lowest dissolution percentage at the highest ZnO

Increase in production and applications of ZnO-NPs is expected to result in its increased release into the environment. Ultimately, the aquatic ecosystems will probably be the main recipients, mainly as a result of industrial and domestic wastewaters [38]. As one of the most produced nanoparticles in the European Union, with an estimated production of around 1600 t [39], ZnO-NPs environmental release may occur as early as their production, production of products containing nanoparticles, during their use and end of life of those products. Despite the knowledge that nanoparticles are increasingly being used in different fields of human activity, the quantification of their release in the environment, at any given time, is quite challenging due to the limited data on their current and expected prevalence in commercial products [40–42]. The technical difficulties associated with quantification of ZnO-NPs levels in the environment led to the need to predict environmental concentrations based on market penetration of nanomaterials, known usage of the products as well as fate/behavior. According to the available data, the theoretically predicted average environmental concentration of ZnO-NPs in European surface waters is 0.09 μg L−1 (with 85% confidence intervals: 0.05–0.29) [39]. No standards have yet been established for permissible levels of nanoparticles in the environment. Nonetheless, in addition to dose, physicochemical properties of nanoparticles (e.g., size, shape, chemical composition, aggregation) as well as ionic strength and pH of receiving media play determinant roles on their behavior, bioavailability, and biological effects of nanoparticles [43]. Once released into the environment, ZnO-NPs may display different behaviors. Nanoparticles in the environment may stay in suspensions as individual particles; dissolve; aggregate; form larger particles and ultimately sediment; adsorb onto water constituents (e.g., dissolved organic matter); transform chemically (e.g., due to redox reactions) or biologically (e.g., in the presence of microorganisms) in the marine environment [44]. Once in the environment, most ZnO-NPs are likely to precipitate due to its poor colloidal stability [45]. The available studies indicate that aggregation of ZnO-NPs, as for other nanoparticles, increases with ionic strength. In high ionic strength environments, reduction in electrostatic repulsion forces between the nanoparticles occurs, promoting aggregation and sedimentation. However, the presence of natural substances such as humic acids may help to steric and electrostatic stabilization of ZnO-NPs, aiding in their transport, mobility, and dispersion [46]. Other highly relevant alterations that may occur in the environment and lead to toxic effects of ZnO-NPs are the dissolution and redox transformations. ZnO-NPs may dissolve, releasing Zn ions which may induce toxic effects [47], with reported faster dissolutions at smaller sizes [48]. Redox reactions on ZnO-NPs' surface may lead to the production of ROS, which are able to oxidize organic compounds and lead to oxidative stress. Thus, for risk assessments of nanoparticles such as ZnO-NPs, different factors have to be taken into account based on the wide variety of Among aquatic species, fish have been considered as the ideal sentinels to detect toxicological effects, due to their wide distribution, known physiology, and sensitivity to exposure to contaminants via food or water. In this chapter, some of the available studies with fish at different life stages (embryos, juveniles, and adults) are presented in **Table 1**.



effect of different *idella* (Grass carp) (4% body weight performance, food growth performance


**Primary particles**

Size: 20 nm

Size: 30 nm

Size: 30 nm

Size: 15– 350 nm

Size: 50– 60 nm

To study Zn accumulation and the mechanism of

hepatic

ions

detoxification in comparison with bulk ZnO and Zn2+

To compare the effects of zinc compounds in the form of nano-, microparticles and

To compare the effect of different

To study stress proteomic responses

To evaluate bioaccumulation and subacute toxicity compared with bulk particles

**Study aim Test organisms Exposure**

88 Toxicology - New Aspects to This Scientific Conundrum

*Oryzias melastigma* (Medaka fish)

*Cyprinus carpio* (Juvenile carp)

*Carassius auratus* (Gold fish)

*Brachydanio* (Danio) *rerio* (zebrafish)

*Ctenopharyngodon idella* (Grass carp) **protocol**

Waterborne exposure to 4 and 40 mg L−1 for 96 h

Waterborne exposure (5–1000

Waterborne exposure for 30 days to 2 mg L−1

Waterborne exposure to 0.2, 2, 10 and 20 mg L −1 for 120 h

Diet exposure (4% body weight

) for 30

mg L−

days

**Assessed endpoints**

Molecular biomarkers (SOD, MT and HSP70)

+ /K+

SOD)

Accumulation in different tissues (gill, liver, intestine, muscle, and brain); histopathological changes; enzyme activities (e.g., Na

ATPase, and

nonenzymatic antioxidants and oxidative damage

Zn concentration and its subcellular distribution gills, liver, gut, and muscle

Embryo/larvae survival, hatching, and malformation

Lethality, growth performance, food

rates

**Main effects References**

[54]

[55]

[56]

[57]

[58]

lipid peroxidation

Upregulation of

bioaccumulation, oxidative effect, and histopathological changes than bulk

Tissue-specific bioaccumulation dependent on the exposed material

Retardation of hatching and deviations in embryonic development; adherence of the particles on the egg surface at high ZnO-NPs concentrations

Improvement in growth performance

products

HSP70

Higher

ZnO


Abbreviations: HSP70: heat shock protein 70; MT: metallothionein; NPs: nanoparticles; ROS: reactive oxygen species; SOD: superoxide dismutase.

**Table 1.** Examples of research studies on the effects of ZnO-NPs on fish.

As shown in **Table 1**, examples of recent research on ZnO-NPs interaction with fish have essentially focused on freshwater organisms such as *Cyprinus carpio, Oreochromis mossambicus, Tilapia zillii, Oreochromis niloticus, Ctenopharyngodon idella, Carassius auratus*, and *Danio rerio*. Several techniques and approaches were used in those studies including, mainly, analytical methods for detection of metal in tissues as well histopathological, hematological, and oxidative stress endpoints to study the effects of ZnO-NPs, and histopathological, hematological, and oxidative stress as relevant endpoints to evaluate the possible interrelationships.

The effects of ZnO-NPs have been associated with two main mechanisms: oxidative stress and nanoparticle-protein interactions [52]. Although some of the effects have been associated with the release of Zn ions as a result of particle dissolution, not all studies confirm that the toxic effects are due to dissolution. Higher bioaccumulation and effects have been observed after exposure to nanoparticle form when compared to bulk form, confirming the complexity and specificity of mechanisms associated with experimental conditions. Nonetheless, it is clear that more studies are needed, with lower concentrations and longer exposure periods, representing a more environmentally relevant scenario. There is also a clear need for studies on combined effects of abiotic factors variation (e.g., temperature, salinity, UV radiation), classical environmental contaminants (e.g., organic compounds, pesticide and pharmaceuticals), and ZnO-NPs, to represent an environmentally relevant scenario. There is also a need to assess the effects of ZnO-NPs in organisms present in high ionic strength environment (e.g., estuaries and marine environments). The information obtained in trophic transfer studies supports the concerns of potential effects of nanoparticles, to higher trophic levels in which humans may also be a target.

Considering the tested endpoints, the available data revealed that histological and hematological responses occur. After ZnO-NPs exposure, both juvenile and/or adult fish have shown its accumulation on tissues such as brain, liver, muscle, and gills [55, 56]. Hao et al. [55] reported ZnO-NPs accumulation on tissues of juvenile carp (*Cyprinus carpio*), and cellular oxidative stress response was denoted as the main toxic mechanism of nano-ZnO. The bioaccumulative behavior of ZnO-NPs and their potential trophic transfer from *Daphnia magna* to zebrafish (*Danio rerio*) was reported by Skjolding et al. [62].

Several parameters such as hematological approaches have been used for the monitoring of health conditions of fish [58, 59]. Blood parameters as red and white blood cells count, hemoglobin content, and hematocrit value, and red blood cell indices are usually assessed on some toxicological studies. Although enhancement on red blood cell count was reported on the grass carp *Ctenopharyngodon idella* with supplementation of NPs from [58], deleterious changes on blood parameters were documented on Tilapia *Oreochromis mossambicus* exposed for 96 h to ZnO-NPs (100–110 mg L−1) [59].

Additionally, histology is also an important tool for the evaluation of fish health, showing the initial signs of lesions not easily noticeable during the macroscopic observation of tissues and organs [55]. Gills are vital organs for respiration and osmoregulation. Gill histopathological alterations can be considered as indicators of the ZnO-NPs–induced toxicity in the common carp [60]. Among other relevant organs, hepatic histopathological changes were documented as a result of exposure to ZnO-NPs [55, 61, 63].

As reported in **Table 1**, the ecotoxicological impacts of ZnO-NPs on fish include in most studies histopathological, hematological, and oxidative stress under different doses, protocols, and exposure [53, 55]. Tomilina et al. [57] reported decreased motility and increased curvature of tail in *Brachydanio* (*Danio*) *rerio* embryos exposed for 24 h to 0.01 mg L−1 ZnO-NPs and affected dynamics of hatching of *Brachydanio* (*Danio*) *rerio* prelarvae at higher concentrations.

In contrast, scarce data have demonstrated positive biological effects of ZnO-NPs. Reports on fish (*C. idella*) growth performance improvement after 90 days ZnO-NPs exposure, through diet, compared to oxide and sulfate form of Zn were recently published [58], suggesting a potential application of these particles on aquaculture.

## **4. Potential impacts on public health**

**Primary particles**

Size: 50 nm

To determine lethal concentration and histopathological

90 Toxicology - New Aspects to This Scientific Conundrum

lesions

SOD: superoxide dismutase.

also be a target.

**Study aim Test organisms Exposure**

*Cyprinus carpio* (Carp)

**Table 1.** Examples of research studies on the effects of ZnO-NPs on fish.

**protocol**

Waterborne exposure for 24 h to 10–50 mg L−1

Abbreviations: HSP70: heat shock protein 70; MT: metallothionein; NPs: nanoparticles; ROS: reactive oxygen species;

As shown in **Table 1**, examples of recent research on ZnO-NPs interaction with fish have essentially focused on freshwater organisms such as *Cyprinus carpio, Oreochromis mossambicus, Tilapia zillii, Oreochromis niloticus, Ctenopharyngodon idella, Carassius auratus*, and *Danio rerio*. Several techniques and approaches were used in those studies including, mainly, analytical methods for detection of metal in tissues as well histopathological, hematological, and oxidative stress endpoints to study the effects of ZnO-NPs, and histopathological, hematological, and oxidative stress as relevant endpoints to evaluate the possible interrelationships.

The effects of ZnO-NPs have been associated with two main mechanisms: oxidative stress and nanoparticle-protein interactions [52]. Although some of the effects have been associated with the release of Zn ions as a result of particle dissolution, not all studies confirm that the toxic effects are due to dissolution. Higher bioaccumulation and effects have been observed after exposure to nanoparticle form when compared to bulk form, confirming the complexity and specificity of mechanisms associated with experimental conditions. Nonetheless, it is clear that more studies are needed, with lower concentrations and longer exposure periods, representing a more environmentally relevant scenario. There is also a clear need for studies on combined effects of abiotic factors variation (e.g., temperature, salinity, UV radiation), classical environmental contaminants (e.g., organic compounds, pesticide and pharmaceuticals), and ZnO-NPs, to represent an environmentally relevant scenario. There is also a need to assess the effects of ZnO-NPs in organisms present in high ionic strength environment (e.g., estuaries and marine environments). The information obtained in trophic transfer studies supports the concerns of potential effects of nanoparticles, to higher trophic levels in which humans may

Considering the tested endpoints, the available data revealed that histological and hematological responses occur. After ZnO-NPs exposure, both juvenile and/or adult fish have shown its accumulation on tissues such as brain, liver, muscle, and gills [55, 56]. Hao et al. [55] reported ZnO-NPs accumulation on tissues of juvenile carp (*Cyprinus carpio*), and cellular oxidative stress response was denoted as the main toxic mechanism of nano-ZnO. The bioaccumulative

**Assessed endpoints**

Lethal

concentrations and histopathological alterations in gills, liver, kidney, and pancreas

**Main effects References**

[63]

Histopathological lesions in the kidney and gills; necrosis in liver, and hemorrhage

in pancreas

The hazard potential of ZnO-NPs to humans in comparison with microparticulate and dissolved Zn has been evaluated in the context of major accident prevention [64]. Based on the analysis of endpoints of subtoxic events (inflammation, oxidative stress response, or gene expression profiling) over different timescales, the authors concluded that the hazard potentials of nano- and microparticles of ZnO are identical during acute (medium) and chronic (low) toxicity. Inhalation of ZnO fume and dust over the permissible exposure limit of 5 mg m−3 appears to be the riskiest toxic exposure, since Zn fume fever could be lethal [65]. ZnO is quite soluble in acids and alkalies, thereby the toxicity of ZnO-NPs was also compared to that of zinc(II) ions. When considering the concentration of dissolved Zn, no significant differences between exposure to ZnO and ZnO-NPs have been found in EC50 and LC50 values, using *Daphnia* and fish, respectively, as testing organisms. Clinical reports on human intoxication by ZnO-NPs are hardly any in the literature. Conversely, information regarding the toxicity of Zn ions for humans has been gathered during the last 60 years, since the first clinical reports on Zn fume fever [66].

Zn is an essential trace element and micronutrient for humans. Under physiological conditions, it exists as a redox neutral divalent cation that is reactive as a Lewis acid. The continuous external supply of this metal ion is vital for many metabolic pathways, given that there is no body storage depot for zinc and its exchange between tissues is limited. The man body Zn content is approximately 2 g, and the recommended daily intake is 8–11 mg (tolerable upper intake level, 40 mg day−1). Bones and skeletal muscles contain more than half of the total body Zn, while the highest concentrations (>1 mg g−1 dry weight) are achieved in prostate gland [67]. Around 0.1% of the total body zinc is replenished daily through diet, and it is equivalent to the percentage that is hold in blood serum (90 μg dL−1) [68].

Zinc homeostasis is tightly controlled at the whole body down to the subcellular level. In cells, half of the Zn content is in the cytoplasm, while nucleus and plasma membrane accounts for, respectively, 35 and 10% of the total cellular Zn [69]. Since this metal ion is mainly bound to proteins (i.e., metallothioneins) and sequestered in organelles (i.e., mitochondria, endoplasmic reticulum, Golgi apparatus, secretory granules, and other vesicular compartments), the cytosolic free Zn concentration is in the picomolar/nanomolar range. In the cytosol, Zn concentrations fluctuate in wave and spark manners, of which regulatory mechanism still not completely understood. Zinc transporters (ZnTs) are of outstanding importance for the cellular and subcellular zinc homeostasis (**Table 2**), since ions cannot be synthesized or broken down by cells. ZnT transporters (ZnT1-ZnT10) belong to the Solute Carrier Family 30A (SLC30A). This protein "Family" also comprises another group of proteins that translocate Zn across membranous barriers, the ZIP transporters (ZIP1-ZIP14). However, ZIP transporters are involved not only in Zn transport but also in the homeostasis of cadmium, manganese, iron, and calcium [70].



Zn is an essential trace element and micronutrient for humans. Under physiological conditions, it exists as a redox neutral divalent cation that is reactive as a Lewis acid. The continuous external supply of this metal ion is vital for many metabolic pathways, given that there is no body storage depot for zinc and its exchange between tissues is limited. The man body Zn content is approximately 2 g, and the recommended daily intake is 8–11 mg (tolerable upper intake level, 40 mg day−1). Bones and skeletal muscles contain more than half of the total body Zn, while the highest concentrations (>1 mg g−1 dry weight) are achieved in prostate gland [67]. Around 0.1% of the total body zinc is replenished daily through diet, and it is equivalent to

Zinc homeostasis is tightly controlled at the whole body down to the subcellular level. In cells, half of the Zn content is in the cytoplasm, while nucleus and plasma membrane accounts for, respectively, 35 and 10% of the total cellular Zn [69]. Since this metal ion is mainly bound to proteins (i.e., metallothioneins) and sequestered in organelles (i.e., mitochondria, endoplasmic reticulum, Golgi apparatus, secretory granules, and other vesicular compartments), the cytosolic free Zn concentration is in the picomolar/nanomolar range. In the cytosol, Zn concentrations fluctuate in wave and spark manners, of which regulatory mechanism still not completely understood. Zinc transporters (ZnTs) are of outstanding importance for the cellular and subcellular zinc homeostasis (**Table 2**), since ions cannot be synthesized or broken down by cells. ZnT transporters (ZnT1-ZnT10) belong to the Solute Carrier Family 30A (SLC30A). This protein "Family" also comprises another group of proteins that translocate Zn across membranous barriers, the ZIP transporters (ZIP1-ZIP14). However, ZIP transporters are involved not only in Zn transport but also in the homeostasis of cadmium, manganese, iron,

**Name Main functions at subcellular level Entries: Protein/Gen\***

Zn2+ transmembrane transport (accumulation in endosomes, lysosomes, and secretory vesicles in mammary epithelial cells). Regulation of

endosomes, and lysosomes). Regulation of sequestering of and response to

in endosomes, lysosomes, secretory vesicles, and trans-Golgi network)

Q9Y6M5

SLC30A2

SLC30A3

SLC30A4

Q8TAD4

(ZNT5\_HUMAN)/ SLC30A5

(ZNT1\_HUMAN)/ SLC30A1

Q9BRI3 (ZNT2\_HUMAN)/

Q99726 (ZNT3\_HUMAN)/

O14863 (ZNT4\_HUMAN)/

**ZnT1** Zn2+ efflux through plasma membrane. Negative regulation of Zn2+ and Ca2+ transmembrane import and neurotransmitter secretion

sequestering of and response to Zn2+

**ZnT3** Zn2+ transporting ATPase (accumulation in synaptic vesicles, late

Zn2+ transmembrane transport into lumens of the Golgi apparatus and early compartments of the secretory pathway such as COPII-coated vesicles (putative transporter of Zn2+ into β cells in order to form insulin crystals). Required with

**ZnT4** Zn2+ transmembrane transport (transport out of the cytosol—accumulation

the percentage that is hold in blood serum (90 μg dL−1) [68].

92 Toxicology - New Aspects to This Scientific Conundrum

and calcium [70].

Zn2+

**Transporters**

**ZnT2 (2 isoforms)**

**ZnT5 (4 isoforms)**




**Transporters**

**ZIP8 (3 isoforms)**

**ZIP9 (3 isoforms)**

**ZIP10 (2 isoforms)**

**ZIP11 (3 isoforms)**

**ZIP12 (5 isoforms)**

**ZIP13 (2 isoforms)**

**ZIP14 (3 isoforms)**

**Family; subfamily**

**ZNF593/ BUD20 C2H2**

**Zn-finger proteins**

Oct-2

process

94 Toxicology - New Aspects to This Scientific Conundrum

cytosol)

**Name Main functions at subcellular level Entries: Protein/Gen\***

Q9C0K1

SLC39A8

Q9NUM3 (S39A9\_HUMAN)/

SLC39A9

Q9ULF5

Q9NUM3 (S39A9\_HUMAN)/

SLC39A9

Q504Y0

SLC39A14

(S39A8\_HUMAN)/

(S39AA\_HUMAN)/ SLC39A10

(S39AC\_HUMAN)/ SLC39A12

(S39AD\_HUMAN)/ SLC39A13

Q15043 (S39AE\_HUMAN)/

**entry\***

O00488

Q9NRE2

P0CG40

Q9BWW7

Q9UJQ4

Zn2+ transport through the plasma membrane and endosomal and

Zn2+ transport through the plasma membrane and trans-Golgi network

Zn2+ transport through the plasma membrane (influx to cytosol). Positive regulation of B cell proliferation, B cell receptor signaling pathway, and protein tyrosine phosphatase. Negative regulation of B cell apoptotic

Zn2+ transport through the trans-Golgi network membrane (release to

Zn2+ transport at the plasma membrane, nucleus, and Golgi apparatus (influx and release to cytosol). Regulation of microtubule polymerization,

Zn2+ transport through the plasma membrane (influx to cytosol). Broadscope metal ion transporter with a preference for Zn2+ uptake (cellular

**Teashirt C2H2** Teashirt homolog 2 is a putative transcriptional regulator in developmental processes,

**Sp1 C2H2** Transcription factor Sp9 positively regulates FGF8 expression in the apical ectodermal

**Snail C2H2** Transcriptional repressor scratch 1 binds E-box motif CAGGTG and modulates the basic helix-loop-helix transcription factors during neuronal differentiation

**Sal C2H2** Sal-like protein 4 is an important transcription factor in the maintenance and self-renewal

ridge and contributes to limb outgrowth in embryos

of embryonic and hematopoietic stem cells

Zn2+ transmembrane transport in the Golgi apparatus (release to cytosol) Q96H72

**Representative protein and its function Protein**

Zinc finger protein 593 negatively modulates the transcriptional regulatory activity of

neuron projection development, and signal transduction

uptake of nontransferrin-bound Fe)

acting as transcriptional repressor

lysosomal membranes (influx and release to cytosol)

membrane (influx and release to cytosol)




**Enzymes**

**Zn-finger proteins**

transcriptional activity

96 Toxicology - New Aspects to This Scientific Conundrum

PRC2 complex activity

**Recommended name EC**

Peptide-methionine (R)-S-oxide

**Representative protein and its function Protein**

coregulators NAB1 and NAB2, whose sumoylation downregulates EGR2 own

spermatogenesis and primary cilia formation through Hedgehog signaling pathway

Zinc finger E-box-binding homeobox 1 is a transcriptional repressor that positively

remodeling, interchromosomal association, regulation of epigenetic modifications, oocyte and preimplantation embryo development. It is also a putative tumor suppressor

Zinc finger protein AEBP2 is a DNA-binding transcriptional repressor that stimulates

**Comments**

Formaldehyde dismutase 1.2.98.1 Contains a tightly but noncovalently bound NADP(H) cofactor,

Superoxide dismutase 1.15.1.1 Degradation of reactive oxygen species and superoxide radicals

Protein geranylgeranyltransferase type I 2.5.1.59 Zn metalloenzyme. Zn2+ is required for peptide, but not for

Riboflavin kinase 2.7.1.26 Mg2+ is preferentially required for activity. Essential in

muscle cells

Histone acetyltransferase 2.3.1.48 Different specificities toward histone acceptors

RING-type E3 ubiquitin transferase 2.3.2.27 Degradation of misfolded protein

Tyrosine transaminase 2.6.1.5 Involved in multiple metabolic pathways

as well as Zn2+ and Mg2+

peptides that had been damaged

1.8.4.12 Selenoprotein. Prevention of oxidative stress damage caused by reactive oxygen species by reducing the oxidized form of methionine back to methionine and thereby reactivating

> isoprenoid, substrate binding. Inhibition induces simultaneous p53-dependent apoptosis and autophagy in airway smooth

> recruiting Nox1 to death receptor4/5, critical role in the KD548-

**DZIP C2H2** Zinc finger protein DZIP1 interaction with DAZ supports the participation in

regulates neuronal differentiation and promotes tumorigenicity

**number\*\***

D-Lactate dehydrogenase (acceptor) 1.1.99.6 Alanine metabolism

**CTCF** Transcriptional repressor CTCF plays important roles in gene silencing, chromatin

**entry\***

Q86YF9

P37275

P49711

Q6ZN18

**Family; subfamily**

**Delta-EF1/ ZFH-1 C2H2**

**AEBP2/jing C2H2**

**Enzymes**

reductase

\*UniProtKB/Swiss-Prot—European Bioinformatics Institute.

\*\*Enzyme Database—BRENDA and IUBMB Enzyme Nomenclature.

**Table 2.** Illustrative examples of human Zn transporters, Zn-finger proteins, and enzymes that require Zn.

Zn ions are important for the regulation of central biochemical processes (gene transcription and the metabolism of lipids, proteins, and nucleic acids), which impacts a variety of physiological functions (e.g., neuronal, endocrine, skeletal, reproductive, immune, and healing). It is estimated that the 10% of the proteins (around 3000 proteins) encoded in the human genome are zinc proteins [71]. According to the last release of the UniProtKB/Swiss-Prot database, more than one hundred of human proteins are zinc finger (ZnF) macromolecules (**Table 2**). Apart from the structural role of Zn in ZNFs [72], which contributes to shape the zinc-binding repeats as molecular scaffolds for tight binding of their target molecules (DNA, RNA, other proteins, or lipids), this metal ion is also essential for enzyme catalysis and cell signaling (**Table 2**) [73, 74]. In enzyme catalytic centers, Zn often promotes substrate activation by stabilizing negative charges due to strong Lewis acid properties. The metal ion acts as endocrine, paracrine, autocrine, and intracrine mediator. In cells, the distribution of Zn is modified by the stimulation that triggers its release in the central nervous and neuroendocrine systems. Zn is also a ubiquitous cytosolic second messenger, leading to fast alteration of signaling enzyme activities (i.e., phosphodiesterases, mitogen-activated protein kinase, protein kinase C, protein tyrosine phosphatases, calcineurin, caspases) and afterward to the biosynthesis of proteins that control its cytosolic concentrations. Interestingly, Zn accumulation in specific subcellular compartments appears to occur during both physiological and pathological conditions. For instance, the Zn spark that follows the calcium wave during fertilization is thought to be crucial for further cell cycle resumption in eggs [75]. Accumulation of Zn in lysosomes is a common observation during neurodegenerative processes and intoxication [76].

In humans, symptoms of Zn deficiency include: severe anemia, persistent diarrhea, immune insufficiency ensuing recurrent inflammations and impairment of wound healing, growth retardation, hypogonadism, skin and eyes abnormalities, baldness mental lethargy, brain dysfunctions, and behavioral changes. Zn deficiency still continues a global public health concern, particularly in developing countries where it causes mortality among young children [77]. Conversely, minor Zn deficiency among elderly population and individuals who undergo gastric bypass surgery for obesity seems to be increasing in industrialized countries. Thereby, development of high efficient Zn-enriched nutritional supplements could be advantageous in decreasing the incidence of degenerative and immunodeficiency disorders, infections, and persistent diarrhea. Current Zn formulations better absorbed through supplements are in the form of picolinate or chelates of amino acids. Duodenum is the principal site for Zn absorption. Therefore, intestinal pathologies that cause poor micromineral absorption, such as Crohn's disease, can also induce Zn malabsorption. Other metal ions, such as Ca2+, Fe2+, and mostly Cu2+ compete with Zn2+ during translocation from the apical surface of the villae to the basolateral surface of enterocytes. In matter of fact, Zn intake close to the recommended dietary allowance (15 mg/day) may cause copper and iron deficiency and adversely affect HDL cholesterol concentrations. Overt symptoms of Zn poisoning (i.e., nausea, vomiting, epigastric pain, lethargy, and fatigue) usually occur only after exposure to extremely high Zn levels [78].

Zn-related diseases might be prompted either when its scarcity and overload go beyond the limited cellular Zn buffering capacity, which seems to be rather sensitive to environmental factors. Apparently, healthy individuals tolerate up to 10-fold changes of the recommended daily intake of Zn [79]. Whether exposure to Zn concentrations outside the physiological range promotes salutary or injurious effects depends on Zn concentration. Different ranges of Zn concentration appear to be required for exhibiting beneficial properties as antioxidant, antiinflammatory, and antiapoptotic agent [80]. Intriguingly, both Zn excess and scarcity undermine the loosely equilibrium toward prooxidant, proinflammatory and proapoptotic actions.

Zn ions are important for the regulation of central biochemical processes (gene transcription and the metabolism of lipids, proteins, and nucleic acids), which impacts a variety of physiological functions (e.g., neuronal, endocrine, skeletal, reproductive, immune, and healing). It is estimated that the 10% of the proteins (around 3000 proteins) encoded in the human genome are zinc proteins [71]. According to the last release of the UniProtKB/Swiss-Prot database, more than one hundred of human proteins are zinc finger (ZnF) macromolecules (**Table 2**). Apart from the structural role of Zn in ZNFs [72], which contributes to shape the zinc-binding repeats as molecular scaffolds for tight binding of their target molecules (DNA, RNA, other proteins, or lipids), this metal ion is also essential for enzyme catalysis and cell signaling (**Table 2**) [73, 74]. In enzyme catalytic centers, Zn often promotes substrate activation by stabilizing negative charges due to strong Lewis acid properties. The metal ion acts as endocrine, paracrine, autocrine, and intracrine mediator. In cells, the distribution of Zn is modified by the stimulation that triggers its release in the central nervous and neuroendocrine systems. Zn is also a ubiquitous cytosolic second messenger, leading to fast alteration of signaling enzyme activities (i.e., phosphodiesterases, mitogen-activated protein kinase, protein kinase C, protein tyrosine phosphatases, calcineurin, caspases) and afterward to the biosynthesis of proteins that control its cytosolic concentrations. Interestingly, Zn accumulation in specific subcellular compartments appears to occur during both physiological and pathological conditions. For instance, the Zn spark that follows the calcium wave during fertilization is thought to be crucial for further cell cycle resumption in eggs [75]. Accumulation of Zn in lysosomes is a common

98 Toxicology - New Aspects to This Scientific Conundrum

observation during neurodegenerative processes and intoxication [76].

In humans, symptoms of Zn deficiency include: severe anemia, persistent diarrhea, immune insufficiency ensuing recurrent inflammations and impairment of wound healing, growth retardation, hypogonadism, skin and eyes abnormalities, baldness mental lethargy, brain dysfunctions, and behavioral changes. Zn deficiency still continues a global public health concern, particularly in developing countries where it causes mortality among young children [77]. Conversely, minor Zn deficiency among elderly population and individuals who undergo gastric bypass surgery for obesity seems to be increasing in industrialized countries. Thereby, development of high efficient Zn-enriched nutritional supplements could be advantageous in decreasing the incidence of degenerative and immunodeficiency disorders, infections, and persistent diarrhea. Current Zn formulations better absorbed through supplements are in the form of picolinate or chelates of amino acids. Duodenum is the principal site for Zn absorption. Therefore, intestinal pathologies that cause poor micromineral absorption, such as Crohn's disease, can also induce Zn malabsorption. Other metal ions, such as Ca2+, Fe2+, and mostly Cu2+ compete with Zn2+ during translocation from the apical surface of the villae to the basolateral surface of enterocytes. In matter of fact, Zn intake close to the recommended dietary allowance (15 mg/day) may cause copper and iron deficiency and adversely affect HDL cholesterol concentrations. Overt symptoms of Zn poisoning (i.e., nausea, vomiting, epigastric pain, lethargy, and fatigue) usually occur only after exposure to extremely high Zn levels [78].

Zn-related diseases might be prompted either when its scarcity and overload go beyond the limited cellular Zn buffering capacity, which seems to be rather sensitive to environmental factors. Apparently, healthy individuals tolerate up to 10-fold changes of the recommended

Lung toxicity after inhalation of ZnO-NPs is likely the most documented deleterious effect in the literature. The severity of ZnO-NPs-induced inflammatory condition is significantly correlated with the mass and surface area of the nanoparticles, suggesting that the toxic effect of ZnO-NPs is mainly caused by the release of Zn ions [81]. It is also claimed that intact ZnO-NPs have a unique way of inducing inflammatory effects compared with dissolved Zn ions [82]. For example, ZnO-NPs may either stimulate the production of IFN-γ and subsequent macrophage activation, neutrophilic infiltration, and fibrosis (Th1 inflammatory response) or cause a mixed inflammatory cell immune response by triggering a Th2 response [83]. Inhaled ZnO-NPs, through the olfactory bulb–brain translocation pathway, could also induce neurotoxic effects by activation of astrocytes and microglia, which causes neuroinflammation [84, 85].

Recently, a battery of tests, including hemolytic and oxidative stress markers, *in vitro* ROS generation and the comet assay, has been applied to evaluate cytotoxic and genotoxic effects of ZnO-NPs on human erythrocytes and lymphocytes [86]. The authors concluded that, similarly to dissolved Zn, ZnO-NPs concentrations above 50 ppm. are cytotoxic and genotoxic, due to the enhancement of oxidative stress induced by ROS generation. In addition to disruption of cellular Zn homeostasis, alteration of multiple enzymatic activities, and interaction with biomolecules, exacerbation of oxidative stress is the most recognized mechanism through which ZnO-NPs induce toxic effects. Accordingly, it has been demonstrated that the cytotoxic effect of ZnO-NPs is more pronounced in human cells previously exposed transiently to sublethal doses of H2O2, a standard oxidative stress-inducing agent [87]. One important scenario of the consequences of human exposure to ZnO-NPs was anticipated by the authors: individuals who suffer from diseases associated with increased oxidative stress (i.e., asthma, atherosclerosis, cardiovascular diseases, chronic obstructive pulmonary disease, and neurodegenerative diseases) should be considered at additional risk upon exposure to ZnO-NPs.

The pharmaceutical industry makes use of Zn(II) compounds (ZnCl2, ZnO, zinc pyrithione, Zn(CH3CO2)2) as active substances, lubricants, and emollients for a long time [88]. Usually, zinc-containing medicines are intended to topic application as wound healings, anti-infectious, disinfectants, and lubricants (**Table 3**). Apart from ZnO-NPs use in pharmaceutical formulations (i.e., drug delivery) and medicine (i.e., bioimaging), they are also present in a large number of consumer products (protective sunscreens, hair care formulations, cosmetics, supplements, food additives, etc.), as already mentioned above (Section 1). Recently, the European Council on Cosmetic Products (The European Commission (2016), Commission Regulation (EU) 2016/621 of 21 April 2016) restricted the use of ZnO-NPs in spray products because it could lead to exposure of the consumer's lungs to ZnO-NPs by inhalation, and encouraged only oral and dermal use of ZnO-NPs, up to a maximum concentration in ready for use preparation of 25%, with the following characteristics: (i) purity ≥96%, with *wurtzite* crystalline structure and physical appearance as clusters that are rod-like, star-like, and/or isometric shapes, with impurities consisting only of carbon dioxide and water, while any other impurities are less than 1% in total, (ii) median diameter of the particle number size distribution D50 (50% of the number below this diameter) >30 nm and D1 (1% below this size) >20 nm; (iii) water solubility <50 mg L−1; and (iv) uncoated or coated with triethoxycaprylylsilane, dimethicone, dimethoxy diphenylsilane triethoxycaprylylsilane crosspolymer, or octyl triethoxy silane.

Up to date information highlights not only the genotoxicity and cytotoxicity of ZnO-NPs due dissolution, ROS generation, immunomodulatory, and apoptotic responses, but also their selective cytotoxicity [82, 89–93]. In general, cytotoxicity is thought to be a collateral effect to avoid. To evaluate the specific risks and benefits of human exposure to ZnO-NPs, its size, shape, degradability, agglomeration/aggregation propensity, adsorption ability, specific surface area, and interfacial chemical and physical reactivity should be considered intrinsic properties, which likely influence their biopersistence, cellular interactions, and bioactivities when compared with microparticulate and dissolved Zn during intentional and unintentional human exposures. For instance, ZnO-NPs sized 20–25 nm appear to exhibit higher antibacterial and antifungal activity than other ZnO forms [15]. Sublethal concentrations of ZnO-NPs (surface area of 10.7 ± 0.7 nm) reduce the mitochondrial membrane potential, leading to a dosedependent increase in gluconeogenesis and glycogenolysis, which could not be only attributed to dissolution of ZnO-NPs in extracellular fluids [94]. Kao et al. [85] proposed that ZnO-NPs are mainly internalized by endocytosis and dissolved in endosomes, raising the cytosolic free Zn2+ concentration, which is further sequestrated by mitochondria leading to cell apoptosis, due to mitochondrial dysfunction and caspases activation. ZnO-NPs readily dissolve in artificial lysosomal fluid (pH 4.5), but form aggregates and precipitates in the slight alkaline interstitial fluid [95].



**Table 3.** Commonly used medications that contain Zn for topical application.

for use preparation of 25%, with the following characteristics: (i) purity ≥96%, with *wurtzite* crystalline structure and physical appearance as clusters that are rod-like, star-like, and/or isometric shapes, with impurities consisting only of carbon dioxide and water, while any other impurities are less than 1% in total, (ii) median diameter of the particle number size distribution D50 (50% of the number below this diameter) >30 nm and D1 (1% below this size) >20 nm; (iii) water solubility <50 mg L−1; and (iv) uncoated or coated with triethoxycaprylylsilane, dimethicone, dimethoxy diphenylsilane triethoxycaprylylsilane crosspolymer, or octyl triethoxy

Up to date information highlights not only the genotoxicity and cytotoxicity of ZnO-NPs due dissolution, ROS generation, immunomodulatory, and apoptotic responses, but also their selective cytotoxicity [82, 89–93]. In general, cytotoxicity is thought to be a collateral effect to avoid. To evaluate the specific risks and benefits of human exposure to ZnO-NPs, its size, shape, degradability, agglomeration/aggregation propensity, adsorption ability, specific surface area, and interfacial chemical and physical reactivity should be considered intrinsic properties, which likely influence their biopersistence, cellular interactions, and bioactivities when compared with microparticulate and dissolved Zn during intentional and unintentional human exposures. For instance, ZnO-NPs sized 20–25 nm appear to exhibit higher antibacterial and antifungal activity than other ZnO forms [15]. Sublethal concentrations of ZnO-NPs (surface area of 10.7 ± 0.7 nm) reduce the mitochondrial membrane potential, leading to a dosedependent increase in gluconeogenesis and glycogenolysis, which could not be only attributed to dissolution of ZnO-NPs in extracellular fluids [94]. Kao et al. [85] proposed that ZnO-NPs are mainly internalized by endocytosis and dissolved in endosomes, raising the cytosolic free Zn2+ concentration, which is further sequestrated by mitochondria leading to cell apoptosis, due to mitochondrial dysfunction and caspases activation. ZnO-NPs readily dissolve in artificial lysosomal fluid (pH 4.5), but form aggregates and precipitates in the slight alkaline

**Role in formulation Target organ Clinical recommendations**

Mouth/ oropharynx

Anus/lower rectum

Skin Treatment of inflammatory acne,

Skin Treatment of diaper dermatitis

Skin Symptomatic treatment of dry and scaly lesions, especially of ichthyosis, psoriasis, and eczema Disinfection and

characterized by bacterial involvement

Treatment of gingivitis and stomatitis; relief of toothache; oral hygiene

Symptomatic treatment of hemorrhoids

silane.

100 Toxicology - New Aspects to This Scientific Conundrum

interstitial fluid [95].

**Route of administration**

Zinc acetate Topic Active ingredient:

Zinc chloride Topic Active ingredient:

Zinc oxide Topic Active ingredient: adjuvant

antibacterial action

healing actions

of healing

Topic Emollient/lubricant:

Topic Emollient/lubricant:

antibacterial, analgesic, and

antihemorrhoidal action

soothing, smoothing, and moisturizing actions

**Zinc compound**

In contract with small molecules that are translocated across the plasma membrane by passive diffusion or active transport, nanosized particles are internalized by cells mainly through endocytosis. This feature of NPs offers a myriad of opportunities for specific cellular targeting, controlled drug delivery, and bioimaging. The endocytotic capability varies significantly among cellular populations. Phagocytes (i.e., macrophages, monocytes, neutrophils, dendritic, and mast cells) are chemotaxing cells that move toward site of infections causing inflammation, play a central role in innate immunity response, and stimulate lymphocytes to produce antibodies (adaptive immunity) by antigen presentation. ZnO-NPs coating that favors interaction with opsonin, scavenger, or Toll-like receptors should enhance selective internalization by phagocytes. Uncoated ZnO-NPs should also be engulfed by lymphocytes, erythrocytes, fibroblasts, epithelial, and endothelial cells. Given that ZnO-NPs are microscopic particle with at least one dimension less than 100 nm, they can be internalized by any living cell by micro- and pinocytosis. The subcellular availability of ZnO-NPs should greatly depend on the specific endocytotic pathway (i.e., clathrin-dependent, caveolae-dependent, clathrin- and caveolae-independent, receptor-mediated) involved in the internalization process. For instance, ZnO-NPs loaded in caveolae vesicles may reach the endoplasmic reticulum and the nucleus, since caveosomes are pH neutral multivesicular bodies [96]. Conversely, internalization of ZnO-NPs through LDL receptor-mediated endocytosis should raise the cytosolic zinc ion significantly, due to dissolution of ZnO in the acidic lysosomal environment. Accordingly, it has been experimentally demonstrated by using ICP-MS and fluorescent-labeled ZnO dissolution occurs in endosomes, and that nondissolved ZnO-NPs enter caveolae in BEAS-2B cells (human bronchial epithelial cells) and enter lysosomes in RAW 264.7 cells (mouse leukemic monocyte macrophage cell) in which smaller particle remnants dissolve [90]. In the support of cell-specific behavior of stable aqueous solutions of monodispersed ZnO-NPs is the fact that ZnO-NPs doses exhibiting negligible cytotoxic effects to osteogenically differentiated mesenchymal stem cells were lethal to proliferating pluripotent mesenchymal stem cells [97]. As already mentioned above (Section 1), ZnO-NPs selectively induce apoptosis, mediated by reactive oxygen species via p53 pathway, in cancer cells (human hepatocellular carcinoma HepG2, human lung adenocarcinoma A549, and human bronchial epithelial BEAS-2B), but did not affect normal astrocytes and hepatocytes [98]. Thereby, to better explore therapeutic advantages and prevent unwanted cytotoxic effects and the potential of ZnNPs in terms of clinical diagnosis, it is important to perform a holistic analysis of the characteristics of ZnNPs, the administration route, the zinc body burden, the target cells, and the relevant physiological processes in each specific case.

## **5. Conclusions, next steps, and opportunities**

The ever increasing literature on ZnO-NPs clearly demonstrates the current value and applications of these particles and tremendous potential for future applications. There are a large number of challenges associated with its safe use, when compared with commonly tested substances. As for other nanoparticles, the advantages have to be carefully weight against potential pernicious effects. The currently available data clearly demonstrate the ability of ZnO-NPs to induce acute effects on fish, although at concentrations higher than those estimated to be present in the environment. Nonetheless, the long-term effects are yet to be explored. The considerable lack of information in terms of how these particles are released in the environment, at which levels, and in what form make the establishment of maximum allowed concentration a difficult task, based on available toxicity tests. More studies have to be conducted to explore the behavior of particles upon alterations of receiving media characteristics (e.g., ionic strength UV/radiation) and their fate. This information is essential for environmentally relevant ecotoxicological studies.

It is expected that, in the very near future, advances in analytical techniques allow quantification and accurate characterization of nanoparticles in environmental matrices which will allow the establishment of potentially impacted areas, monitoring of levels and effects on biota from those sites. Also, the need to the development of more effective wastewater treatments will potentially reduce the risk of the increased production of nanoparticle containing materials.

As can be seen from the literature, a broad range of applications of nanomaterials, in particular ZnO-NPs, exists on human activities. In this chapter, the benefits of ZnO nanomaterials are clearly recognized on a myriad of applications, having a great potential for the diagnosis, imaging, drug delivery, and treatment of several pathologies. Other areas within agricultural domain and energy resources have also relevant applications. Moreover, great potentials for their applications on aquaculture improving fish growth were documented.

The disposal and fate of ZnO-NPs into the environment may represent a risk to aquatic biota. This chapter highlights the significance in considering their fate and behavior into water bodies and its role on aquatic organisms, particularly fish. The published literature undoubtedly illustrates that ZnO-NPs have different toxic effects on microorganisms, rodents, human cells, and fish depending on their physicochemical features. In addition, the trophic transfer of these nanomaterials to humans through diet (i.e., by consuming contaminated fish) warrants special care. Therefore, disposal of ZnO-NPs deserves more attention since bioaccumulation of these elements may occur on aquatic species with impact on both human and environmental health. Precaution and more strict rules must be delimited for disposal of ZnO-NPs into the aquatic environments.

Zinc ion homeostasis is vital for humans and is closely linked with the homeostasis of other metal ions, particularly iron and copper. Nowadays, hypozincemia and hyperzincemia are two pathophysiological conditions of which enduring prevalence is also related to malnutrition during aging and emerging lifestyle diseases (i.e., obesity) in industrialized countries. While the risks of using ZnO-NPs are not fully understood, the advantages of its emerging applications, including in the therapeutic and diagnostic areas, are already widely recognized. Probably, the toxicity of ZnO-NPs for man is not superior to the zinc ion itself, and nanoparticulate forms appear to enable interaction with specific cell cycle states (i.e., proliferating cells) and selective interference with important physiological processes, allowing not only selection of the administration route of ZnO-NPs but also the cellular internalization pathway and further intracellular distribution.

The balance of the positive aspects of these nanomaterials and risks caused in some aquatic species, particularly on fish, targeting possible implications for human health deserve a continuous monitoring. Although safety measures have been assumed during industrial production, storage, and removal of these nanomaterials, a constant monitoring of possible risks for aquatic life and ultimately humans is needed.

As a general conclusion, it is expected that in the near future, there is an increase in the use of ZnO-NPs for various purposes. Comprehensive understanding of their toxic effect is needed for their prolonged use.

## **Acknowledgements**

the administration route, the zinc body burden, the target cells, and the relevant physiological

The ever increasing literature on ZnO-NPs clearly demonstrates the current value and applications of these particles and tremendous potential for future applications. There are a large number of challenges associated with its safe use, when compared with commonly tested substances. As for other nanoparticles, the advantages have to be carefully weight against potential pernicious effects. The currently available data clearly demonstrate the ability of ZnO-NPs to induce acute effects on fish, although at concentrations higher than those estimated to be present in the environment. Nonetheless, the long-term effects are yet to be explored. The considerable lack of information in terms of how these particles are released in the environment, at which levels, and in what form make the establishment of maximum allowed concentration a difficult task, based on available toxicity tests. More studies have to be conducted to explore the behavior of particles upon alterations of receiving media characteristics (e.g., ionic strength UV/radiation) and their fate. This information is essential for

It is expected that, in the very near future, advances in analytical techniques allow quantification and accurate characterization of nanoparticles in environmental matrices which will allow the establishment of potentially impacted areas, monitoring of levels and effects on biota from those sites. Also, the need to the development of more effective wastewater treatments will potentially reduce the risk of the increased production of nanoparticle containing materials. As can be seen from the literature, a broad range of applications of nanomaterials, in particular ZnO-NPs, exists on human activities. In this chapter, the benefits of ZnO nanomaterials are clearly recognized on a myriad of applications, having a great potential for the diagnosis, imaging, drug delivery, and treatment of several pathologies. Other areas within agricultural domain and energy resources have also relevant applications. Moreover, great potentials for

The disposal and fate of ZnO-NPs into the environment may represent a risk to aquatic biota. This chapter highlights the significance in considering their fate and behavior into water bodies and its role on aquatic organisms, particularly fish. The published literature undoubtedly illustrates that ZnO-NPs have different toxic effects on microorganisms, rodents, human cells, and fish depending on their physicochemical features. In addition, the trophic transfer of these nanomaterials to humans through diet (i.e., by consuming contaminated fish) warrants special care. Therefore, disposal of ZnO-NPs deserves more attention since bioaccumulation of these elements may occur on aquatic species with impact on both human and environmental health. Precaution and more strict rules must be delimited for disposal of ZnO-NPs into the aquatic

Zinc ion homeostasis is vital for humans and is closely linked with the homeostasis of other metal ions, particularly iron and copper. Nowadays, hypozincemia and hyperzincemia are two

their applications on aquaculture improving fish growth were documented.

processes in each specific case.

102 Toxicology - New Aspects to This Scientific Conundrum

**5. Conclusions, next steps, and opportunities**

environmentally relevant ecotoxicological studies.

environments.

This chapter was funded by project CICECO – Aveiro Institute of Materials, POCI-01-0145- FEDER-007679 (Foundation for Science and Technology—FCT Ref. UID/CTM/50011/2013), financed by national funds through the FCT/MEC and when appropriate cofinanced by FEDER under the PT2020 Partnership Agreement; also CICECO scholarships were given to A. Begam and P. Prasad, respectively, (BIC/UI89/7424/2016; BIC/UI89/7426/2016) added to the support of Mahatma Gandhi University, Kerala, India.

## **Author details**

Asfina Beegam1 , Parvathy Prasad1 , Jiya Jose1 , Miguel Oliveira2 , Fernando G. Costa3 , Amadeu M.V.M. Soares2 , Paula P. Gonçalves2 , Tito Trindade4 , Nandakumar Kalarikkal1 , Sabu Thomas1 and Maria de Lourdes Pereira5\*

\*Address all correspondence to: mlourdespereira@ua.pt

1 International and Inter University Centre for Nanoscience and Nanotechnology (IIUCNN), Mahatma Gandhi University, Kottayam, Kerala, India

2 Department of Biology, CESAM, University of Aveiro, Aveiro, Portugal

3 Department of Morphology and Function, CIISA Interdisciplinary Centre of Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal

4 Department of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal

5 Department of Biology, CICECO – Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal

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4 Department of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro,

5 Department of Biology, CICECO – Aveiro Institute of Materials, University of Aveiro,

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110 Toxicology - New Aspects to This Scientific Conundrum

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112.003038

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#### **Microplastics in Aquatic Environments and Their Toxicological Implications for Fish Microplastics in Aquatic Environments and Their Toxicological Implications for Fish**

Cristóbal Espinosa, M. Ángeles Esteban and Alberto Cuesta Cristóbal Espinosa, M. Ángeles Esteban and Alberto Cuesta

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

http://dx.doi.org/10.5772/64815

#### **Abstract**

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112 Toxicology - New Aspects to This Scientific Conundrum

10.1186/1743-8977-11-15

IJN.S29129

The intensive use of plastics and derivatives during the last century has increased the contamination of animal habitats. The breakdown of these primary plastics in the environment results in microplastics (MP), small fragments of plastic typically <1–5 mm in size. Apart from the potential negative effects of the MPs *per se*, it is generally assumed that microplastics may increase the exposure of marine aquatic organisms to chemicals associated with the plastics. In addition, to enhance the performance of plastics, additives are added during manufacture. Furthermore, they are active in absorbing other contaminants and be used as vectors of highly and well‐documented persistent contaminants. Finally, these small MPs are easily ingested by animals and affect their physiology and behaviour. Thus, aquatic living organisms are continuously exposed to these MPs, and associated contaminants, and could suffer from its contamination but also introduce them into the food chain.

**Keywords:** Microplastics, toxicants, aquatic environment, fish

## **1. Introduction**

The production of synthetic polymers has increased more than 100‐fold since the middle of the twentieth century to reach the 280 million tonnes of plastics produced annually worldwide most of which is destined for disposable use [1]. High production coupled with the physical characteristics of most plastics, such as their chemical inertness and very slow biodegradation rates, results in an accumulation of plastic debris in the environment [2]. Routes of discharge such as improper waste disposal, insufficient waste management and urban run‐offs [3] may

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

lead to significant amounts of these plastics entering the aquatic environment [4, 5]. It is a long‐ recognized fact that marine plastic debris contaminates the oceans and seas of all the world [3, 6, 7]. In the marine environment, plastics undergo a process of weathering and fragmen‐ tation that breaks down macrodebris into smaller micro‐ and nanodebris. This fragmentation of plastic is caused by a combination of mechanical forces, for example waves and/or photo‐ chemical processes triggered by sunlight. Some 'degradable' plastics are even designed to fragment quickly into small particles, although the resulting material does not necessarily biodegrade [8].

The terms 'microplastics' (MP) and 'microlitter' have been defined differently by various researchers. Gregory and Andrady [9] defined microlitter as the barely visible particles that pass through a 500‐µm sieve but are retained by a 67‐µm sieve (≈0.06–0.5 mm in diameter), while particles larger than this were called mesolitter. Others [10–12] defined the MPs as being in the size range <5 mm (recognizing 333 µm as a practical lower limit when neuston nets are used for sampling). Microplastic particles may further fragment into 'nanoplastics', a term that has not been defined uniformly in the literature, and may refer to <100‐µm particles of plastic [13].

Microplastics have been accumulating in the environment for nearly half a century and are found in oceans worldwide [3] including in the Antarctic [7]. Despite this worldwide dissem‐ ination of plastic fragments, the global load of plastics on the open ocean surface has been estimated to be far less than might be expected, but nevertheless increasing. Thus, the potential effects of microplastics on marine ecosystems are still far from being well understood [14]. It is believed that the virging MPs are not chemical contaminants to marine organism, but they can produce physical problems such as digestive congestion. However, they can be loaded with many substances to fit the virgin MPs to industry and consumer demand (e.g. additives, preservatives, etc.). In addition, these MPs can also adsorb contaminants present in the environment and act as vectors. Therefore, in this chapter, we shall summarize some important aspects of the microplastics found in the marine environments and some of the effects described in fish biota.

## **2. Chemical nature**

Plastics are usually synthesized from fossil fuels, but biomass can also be used as feedstock. The most commonly used plastic materials, the also called virgin plastics, are polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS) and polyethylene terephthalate (PET), which, together, represent approximately 90% of total world plastic production [15]. They are elements of high molecular weight and are non‐biodegradable and therefore extremely persistent in the environment. PE, PP, PVC, PS, PET and polyurethane (PUR) are widely used resins (29, 19, 12, 8, 6, and 7% of global production, respectively) [16]. Plastics present many advantages since they are inexpensive, water‐ and corrosion‐resistant, chemically inert, easily moulded and exhibit good thermal and electrical insulating properties. However, plastics also present many disadvantages, being non‐renewable resources and sources of contamination by additive compounds; they suffer embrittlement at low tempera‐ tures and deformation under loads; they need costly recycling processes and are highly resistant to degradation, etc. The behaviour of plastics in the environment will differ according to their chemical nature and physical properties. A reflection of this is the description of microplastics found in marine environments in different studies (**Table 1**).


**Table 1.** Frequency of occurrence of different polymer types in microplastic debris sampled at sea or in marine sediments [17].

## **3. Sources**

lead to significant amounts of these plastics entering the aquatic environment [4, 5]. It is a long‐ recognized fact that marine plastic debris contaminates the oceans and seas of all the world [3, 6, 7]. In the marine environment, plastics undergo a process of weathering and fragmen‐ tation that breaks down macrodebris into smaller micro‐ and nanodebris. This fragmentation of plastic is caused by a combination of mechanical forces, for example waves and/or photo‐ chemical processes triggered by sunlight. Some 'degradable' plastics are even designed to fragment quickly into small particles, although the resulting material does not necessarily

The terms 'microplastics' (MP) and 'microlitter' have been defined differently by various researchers. Gregory and Andrady [9] defined microlitter as the barely visible particles that pass through a 500‐µm sieve but are retained by a 67‐µm sieve (≈0.06–0.5 mm in diameter), while particles larger than this were called mesolitter. Others [10–12] defined the MPs as being in the size range <5 mm (recognizing 333 µm as a practical lower limit when neuston nets are used for sampling). Microplastic particles may further fragment into 'nanoplastics', a term that has not been defined uniformly in the literature, and may refer to <100‐µm particles of plastic

Microplastics have been accumulating in the environment for nearly half a century and are found in oceans worldwide [3] including in the Antarctic [7]. Despite this worldwide dissem‐ ination of plastic fragments, the global load of plastics on the open ocean surface has been estimated to be far less than might be expected, but nevertheless increasing. Thus, the potential effects of microplastics on marine ecosystems are still far from being well understood [14]. It is believed that the virging MPs are not chemical contaminants to marine organism, but they can produce physical problems such as digestive congestion. However, they can be loaded with many substances to fit the virgin MPs to industry and consumer demand (e.g. additives, preservatives, etc.). In addition, these MPs can also adsorb contaminants present in the environment and act as vectors. Therefore, in this chapter, we shall summarize some important aspects of the microplastics found in the marine environments and some of the effects

Plastics are usually synthesized from fossil fuels, but biomass can also be used as feedstock. The most commonly used plastic materials, the also called virgin plastics, are polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS) and polyethylene terephthalate (PET), which, together, represent approximately 90% of total world plastic production [15]. They are elements of high molecular weight and are non‐biodegradable and therefore extremely persistent in the environment. PE, PP, PVC, PS, PET and polyurethane (PUR) are widely used resins (29, 19, 12, 8, 6, and 7% of global production, respectively) [16]. Plastics present many advantages since they are inexpensive, water‐ and corrosion‐resistant, chemically inert, easily moulded and exhibit good thermal and electrical insulating properties. However, plastics also present many disadvantages, being non‐renewable resources and

biodegrade [8].

114 Toxicology - New Aspects to This Scientific Conundrum

described in fish biota.

**2. Chemical nature**

[13].

Microplastics comprise a very heterogeneous assemblage of particles that vary in size, shape, colour, chemical composition, density, and other characteristics. They can be subdivided according to their usage and source into (i) 'primary' MPs, produced either for indirect use as precursors (nurdles or virgin resin pellets), for the production of polymer consumer products or for direct use, for example in cosmetics, scrubs and abrasives and (ii) 'secondary' MPs, which result from the breakdown of larger plastic material into smaller fragments [18].

#### **3.1. Primary: common consumer products**

Microplastics (e.g. PE spheres) are used in personal care products such as toothpaste, facial and exfoliating creams, even though many consumers are not aware of this. In some cases, these MPs have replaced natural materials, such as seeds, shells or ground pumice ingredients. Usually, they are not filtered during wastewater treatment and are usually released directly into the sea or other water bodies such as lakes and rivers. Microplastics are also found in synthetic textiles: wastewaters from washing synthetic clothes, such as shirts, contain more than 100 fibres per litre of water. According to a study by Browne et al. [19], on average, about 1900 MP fibres can be released in a single machine wash. Similar fibres have been observed in wastewater effluent and sludge near large urban centres.

## **3.2. Primary: industrial sources of microplastics**

Plastic pellets are the raw material of plastic products. They are typically spherical or cylin‐ drical in shape and millimetres in diameter. In addition, pellets are used in various industrial applications, including as ingredients of printing inks, paints spray, injection mouldings and abrasives [20]. A proportion of MPs used in these industrial applications enters the environ‐ ment. The improvement in the management of operations in which plastic pellets are used could be a clear way to prevent them from entering the environment.

#### **3.3. Secondary: plastic waste as a source of microplastics**

Secondary microplastics are formed when larger plastic items are broken down. The rate at which fragmentation occurs is highly dependent on environmental conditions, especially temperature and the amount of UV light available [20]. Plastic debris can enter the ocean directly or can reach it through other water bodies or the atmosphere. The key to stopping plastic 'ocean trash' is to prevent such waste from entering the environment in the first place. Obviously, larger objects are easier to identify and control than smaller objects. About half of the world's population lives within 100 km of the coast, with an increasing population in that area. It is therefore highly likely that the amount of plastic waste entering the ocean from land‐ based sources will increase if significant changes are not made in the waste management on land.

## **4. Ecosystem distribution**

The accuracy of MP emission estimates is currently hindered by lack of data. More specifically, information on MP transport efficiency in run‐off and streams is missing. This is despite the large number of qualitative studies on microplastics in rivers and sediments [21–24]. Similarly, only limited assessments of MPs from sewage and canalizations, their retention by wastewater treatment plants and release by effluents are available [25, 26].

One of the most important factors affecting microplastic distribution in marine waters is the density of the materials (**Table 2**). Materials whose specific density is less than that of marine water (∼1.02) may be located on the surface, while materials with a specific density greater than that of marine water may be sink (**Table 2**). Thus, being buoyant in water, PE and PP float in seawater and mainly affect ocean surfaces and deposits ashore [27, 28], while PVC, which is denser than seawater, affects the seabed, often next to the source [27].


\* Specific density expressed in g/cm3 . The density of plastics may change depending on additives and environmental processes. Based on References [5, 29, 30].


**Table 3.** Microplastic concentrations observed in oceans of the world.

into the sea or other water bodies such as lakes and rivers. Microplastics are also found in synthetic textiles: wastewaters from washing synthetic clothes, such as shirts, contain more than 100 fibres per litre of water. According to a study by Browne et al. [19], on average, about 1900 MP fibres can be released in a single machine wash. Similar fibres have been observed in

Plastic pellets are the raw material of plastic products. They are typically spherical or cylin‐ drical in shape and millimetres in diameter. In addition, pellets are used in various industrial applications, including as ingredients of printing inks, paints spray, injection mouldings and abrasives [20]. A proportion of MPs used in these industrial applications enters the environ‐ ment. The improvement in the management of operations in which plastic pellets are used

Secondary microplastics are formed when larger plastic items are broken down. The rate at which fragmentation occurs is highly dependent on environmental conditions, especially temperature and the amount of UV light available [20]. Plastic debris can enter the ocean directly or can reach it through other water bodies or the atmosphere. The key to stopping plastic 'ocean trash' is to prevent such waste from entering the environment in the first place. Obviously, larger objects are easier to identify and control than smaller objects. About half of the world's population lives within 100 km of the coast, with an increasing population in that area. It is therefore highly likely that the amount of plastic waste entering the ocean from land‐ based sources will increase if significant changes are not made in the waste management on

The accuracy of MP emission estimates is currently hindered by lack of data. More specifically, information on MP transport efficiency in run‐off and streams is missing. This is despite the large number of qualitative studies on microplastics in rivers and sediments [21–24]. Similarly, only limited assessments of MPs from sewage and canalizations, their retention by wastewater

One of the most important factors affecting microplastic distribution in marine waters is the density of the materials (**Table 2**). Materials whose specific density is less than that of marine water (∼1.02) may be located on the surface, while materials with a specific density greater than that of marine water may be sink (**Table 2**). Thus, being buoyant in water, PE and PP float in seawater and mainly affect ocean surfaces and deposits ashore [27, 28], while PVC, which

wastewater effluent and sludge near large urban centres.

could be a clear way to prevent them from entering the environment.

**3.3. Secondary: plastic waste as a source of microplastics**

treatment plants and release by effluents are available [25, 26].

is denser than seawater, affects the seabed, often next to the source [27].

land.

**4. Ecosystem distribution**

**3.2. Primary: industrial sources of microplastics**

116 Toxicology - New Aspects to This Scientific Conundrum

Moreover, the colonization of MPs by microalgae and other microorganisms increases plastic density, which has been shown to affect the vertical transport of MPs in an aquatic environment and their long‐term distribution [31]. However, the chemical composition, particularly as a result of low amounts of additives, may partially explain the changes in microbiological colonization from one type of polymer to another. Also, for the same type of polymer, the chemical composition can vary considerably depending on chemical additives and the time passed in the environment. Hence, the distribution of MPs in the ecosystems may change according to these parameters, too. Long et al. [32] recently showed that MPs could be incorporated in microalgal homo‐aggregates, demonstrating the existence of a pathway of vertical transport of MP from the surface layer to the floor of the ocean.

In addition, MP concentrations and/or quantities differ between sampling sites (**Table 3**). A significant variation between the microplastics sampled in different oceans is evident, but there are also differences between the areas of the same ocean or sea. Published works have detected different concentrations of MPs depending on the proximity to populated and/or contaminat‐ ed areas.

## **5. Absorption of toxicants**

To enhance the performance of plastics, additives are added during manufacture, such as reinforcing fibres, fillers, coupling agents, plasticizers, colorants, stabilizers (halogen stabiliz‐ ers, antioxidants, ultraviolet absorbers and biological preservatives), adsorbed chemicals, and unreacted starting materials (monomers), processing aids (lubricants and flow control), flame retardants, peroxide, antistatic agent, and plasticizers [16, 42], which may leach out under conditions of use and accumulate in the environment [43]. Apart from the potential negative effects of the MPs *per se*, it is generally assumed that microplastics may increase the exposure of marine aquatic organisms to chemicals associated with the plastics, such as persistent organic pollutants (POPs) or plastic additives [44–47]. Thus, analytical study of marine MPs has revealed the composition of many toxicants adsorbed to them.



In addition, MP concentrations and/or quantities differ between sampling sites (**Table 3**). A significant variation between the microplastics sampled in different oceans is evident, but there are also differences between the areas of the same ocean or sea. Published works have detected different concentrations of MPs depending on the proximity to populated and/or contaminat‐

To enhance the performance of plastics, additives are added during manufacture, such as reinforcing fibres, fillers, coupling agents, plasticizers, colorants, stabilizers (halogen stabiliz‐ ers, antioxidants, ultraviolet absorbers and biological preservatives), adsorbed chemicals, and unreacted starting materials (monomers), processing aids (lubricants and flow control), flame retardants, peroxide, antistatic agent, and plasticizers [16, 42], which may leach out under conditions of use and accumulate in the environment [43]. Apart from the potential negative effects of the MPs *per se*, it is generally assumed that microplastics may increase the exposure of marine aquatic organisms to chemicals associated with the plastics, such as persistent organic pollutants (POPs) or plastic additives [44–47]. Thus, analytical study of marine MPs

**Additive CAS Log** *K***OW Water solubility (mg/L)** 

Benzophenone 119‐61‐9 3.18 None Benzotriazol 95‐14‐7 1.44 1.98 × 104

Irganox 1024 32687‐78‐8 7.79 <1 Irganox 1098 23128‐74‐7 – 0.1 Irganox 1076 2082‐79‐3 <6 <0.01 Irganox 1010 6683‐19‐8 ≈23 <0.01 Irganox 168 31570‐04‐4 >6 <0.005

Dimethyl phthalate 131‐11‐3 1.61 4.2 × 104 Diethyl phthalate 84‐66‐2 2.38 1.1 × 104 Di‐n‐butyl phthalate 84‐74‐2 4.45 112 Butylbenzyl phthalate 85‐68‐7 4.59 2.7 Bis(2‐ethylhexyl) phthalate 17‐81‐7 7.5 0.003 Di‐n‐octyl phthalate 3‐1307 8.06 0.02

has revealed the composition of many toxicants adsorbed to them.

ed areas.

*UV stabilizers*

*Antioxidants*

*Plasticisers*

*Lubricants*

**5. Absorption of toxicants**

118 Toxicology - New Aspects to This Scientific Conundrum


**Table 4.** Log *K*OW and water solubility of main additives of microplastics. Based on References [53–62].

In recent model analyses, however, it was shown that the effects of plastic on the bioaccumu‐ lation of POPs may be small, due to a lack of gradient between POPs in plastic and biota lipids, and that a cleaning mechanism is likely to dominate at higher log *K*OW (octanol/water partition coefficient) values [44, 48, 49] (**Table 4**). In the case of additives, monomers or oligomers, which are components of the plastics, this issue has hardly been addressed. Many substances such as plasticizers may have biological effects even at low concentrations in the ng/L or µg/L range [50]. Although it has been argued that exposure to additives will probably be low because of the low diffusivities of the chemicals, bioaccumulation could increase the concentration in animal tissues. Moreover, POPs, like the bisphenols or nonylphenols found in plastics, have been suggested to be a relevant environmental problem [51]. It has been reported that the concentrations of bisphenol A in wild freshwater fishes oscillated from undetected to 25.2 µg/ kg biomass, while nonylphenol levels varied from 1.01 to 277 µg/kg [52]. So, the substances can enter and be accumulated by animals, and the log *K*OW could give an idea of the behaviour of additives in aquatic environments and their solubility in water (**Table 4**).

## **5.1. Hydrocarbons**

**Additive CAS Log** *K***OW Water solubility (mg/L)** 

‐pentaBDE 32534‐81‐9 6.57 13.3 × 10‐3 ‐hexaBDE 36483‐60‐0 6.86–7.92 4.2 × 10‐6 ‐heptaBDE 68928‐80‐3 9.44 2.2 × 10‐7 ‐octaBDE 32536‐52‐0 6.29 5 × 10‐4 ‐nonaBDE 63936‐56‐1 11.22 5.6 × 10‐10 ‐decaBDE 1163‐19‐5 6.265 – α‐HBCD 134237‐50‐6 5.07 48.8 β‐HBCD 134237‐51‐7 5.12 14.7 γ‐HBCD 134237‐52‐8 5.47 2.1 TBBP‐A 79‐94‐7 4.5 720 BTBPE 37853‐59‐1 7.88 19 DBDPE 84852‐53‐9 11.1 21 Anti‐DP syn‐DP 13560‐89‐9 9.3 250

120 Toxicology - New Aspects to This Scientific Conundrum

7,9‐Di‐tert‐butyl‐1‐oxaspiro(4,5)deca‐6,9‐diene‐2,8‐dione 82304‐66‐3 3.59 15.5 Glycerol 1‐palmitate 32899‐41‐5 6.17 0.1252 (Z)‐13‐docosenamide 112‐84‐5 5.3 0.2 Di‐tert‐dodecyl disulfide 27458‐90‐8 6.1 None 1‐Hexadecanol 36653‐82‐4 6.83 4.1 × 10‐2 Oleic acid eicosyl ester 22393‐88‐0 13.609 –

Octadecanoic acid 57‐11‐4 8.23 0.568–0.597

**Table 4.** Log *K*OW and water solubility of main additives of microplastics. Based on References [53–62].

In recent model analyses, however, it was shown that the effects of plastic on the bioaccumu‐ lation of POPs may be small, due to a lack of gradient between POPs in plastic and biota lipids, and that a cleaning mechanism is likely to dominate at higher log *K*OW (octanol/water partition coefficient) values [44, 48, 49] (**Table 4**). In the case of additives, monomers or oligomers, which are components of the plastics, this issue has hardly been addressed. Many substances such as plasticizers may have biological effects even at low concentrations in the ng/L or µg/L range [50]. Although it has been argued that exposure to additives will probably be low because of the low diffusivities of the chemicals, bioaccumulation could increase the concentration in animal tissues. Moreover, POPs, like the bisphenols or nonylphenols found in plastics, have

Octadecanoic acid 4‐hydroxy‐methyl ester 2420‐38‐4 – – Tridecanoic acid 4,8,12‐trimethyl‐methyl ester 5129‐58‐8 – – Succinic acid 110‐15‐6 ‐0.59 8.32 × 104 Triclosan 3380‐34‐5 4.76 12

*Others*

Hydrocarbons are organic compounds comprising only carbon and hydrogen atoms. The molecular structure comprises a frame of carbon and hydrogen atoms and grouped into saturated (straight, substituted and cyclic alkanes), unsaturated (alkenes with straight, branched and cyclic), halogenated and aromatic hydrocarbons. The hydrocarbons can be classified into two types—aliphatic and aromatic. Aliphatic hydrocarbons in turn can be classified into alkanes, alkenes and alkynes as link types that bind the carbon atoms. The general formulas of alkanes, alkenes and alkynes are C*n*H2*n*+2, C*n*H2*n* and C*n*H2*n*‐2, respectively. Many alkanes with a chain length varying from C‐11 to C‐31 have been found in plastics from coastal debris [16]. These are other oligomers originating from polyolefins (polypropylene, polyethylene and poly(acetylene: styrene)) during recycling [63]. Octadecane (*n* = 20/43), hexadecane (*n* = 19), eicosane (*n* = 18), tetradecane (*n* = 18), heptacosane (*n* = 14), heptadecane (*n* = 13), pentadecane (*n* = 11), tetracosane (*n* = 10), docosane (*n* = 8), dodecane (*n* = 7), hexaco‐ sane (*n* = 7), 2,6,10‐trimethyl‐tetradecane (*n* = 10) and heptadecane, 3‐methyl‐ (*n* = 6) were the most frequently detected in the plastic debris from near the coasts [16].

Linear alkanes, together with iso‐alkanes, originate from the paraffin wax that is used as an external lubricant in PVC and other polymers, where they help the polymers to slide over other surfaces. Alkanes are also used as a solvents, such as hexane and heptane. Alkenes (squalene and others) and cycloalkenes are used as starting compounds for several additives and polymers and are formed as by‐products during olefin polymerization.

Aromatic hydrocarbons, such as benzene and anthracene derivatives, have also been found in MP debris. Benzene is an important organic chemical compound used mainly as an inter‐ mediate to make other chemicals, mainly ethylbenzene, cumene, cyclohexane, nitrobenzene, and alkylbenzene. More than half of the entire benzene production is processed into ethyl‐ benzene, a precursor of styrene, which is used to make polymers and plastics like polystyrene and expanded polystyrene. Around 20% of benzene production is used to manufacture cumene, which is needed to produce phenol and acetone for resins and adhesives. The plastics may also carry halogenated hydrocarbons, which have been considered as POPs and are of proven toxicity [64, 65].

## **5.2. UV stabilizers/absorbers**

Benzophenone and its derivatives are used as photo‐initiators in the UV curing of inks and as UV absorbers. These compounds absorb the harmful UV light that would eventually change the physical and optical properties of the polymer and make the material lose colour or fade. This substance can also be added to plastic packaging as a UV blocker to prevent photo‐ degradation of the packaging polymers or contents. Its use allows manufacturers to package the product in clear glass or plastic [66] since, without the UV blocker, opaque or dark packaging would be required. These plastic additives are used in PP, PE (2–3%) and acryloni‐ trile, butadiene and styrene (ABS) copolymer products. Benzotriazole UV stabilizers (BUVS) are emerging contaminants that are mutagenic, toxic, pseudopersistent, bioaccumulated and show significant estrogenic activity [67–70]. Great amounts of BUVS have been detected in rivers from Japan and China coming from wastewater treatment plants [71–73]. Due to their common use, BUVS have been found in aquatics environments [69, 72, 74], organisms [71, 72, 74, 75], tap water and well water [76]. Recent findings in German rivers and previously reports suggest that BUVSs have a potential of long‐range transport, similar to several POPs [74].

## **5.3. Antioxidants**

Antioxidants are widely used in plastic polymers to delay oxidation and to improve polymer properties [77]. Several types of antioxidants can be used to prevent the aging of plastic, such as phenolic antioxidants, organophosphorus compounds and different amines. However, antioxidants can migrate from the plastics into the food and contaminate it during production or storage, potentially giving rise to food safety issues [78, 79]. Antioxidants are used in almost all commercial polymers in small amounts up to 2% (w/w) (20,000 mg/kg or ppm) [16]. The polymers can be oxidized during synthesis, processing, transfer or final use, resulting in loss of chemical, optical and mechanical properties, among others. Thermal oxidation results in the formation of free radicals that react with oxygen to form hydroperoxides. In order to inhibit the onset of thermal oxidation of polymers and/or slow down degradative processes, the antioxidant additives are added during manufacture, processing and/or during the manufac‐ ture of the products. In the specific case of the polypropylene, antioxidant additives are important because the chemical structure of this type of polyolefin tends to degrade easily. The plastic antioxidants identified in the literature are usually limited to the commonly used Irganox series (including Irganox 1010, Irganox 1076, Irganox 168) [80–84].

#### **5.4. Plasticizers**

Plastic as a material may contain a variety of chemicals, some potentially hazardous. Plasti‐ cizers, which are used to make the plastic soft and flexible, are mainly used in PVC, but they are detected in other polymer plastics. Several types of plasticizers are found in plastic debris, but phthalates predominate [85]. The phthalates found in plastics include dimethyl phthalate (DMP), diethyl phthalate (DEP), di‐n‐butyl phthalate (DBP), butyl benzyl phthalate (BBP), bis(2‐ethylhexyl) phthalate (DEHP) and di‐n‐octyl phthalate (DNOP) [86]. Their concentra‐ tions in different plastics vary widely; for example, in foodstuffs, the content of phthalates varies from 658 to 1610 ng/g fresh weight [87]. Phthalates are produced in large quantities around the world and are also widely used in cosmetics, plastics, carpets, building materials, toys, medical and cleaning products.

Several cross‐sectional and case‐control studies have reported an association between expo‐ sure to phthalates and the development of certain human allergies and respiratory diseases [88]. A recent systematic review based on less than ten relatively small (*N* < 400) studies found that the findings from these studies are inconsistent, with both decreases in birthweight and null associations, and both longer and shorter gestational periods being recorded [89]. A prospective birth cohort study researched the association between butyl benzyl phthalate and an early‐onset eczema, although not the late‐onset eczema, finding that prenatal exposure to butylbenzyl phthalate may influence the risk of developing eczema in early childhood [90]. Three studies reported a positive relation between prenatal exposition and the risk of wheeze, asthma and respiratory infections in children aged 5–11 years [91–93], although, even here, there were inconsistencies concerning the phthalate congeners implicated [94].

On the other hand, phthalates have been related with hormone disorders [95], abortion [96], metabolic diseases [97], hormone disturbances, reprotoxicity or even suspected cancer [98– 100]. Other plasticizers are often used as substitutes for phthalates, but their effects on the health are not always clear, usually because of the limited data available. Therefore, because the amount of plasticizers could increase the 50% of the total weight, and the possibility that these substances will leach when the plastics come into contact with seawater is greater [16], the substances called plasticizers should be considered in a hazard category and need be reviewed.

## **5.5. Lubricants**

degradation of the packaging polymers or contents. Its use allows manufacturers to package the product in clear glass or plastic [66] since, without the UV blocker, opaque or dark packaging would be required. These plastic additives are used in PP, PE (2–3%) and acryloni‐ trile, butadiene and styrene (ABS) copolymer products. Benzotriazole UV stabilizers (BUVS) are emerging contaminants that are mutagenic, toxic, pseudopersistent, bioaccumulated and show significant estrogenic activity [67–70]. Great amounts of BUVS have been detected in rivers from Japan and China coming from wastewater treatment plants [71–73]. Due to their common use, BUVS have been found in aquatics environments [69, 72, 74], organisms [71, 72, 74, 75], tap water and well water [76]. Recent findings in German rivers and previously reports suggest that BUVSs have a potential of long‐range transport, similar to several POPs [74].

Antioxidants are widely used in plastic polymers to delay oxidation and to improve polymer properties [77]. Several types of antioxidants can be used to prevent the aging of plastic, such as phenolic antioxidants, organophosphorus compounds and different amines. However, antioxidants can migrate from the plastics into the food and contaminate it during production or storage, potentially giving rise to food safety issues [78, 79]. Antioxidants are used in almost all commercial polymers in small amounts up to 2% (w/w) (20,000 mg/kg or ppm) [16]. The polymers can be oxidized during synthesis, processing, transfer or final use, resulting in loss of chemical, optical and mechanical properties, among others. Thermal oxidation results in the formation of free radicals that react with oxygen to form hydroperoxides. In order to inhibit the onset of thermal oxidation of polymers and/or slow down degradative processes, the antioxidant additives are added during manufacture, processing and/or during the manufac‐ ture of the products. In the specific case of the polypropylene, antioxidant additives are important because the chemical structure of this type of polyolefin tends to degrade easily. The plastic antioxidants identified in the literature are usually limited to the commonly used

Plastic as a material may contain a variety of chemicals, some potentially hazardous. Plasti‐ cizers, which are used to make the plastic soft and flexible, are mainly used in PVC, but they are detected in other polymer plastics. Several types of plasticizers are found in plastic debris, but phthalates predominate [85]. The phthalates found in plastics include dimethyl phthalate (DMP), diethyl phthalate (DEP), di‐n‐butyl phthalate (DBP), butyl benzyl phthalate (BBP), bis(2‐ethylhexyl) phthalate (DEHP) and di‐n‐octyl phthalate (DNOP) [86]. Their concentra‐ tions in different plastics vary widely; for example, in foodstuffs, the content of phthalates varies from 658 to 1610 ng/g fresh weight [87]. Phthalates are produced in large quantities around the world and are also widely used in cosmetics, plastics, carpets, building materials,

Several cross‐sectional and case‐control studies have reported an association between expo‐ sure to phthalates and the development of certain human allergies and respiratory diseases [88]. A recent systematic review based on less than ten relatively small (*N* < 400) studies found

Irganox series (including Irganox 1010, Irganox 1076, Irganox 168) [80–84].

**5.3. Antioxidants**

122 Toxicology - New Aspects to This Scientific Conundrum

**5.4. Plasticizers**

toys, medical and cleaning products.

Usually, lubricants are used to minimize adhesion and viscosity of plastic polymers. Internal lubricants can facilitate the production process by providing lubrication at molecular level between the polymer chains [101]. Commonly, they are composed of an oil base accompanied by a variety of additives that confer desirable properties. Lubricants are based in one type of base oil, but in commercial requirements, it usually makes that a mixture are used [102]. n‐ Hexadecanoic acid, oleic acid, glycerol tricaprylate, isopropyl myristate, 1‐eicosanol, 2‐hexyl‐ 1‐decanol, octadecanamide, 4‐methyl‐benzenesulfonamide, 1‐hexacosanol and decanedioic acid, bis(2‐ethylhexyl) ester can be found in plastic debris [16]. The transfer of additives such as lubricants to the medium or to the substances which are in contact with the plastics has been reported previously [103].

#### **5.6. Fuel**

Chemicals like pentadactyl ester trichloroacetic acid, 1,10‐[2‐methyl‐2‐(phenylthio) cyclopro‐ penylidene] bisbenzene and 2,4‐dimethyl‐4‐octanol are often found in plastic debris [16]. These substances and others, like hexadecyl ester trichloroacetic acid, have been considered as fuel precursor based on plastic wastes additives, due to the large amount and variety of additives that plastics can contain [63]. Waste plastics are considered a promising source for fuel production because of their high combustion heat and their increasing availability in local communities [104].

#### **5.7. Intermediates**

In manufacture of plastics, it is normal to use stabilizers (DEHA or DEHP) and plasticizers that contain intermediate substances like hexanoic acid 2‐ethyl‐hexadecyl ester (HEHA), 2,3‐ dihydroxypropyl ester hexadecanoic acid, hexadecanoic acid ethyl ester, behenic alcohol, nonanoic acid, pimaric acid, 3,5‐di‐tert‐butyl‐4‐hydroxyphenyl propionic acid, abietic acid and dehydroabietic acid [16]. HEHA has been classified as belonging to reprotoxic category 3 by Council Directive 67/548/EEC [105].

## **5.8. Flame retardants**

Flame retardants are a group of chemical compounds that are used in plastics with the aim of diminishing the flammability of combustible materials, like synthetic polymers and plastics. To make sure that flame retardants remain in the polymers, these compounds are designed to be stable for many years, which means they will remain in the environment long past the time when the material itself was used [106]. Thus, these compounds can enter aquatic environ‐ ments via the atmospheric deposition of fine particles, direct discharges of municipal and industrial wastewater effluents, and through run‐off and other human activities [107]. Flame retardants include α, β, γ‐diastereoisomers of hexabromocyclododecane (HBCD), tetrabro‐ mobisphenol‐A (TBBP‐A), anti‐ and syn‐isomers of dechlorane plus (DP) and two novel compounds, decabromodiphenylethane (DBDPE) and 2‐bis(2,4,6‐tribromophenoxy) ethane (BTBPE). Among the most widely used flame retards are polybrominated diphenyl ethers, which have been in use since the late 1970s. Polybrominated diphenyl ethers are a class of brominated compounds widely used as flame retardants including in polymers such as low density polyethylene or silicone rubber [45, 108]. Polybrominated diphenyl ethers are very hydrophobic, with log *K*OW above 5.5 and molecular weights (MW) in the range of 300–1000  g/mol which means that these compounds are likely to have diffusion coefficients significantly lower than those measured for polycyclic aromatic hydrocarbons and polychlorinated biphenyls. This implies that the polymer diffusion coefficients for these plastic additives used as flame retardants need to be taken into account when considering the risk posed by micro‐ plastic particle ingestion by marine organisms [109]. Many studies on polybrominated diphenyl ethers [110–118] have shown that these compounds are ubiquitous, toxic, persistent and bioaccumulated in the environment. As a result, some flame retardants have been prohibited in the USA and European Union [119, 120], such a penta‐ and octabrominated diphenyl ether. Nevertheless, new compounds have replaced the forbidden polybrominated diphenyl ethers, such as 1,2‐bis(pentabromodiphenyl) ethane, which is used in solid plastics, wire, cable and electronics, high impact polystyrene and thermoplastics [121].

#### **5.9. Monomers and oligomers**

Bisphenol A (2,2‐(4,4‐dihydroxydiphenyl) propane) is used as a monomer in polycarbonate, for the production of polycarbonate plastics and epoxy resins. It has been found in samples of PE, PP and acrylate‐styrene, where it is probably used as chain terminator, to finish the polymerization of polymers or as antioxidant for polymers or plasticizers [16]. Bisphenol A is also used to manufacture a great variety of products, including CDs, food can linings, thermal paper, safety helmets, plastic windows, car parts, adhesives, protective coatings, powder paints, and the sheathing of electrical and electronic parts [122]. As a result of its wide usage, bisphenol A is frequently detected in wastewaters [123].

Bisphenol A has been identified as an endocrine disruptor [124], and several studies have demonstrated reproductive, metabolic and neurodevelopmental problems in animals exposed to environmentally relevant levels of this substance [125–127]. In addition, an increased risk for cardiovascular disease, altered immune system activity, miscarriages, decreased birth‐ weight at term, metabolic problems and diabetes in adults, breast and prostate cancer, reproductive and sexual dysfunctions and cognitive and behavioural development in young children have been associated with the human exposure to bisphenol A [128–134].

It is known that plasticizers may have biological effects even at low concentrations in the ng/L range, especially for molluscs, crustaceans and amphibians [50]. Although it has been argued that one should expect levels of exposure to plastic additives to be low due to the low diffusivities of chemicals like bisphenol A or nonylphenol in plastics [51], as we said above, their bioaccumulation could play an important role, increasing physiological concentrations in the food chain. In an attempt to solve these problems, physicochemical processes for the removal of bisphenol A from wastewaters have been studied [135, 136]. However, possible solutions presented several problems related to the cost of chemicals, the generation of bisphenol A‐containing sludge and the conditions necessary to optimize the bisphenol A elimination process. The most frequently detected metabolic products of the aerobic biode‐ gradation pathway of bisphenol A include 4‐hydroxyacetophenone and 4‐hydroxybenzoic acid [137]. Both bisphenol A and 4‐hydroxybenzoic acids have shown a certain degree of biodegradability [138], and these compounds are not expected to be persistent in an activated sludge system, although the information concerning 4‐hydroxyacetophenone is scarce.

## **5.10. Others**

dihydroxypropyl ester hexadecanoic acid, hexadecanoic acid ethyl ester, behenic alcohol, nonanoic acid, pimaric acid, 3,5‐di‐tert‐butyl‐4‐hydroxyphenyl propionic acid, abietic acid and dehydroabietic acid [16]. HEHA has been classified as belonging to reprotoxic category 3 by

Flame retardants are a group of chemical compounds that are used in plastics with the aim of diminishing the flammability of combustible materials, like synthetic polymers and plastics. To make sure that flame retardants remain in the polymers, these compounds are designed to be stable for many years, which means they will remain in the environment long past the time when the material itself was used [106]. Thus, these compounds can enter aquatic environ‐ ments via the atmospheric deposition of fine particles, direct discharges of municipal and industrial wastewater effluents, and through run‐off and other human activities [107]. Flame retardants include α, β, γ‐diastereoisomers of hexabromocyclododecane (HBCD), tetrabro‐ mobisphenol‐A (TBBP‐A), anti‐ and syn‐isomers of dechlorane plus (DP) and two novel compounds, decabromodiphenylethane (DBDPE) and 2‐bis(2,4,6‐tribromophenoxy) ethane (BTBPE). Among the most widely used flame retards are polybrominated diphenyl ethers, which have been in use since the late 1970s. Polybrominated diphenyl ethers are a class of brominated compounds widely used as flame retardants including in polymers such as low density polyethylene or silicone rubber [45, 108]. Polybrominated diphenyl ethers are very hydrophobic, with log *K*OW above 5.5 and molecular weights (MW) in the range of 300–1000  g/mol which means that these compounds are likely to have diffusion coefficients significantly lower than those measured for polycyclic aromatic hydrocarbons and polychlorinated biphenyls. This implies that the polymer diffusion coefficients for these plastic additives used as flame retardants need to be taken into account when considering the risk posed by micro‐ plastic particle ingestion by marine organisms [109]. Many studies on polybrominated diphenyl ethers [110–118] have shown that these compounds are ubiquitous, toxic, persistent and bioaccumulated in the environment. As a result, some flame retardants have been prohibited in the USA and European Union [119, 120], such a penta‐ and octabrominated diphenyl ether. Nevertheless, new compounds have replaced the forbidden polybrominated diphenyl ethers, such as 1,2‐bis(pentabromodiphenyl) ethane, which is used in solid plastics,

wire, cable and electronics, high impact polystyrene and thermoplastics [121].

bisphenol A is frequently detected in wastewaters [123].

Bisphenol A (2,2‐(4,4‐dihydroxydiphenyl) propane) is used as a monomer in polycarbonate, for the production of polycarbonate plastics and epoxy resins. It has been found in samples of PE, PP and acrylate‐styrene, where it is probably used as chain terminator, to finish the polymerization of polymers or as antioxidant for polymers or plasticizers [16]. Bisphenol A is also used to manufacture a great variety of products, including CDs, food can linings, thermal paper, safety helmets, plastic windows, car parts, adhesives, protective coatings, powder paints, and the sheathing of electrical and electronic parts [122]. As a result of its wide usage,

Council Directive 67/548/EEC [105].

124 Toxicology - New Aspects to This Scientific Conundrum

**5.9. Monomers and oligomers**

**5.8. Flame retardants**

Degradation products, antifogging, antiblocking, colouring, heat stabilizers, fatty acids and their derivatives have also been found in plastics debris [16]. This heterogeneous group includes 7,9‐di‐tert‐butyl‐1‐oxaspiro(4,5)deca‐6,9‐diene‐2,8‐dione, glycerol 1‐palmitate, (Z)‐13‐docose‐ namide, 2,3‐dichloro‐1,10‐biphenyl, trans‐13‐docosenamide, di‐tert‐dodecyl disulfide, 1‐hexa‐ decanol, 2,4‐bis [2‐(4‐methoxyphenyl‐2‐propyl)] methoxybenzene, oleic acid eicosyl ester, octadecanoic acid, octadecanoic acid 4‐hydroxy‐methyl ester, octadecanoic acid 2‐hydroxy‐1‐ (hydroxymethyl)ethyl ester, tridecanoic acid 4,8,12‐trimethyl‐methyl ester, heptanedioic acid 4‐(ethoxycarbonylmethylene)‐diethyl ester and succinic acid [16]. Fatty acids and their esters could originate from several kinds of oils, such as coconut oil (lauric acid) or palm oil (palmitic acid), acids which, along with their esters, are usually used as internal lubricants. Besides, metallic salts of fatty acids are normally used as stabilizers and plasticizers in the production of the plastics.

Other substances can stick or bind to plastics, such as disinfectants, aromatic compounds, soaps used to clean the plastics. In this respect, triclosan (5‐chloro‐2‐[2,4‐dichloro‐phenoxy]‐ phenol) is an additive that has been reported to be toxic [139–141]. Triclosan is an antimicrobial that is effective against bacteria of the adult oral cavity and skin. It is currently used in antibacterial soaps, deodorants, skin creams, toothpastes and plastics. Triclosan is an ionizable chlorinated biphenyl ether of low water solubility, with a p*K*a of 8.1, and a vapour pressure of 4 × 10‐6 mm Hg [139]. Triclosan readily bioaccumulates within aquatic organisms and has been found to be toxic to fish. In larval fishes, exposure to triclosan disrupts a variety of develop‐ mental processes, impairs hatching success, and causes pericardial oedema, having the potential to cause subtle cardiac toxicity [142]. Browne et al. [47] showed that triclosan added to MPs diminished the ability of worms to engineer sediments and caused mortality, each by >55%, while PVC alone made worms >30% more susceptible to oxidative stress. Triclosan persists in water and is difficult to eliminate from wastewaters [143, 144]. The ingestion of MPs by organisms can transfer pollutants and additives (such as triclosan) to their tissues at concentrations sufficient to disrupt ecophysiological functions linked to health and biodiver‐ sity. Biomarkers of endocrine disruption found in fish indicated long‐term exposure to estrogenic chemicals in the wastewater [145].

## **6. Effects on marine fish**

The accumulation of microplastic waste could affect the functioning of marine ecosystems. However, the mechanisms by which these effects will be manifested have not been identified. Impacts on biota and marine environmental quality are well documented [146], with damage for the global economy estimated to be in the range of \$13 billion per year [147].

**Figure 1.** Principal effects of microplastics on fish.

Negative effects include entanglement in plastic wires or nets, or to ingestion, which has been reported in benthic invertebrates, birds, fish, mammals and turtles [148–151]. This is especially true for eggs, embryos and larvae of aquatic organisms, which are particularly vulnerable to water‐borne pollutants owing to their limited ability to regulate their internal environment [152]. In particular, the early life stages of fishes are subjected to strong selection forces, driven by high rates of predator‐induced mortality [153, 154]. So, it has been reported that there is a clear overlap between areas with high levels of microplastics pollution and the feeding grounds of fin whales in the Mediterranean Sea, which could mean that fin whales are subjected to a high level of exposure to MPs ingestion during feeding in the areas [155]. The bioaccumulation of MPs and the substances which they could carry seem to be an increasing problem due to MPs which has been detected from little fish species to the top of food web.

The ingestion of the MPs can influence marine animals in different ways (**Figure 1**). It can affect to the immune system, both chemically (caused by the substances that MPs might contain, absorb or release, which may be toxic) [156] and physically blocking the digestive organs and preventing the animals from feeding [157]. Ecology and behaviour could also be affected.

## **6.1. Immune system**

found to be toxic to fish. In larval fishes, exposure to triclosan disrupts a variety of develop‐ mental processes, impairs hatching success, and causes pericardial oedema, having the potential to cause subtle cardiac toxicity [142]. Browne et al. [47] showed that triclosan added to MPs diminished the ability of worms to engineer sediments and caused mortality, each by >55%, while PVC alone made worms >30% more susceptible to oxidative stress. Triclosan persists in water and is difficult to eliminate from wastewaters [143, 144]. The ingestion of MPs by organisms can transfer pollutants and additives (such as triclosan) to their tissues at concentrations sufficient to disrupt ecophysiological functions linked to health and biodiver‐ sity. Biomarkers of endocrine disruption found in fish indicated long‐term exposure to

The accumulation of microplastic waste could affect the functioning of marine ecosystems. However, the mechanisms by which these effects will be manifested have not been identified. Impacts on biota and marine environmental quality are well documented [146], with damage

for the global economy estimated to be in the range of \$13 billion per year [147].

estrogenic chemicals in the wastewater [145].

126 Toxicology - New Aspects to This Scientific Conundrum

**6. Effects on marine fish**

**Figure 1.** Principal effects of microplastics on fish.

Interactions between plastic microparticles and aquatic organisms have been reported, and several recent studies have addressed the effects of nanoplastic material on different organisms and their health status. This research suggests that nanoplastics can enter different organisms and may interact with the immune system [158–161].

In fish, cellular innate immunity effectors act as one of the first organ defences against various agents, which makes these effectors the possible target for interaction with nanoplastic particles. Neutrophil activation is critical for the host defences, and their function is a valuable tool to assess the health status of individuals and animal populations [162]. So, fish neutrophils can extravasate, migrate chemotactically, degranulate, release neutrophil extracellular traps and phagocytize particulate matter such as bacteria [163]. Hypotheses existed about the interactions between MPs or nanoplastics and the neutrophils until recently, it has been reported that polystyrene and polycarbonate nanoplastic can act as stressors to the innate immune response of fish [164]. Therefore, nanoplastic could potentially interfere with innate immune responses in fish populations by altering organismal defence mechanisms.

In addition, plastic fragments found in the marine habitat have been shown to absorb POPs, so effects on the immune system may be caused by particle toxicity, plastic‐associated chemi‐ cals and absorbed environmental chemicals.

## **6.2. Disrupting effects**

Evidence points to the potential role of microplastics as vectors of chemical pollutants, either used as additives during polymer synthesis, or adsorbed directly from seawater [27, 45, 165]. The hydrophobicity of organic xenobiotics and the surfaces of polymers facilitate the adsorp‐ tion of the chemicals on MPs at concentrations with orders of magnitude higher than those usually detected in seawater [166].

Several of these plastic‐associated chemicals have been linked to endocrine‐disrupting effects in fish. Styrene [167], a monomer of several plastic types including polystyrene, rubber and acrylonitrile–butadiene–styrene, and bisphenol‐A [168] a monomer of polycarbonate, can disrupt the endocrine system function, as mentioned above. In addition, there is evidence that UV stabilizers, phthalates and nonylphenol, additives to plastic, are estrogenic and/or antiandrogenic [169, 170]. Furthermore, chemicals historically known to promote adverse effects in the endocrine system functions, including heavy metals, organochlorine pesticides and petroleum hydrocarbons [171, 172], have been found attached to plastic debris around the world [173, 174].

The ingestion of plastic debris has been documented in fish [175, 176], which may introduce a 'cocktail' of endocrine‐disrupting chemicals [47, 150, 177]. Significantly higher concentrations of several polybrominated diphenyl ethers, such as polychlorinated biphenyl congener (PCB#28) and the polycyclic aromatic hydrocarbon chrysene, have been recorded in Japanese medaka (*Oryzias latipes*) exposed to polyethylene that had been deployed in the marine environment compared to fish exposed to a virgin polyethylene and a control treatment [177].

Fish are useful as sensitive indicators of endocrine‐disrupting chemicals in aquatic habitats, as exposure can result in changes in gonadal growth, gonadal degeneration, sex‐specific gene protein and intersex induction [178]. Finally, recent research showed that ingestion of plastic debris at environmentally relevant concentrations may alter the endocrine system function in adults [179], where the presence of abnormal germ cell proliferation observed may be related to plastic. In this respect, ovary structure protein 1 (OSP1) gene has been proposed as a suitable indicator of the early stages of intersex development and suggested to be a more sensitive early‐warning signal than histopathological observation [180].

## **6.3. Physiological**

It has been shown in various marine organisms that ingestion of MPs occurs in animals with different feeding strategies and may negatively influence both the feeding activity and nutritional value, especially in species which cannot vary their food source [181, 182]. Different studies have pointed to the obstruction and damage of digestive tracts or even animals starving to death caused by stomachs filled with plastic [18]. In addition, MP ingestion by marine biota has been detected in benthic fish species [183, 184], and different sized plastic items were identified in the stomachs of three large pelagic fish in the Mediterranean Sea [185].

In a study made in Spanish coastal waters and which constitutes the first report of MPs ingestion by demersal fishes, red mullets (*Mullus barbatus*) from Barcelona presented the highest abundance of microplastics, followed by dogfish (*Scyliorhinus canicula*) from the Cantabrian coast and the Gulf of Cadiz, whereas dogfish from the Galician coast presented the lowest levels [186]. In agreement with previous studies, the detected MPs were mostly fibres (71%) [174, 184, 187], and the most frequent colour was black (51%) (**Table 5**).

Because of their small size, MPs may be ingested by marine organisms, regardless of their feeding mechanisms, and may enter their circulatory system and accumulate in different types of tissues, as has been proven in laboratory experiments [182]. These reported data, along with the fact that MPs serve as dispersal vectors for invasive species [188] and the toxic and bioaccumulative substances bound to the plastics [149], together with the research that indicates that MPs may have the ability to enter and disseminate though the marine food web [189, 190], suggest grave ecological implications of microplastics across the food web.


**Table 5.** Types of plastics found in fish and their relative abundance in Spanish coastal waters [188].

#### **6.4. Behaviour**

Several of these plastic‐associated chemicals have been linked to endocrine‐disrupting effects in fish. Styrene [167], a monomer of several plastic types including polystyrene, rubber and acrylonitrile–butadiene–styrene, and bisphenol‐A [168] a monomer of polycarbonate, can disrupt the endocrine system function, as mentioned above. In addition, there is evidence that UV stabilizers, phthalates and nonylphenol, additives to plastic, are estrogenic and/or antiandrogenic [169, 170]. Furthermore, chemicals historically known to promote adverse effects in the endocrine system functions, including heavy metals, organochlorine pesticides and petroleum hydrocarbons [171, 172], have been found attached to plastic debris around the

The ingestion of plastic debris has been documented in fish [175, 176], which may introduce a 'cocktail' of endocrine‐disrupting chemicals [47, 150, 177]. Significantly higher concentrations of several polybrominated diphenyl ethers, such as polychlorinated biphenyl congener (PCB#28) and the polycyclic aromatic hydrocarbon chrysene, have been recorded in Japanese medaka (*Oryzias latipes*) exposed to polyethylene that had been deployed in the marine environment compared to fish exposed to a virgin polyethylene and a control treatment [177].

Fish are useful as sensitive indicators of endocrine‐disrupting chemicals in aquatic habitats, as exposure can result in changes in gonadal growth, gonadal degeneration, sex‐specific gene protein and intersex induction [178]. Finally, recent research showed that ingestion of plastic debris at environmentally relevant concentrations may alter the endocrine system function in adults [179], where the presence of abnormal germ cell proliferation observed may be related to plastic. In this respect, ovary structure protein 1 (OSP1) gene has been proposed as a suitable indicator of the early stages of intersex development and suggested to be a more sensitive

It has been shown in various marine organisms that ingestion of MPs occurs in animals with different feeding strategies and may negatively influence both the feeding activity and nutritional value, especially in species which cannot vary their food source [181, 182]. Different studies have pointed to the obstruction and damage of digestive tracts or even animals starving to death caused by stomachs filled with plastic [18]. In addition, MP ingestion by marine biota has been detected in benthic fish species [183, 184], and different sized plastic items were

In a study made in Spanish coastal waters and which constitutes the first report of MPs ingestion by demersal fishes, red mullets (*Mullus barbatus*) from Barcelona presented the highest abundance of microplastics, followed by dogfish (*Scyliorhinus canicula*) from the Cantabrian coast and the Gulf of Cadiz, whereas dogfish from the Galician coast presented the lowest levels [186]. In agreement with previous studies, the detected MPs were mostly fibres

Because of their small size, MPs may be ingested by marine organisms, regardless of their feeding mechanisms, and may enter their circulatory system and accumulate in different types of tissues, as has been proven in laboratory experiments [182]. These reported data, along with

identified in the stomachs of three large pelagic fish in the Mediterranean Sea [185].

(71%) [174, 184, 187], and the most frequent colour was black (51%) (**Table 5**).

early‐warning signal than histopathological observation [180].

world [173, 174].

128 Toxicology - New Aspects to This Scientific Conundrum

**6.3. Physiological**

Behaviour is a crucial determinant for essential parameters such as overall health, growth, reproduction and survival [191]. During the life cycle of fish, a critical point is the early stage of development. Survival depends, in many cases, on the capacity of the organism to evade predators. An innate ability to detect and act accordingly is therefore vital [153, 154, 192].

In this regard, it has been suggest that olfactory sense in fish larvae could suffer damage mediated by an immunological response produced by the pollutant from microplastics. Lönnstedt and Eklöv [193] found that not only was crucial behaviour, such as activity and feeding, affected by microplastics, but that innate responses to olfactory threat cues were also impaired. Such a loss of predator avoidance behaviour greatly increased predator‐induced mortality rates of larvae. Finally, survival of fishes could be seriously affected by the presence of MPs, with their significant impact on the life cycle of the fish.

## **7. Conclusion**

Microplastics in the aquatic environment have been demonstrated to be a significant problem. The great amount of research on this topic, as well as the quantity of the results that describe the problem of MPs and their effects on fishes and aquatic life, have thrown some light on this issue. Among the effects that MPs have are stress, intestinal obstruction and the alteration of health, while further studies are in progress to ascertain the full potential risks of MPs in aquatic organisms with special attention paid to fish. A huge number of substances are added to plastics, which can bioaccumulate throughout the trophic chain. Besides the problems that MPs represent for marine life in general, the MPs could begin act as disruptors of the welfare and health of fishes, both wild and cultivated. This is clearly a growing problem not only for the environment but also for human health. For these reasons, further efforts are needed to know the exact effects that microplastics, and their constitutive and adsorbed contaminants, may have on aquatic environments.

## **Acknowledgements**

Financial support by grants PCIN‐2015‐187‐C03‐02 (MINECO, JPIOceans: Microplastics, EPHEMARE) and 19883/GERM/15 (Fundación Séneca de la Región de Murcia, Spain) is gratefully acknowledged.

## **Author details**

Cristóbal Espinosa, M. Ángeles Esteban and Alberto Cuesta\*

\*Address all correspondence to: alcuesta@um.es

Fish Innate Immune System Group, Department of Cell Biology and Histology, Faculty of Biology, Campus Regional de Excelencia Internacional "Campus Mare Nostrum", University of Murcia, Murcia, Spain

## **References**


[7] Zarfl C, Matthies M. Are marine plastic particles transport vectors for organic pollutants to the Arctic? Mar Pollut Bull. 2010;60:1810–1814. doi:10.1016/j.marpolbul.2010.05.026

**Acknowledgements**

130 Toxicology - New Aspects to This Scientific Conundrum

gratefully acknowledged.

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Cristóbal Espinosa, M. Ángeles Esteban and Alberto Cuesta\*

\*Address all correspondence to: alcuesta@um.es

**Author details**

Financial support by grants PCIN‐2015‐187‐C03‐02 (MINECO, JPIOceans: Microplastics, EPHEMARE) and 19883/GERM/15 (Fundación Séneca de la Región de Murcia, Spain) is

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## **Biotests in Ecotoxicology: Current Practice and Problems Biotests in Ecotoxicology: Current Practice and Problems**

Mariyana Lyubenova and Silvena Boteva Additional information is available at the end of the chapter

Mariyana Lyubenova and Silvena Boteva

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64776

#### **Abstract**

Nowadays ecotoxicology plays the role of a theoretician – methodical unifying centre for the optimization of man – biosphere relations and sustainable existence of life on the Earth. The main basis for its development is the classical toxicology—studies of chemical compounds' effects on man, but ecotoxicology is the original part, following it. According to the modern concept, the ecotoxicology is a science for migration, transformation and utilization of different toxic ingredients (with organic, inorganic or organic-mineral chemical nature; with natural biotic or abiotic origin and artificial, mainly anthropogenic origin) in the environment and their impact on Macro- biological systems with different levels of integration as groups of individuals, population, community, ecosystem, etc. studied in ecology. In this chapter, the types of ecotoxicological tests are discussed in detail with a set of examples about used species, advantages and disadvantages of different types of toxicity testing. The application of exposed natural ecosystems or man-made analogue systems is also commented as the environmentally more realistic approach for ecotoxicological testing. These test systems are increasingly becoming in aquatic ecotoxicology practice, but they are contemporary challenge in terrestrial testing. The development of test systems for realistic assessment of contaminant toxicity is essential for the efficient control of human influence on the environment.

**Keywords:** ecotoxicology, bio tests, acute, chronic, mono-species, multispecies, biomarkers, kits

## **1. Introduction: the contemporary meaning of ecotoxicology as a complex science**

Ecotoxicology is a scientific discipline, which of the modern stage of man-biosphere relations, is developed as the theoretician – methodical unifying centre for the optimization of these

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

relations for the sustainability of life existence on Earth. The main basis for the development of ecotoxicology is classical toxicology—the research of drugs and chemical compounds effects on man. The modern concept of ecotoxicology is that it is the original part following the classical toxicology, which studies migration, transformation, degradation and utilization of toxic ingredients (with organic, inorganic or organic-mineral chemical nature; with natural biotic or abiotic origin and artificial, mainly anthropogenic origin) in the environment and their impact on Macro- biological systems with different levels of integration as groups of individuals, population, community, ecosystem, etc. studied in ecology (MBS) [1, 2] and others.

The main objects of the ecotoxicological studies are the both: (1) toxic ingredients and their "behaviour" in the five main environments such as air, soil, terrestrial, water (incl. sediments) and biotic and (2) the responses of MBS in nature. The studied toxicants can be: (1) by the chemical nature: organic, inorganic or organic-mineral; (2) by the origin: natural (biotic or abiotic) and artificial (mainly anthropogenic origin); (3) by the toxicity: toxicants in the Black list, toxicants in the Grey list, etc. and (4) by the main environment of circulation: air, water, soil or different bio toxicants. The migration, transformation and degradation of toxic ingredients depend on the internal (endogenous) factors, that are, chemical properties of the toxicant and external (exogenous) factors or features of the environment. The studied MBS at different levels of integration can be: diverse individuals as elements of the population; homogeneous and heterogeneous populations as elements of the communities; heterogeneous communities as elements of the bio cenosis; ecosystem as a functional unity between biotope and bio cenosis; landscape, biome and biosphere formed by a corresponding set of ecosystems and their environment. The responses of MBS also depend on the endogenous factors (level of integration and features of the MBS) and exogenous factors (the characteristics of toxicants and habitats). Therefore, according to the used objects, ecotoxicology is an interdisciplinary complex science, developing on the border of chemical, biological, medical, ecological, environmental, economic, social and legal sciences. It can also be considered as applied environmental science studying the biological effects of anthropogenic ingredients. According to the main objects concerned, the main sections of ecotoxicology are *Toxicant dynamics and Bio toxicology.*

*Toxicant dynamics* considers the migration, transformation and utilization of toxicants according to the characteristics of different environmental areas. There is a great difference in the evaluation of contaminants effects in the laboratory and in the environment. The physical and chemical changes of compounds in the environmental migration lead to the changes of their impact dose that vary in different environments. Therefore, the toxicants in nature often have an indirect effect on biosystems, changing the physical and chemical environment to act upon an indirect effect on the survival of organisms. For the terrestrial environment of great importance to toxicant migration is the temperature-precipitation Dynamics of habitat. In climatology, biogeography and ecology, this dynamics is well characterized by "climatic diagram" ("ombro-thermal" diagram) by Lyubenova [3]. The toxicants emission during the period of drought and semi-drought (calculated on the chart curves) poses an extra risk to the environment, because of the bio systems stress state, limited by temperature – precipitation patterns. The characteristics of different environments and the particular environment are important for the sensitivity of bio systems to toxicant. The toxicity and behavior of contaminant in the environment are also dependent on the concentration, but also to a large extent, on the predominant form (molecules, ions, complexes, etc.) of migration in biotope and of taken by bio systems.

relations for the sustainability of life existence on Earth. The main basis for the development of ecotoxicology is classical toxicology—the research of drugs and chemical compounds effects on man. The modern concept of ecotoxicology is that it is the original part following the classical toxicology, which studies migration, transformation, degradation and utilization of toxic ingredients (with organic, inorganic or organic-mineral chemical nature; with natural biotic or abiotic origin and artificial, mainly anthropogenic origin) in the environment and their impact on Macro- biological systems with different levels of integration as groups of individuals,

The main objects of the ecotoxicological studies are the both: (1) toxic ingredients and their "behaviour" in the five main environments such as air, soil, terrestrial, water (incl. sediments) and biotic and (2) the responses of MBS in nature. The studied toxicants can be: (1) by the chemical nature: organic, inorganic or organic-mineral; (2) by the origin: natural (biotic or abiotic) and artificial (mainly anthropogenic origin); (3) by the toxicity: toxicants in the Black list, toxicants in the Grey list, etc. and (4) by the main environment of circulation: air, water, soil or different bio toxicants. The migration, transformation and degradation of toxic ingredients depend on the internal (endogenous) factors, that are, chemical properties of the toxicant and external (exogenous) factors or features of the environment. The studied MBS at different levels of integration can be: diverse individuals as elements of the population; homogeneous and heterogeneous populations as elements of the communities; heterogeneous communities as elements of the bio cenosis; ecosystem as a functional unity between biotope and bio cenosis; landscape, biome and biosphere formed by a corresponding set of ecosystems and their environment. The responses of MBS also depend on the endogenous factors (level of integration and features of the MBS) and exogenous factors (the characteristics of toxicants and habitats). Therefore, according to the used objects, ecotoxicology is an interdisciplinary complex science, developing on the border of chemical, biological, medical, ecological, environmental, economic, social and legal sciences. It can also be considered as applied environmental science studying the biological effects of anthropogenic ingredients. According to the main objects concerned, the main sections of ecotoxicology are *Toxicant dynamics and Bio*

*Toxicant dynamics* considers the migration, transformation and utilization of toxicants according to the characteristics of different environmental areas. There is a great difference in the evaluation of contaminants effects in the laboratory and in the environment. The physical and chemical changes of compounds in the environmental migration lead to the changes of their impact dose that vary in different environments. Therefore, the toxicants in nature often have an indirect effect on biosystems, changing the physical and chemical environment to act upon an indirect effect on the survival of organisms. For the terrestrial environment of great importance to toxicant migration is the temperature-precipitation Dynamics of habitat. In climatology, biogeography and ecology, this dynamics is well characterized by "climatic diagram" ("ombro-thermal" diagram) by Lyubenova [3]. The toxicants emission during the period of drought and semi-drought (calculated on the chart curves) poses an extra risk to the environment, because of the bio systems stress state, limited by temperature – precipitation patterns. The characteristics of different environments and the particular environment are

population, community, ecosystem, etc. studied in ecology (MBS) [1, 2] and others.

148 Toxicology - New Aspects to This Scientific Conundrum

*toxicology.*

The development of analytical methods is very important in this division for solving the series of toxicological problems. For example, with the appearance of inductively coupled plasma mass spectrometry (ICP/MS) as a method of measurement, it is possible to separate and determinate the toxicity of various forms of each toxicant. Therefore, the chemical and physical measurements for assessing the toxicity of the substances and their forms are important for determining the valid toxic concentration in the bioassay and complementary test system reply.

*Bio toxicology* examines the effects of toxicants on sensitive test systems and further on MBS in nature. For the manifestation of this effects is necessary to consider four main phases in passing of toxicants in bio systems, namely biological absorption and assimilation, metabolism, transport and excretion from the system. At the MBS level, biological uptake and accumulation from the environment (bioaccumulation) in the food chains (biomagnification) and accumulation in different organs or elements of bio system (bioconcentration) can be characterized by calculating several geochemical coefficients [4]: biological absorption coefficient (BAC); relative absorption coefficient, (RAC); acropetal coefficient (AC); temporal absorption coefficient (TAC); litter-mulch coefficient, A; and relative rate of transformation of the organic matter (RRTOM).

There are few main differences between classical toxicology and ecotoxicology: (a) the usage of bio-test, including selected for this purpose; (b) the main objects for acute toxicity measurement are different – *Daphnia spp*., or laboratory rat (c) the reference of induced toxicological effects on sensitive/representative test objects to MBS in nature; (d) the usage of standardized methods and indicators (good laboratory practice) guaranteed the results recognition everywhere, etc. The requirements for the test bio systems are: susceptibility of cultivation and maintenance in laboratory, low-cost, highly responsiveness to the toxicant, mass usage and vast database available, representativeness of the exposed species to MBS, low correlation with other assessments in the same trophic level and strong correlation with a series of changes in ecosystems [1]. Today, in laboratory tests with test objects, most often are reported mortality, reproductive capacity, changes in growth, development, behaviour, biochemical, genetic changes and other.

The MBS are characterized by a complex structural and functional organization and the specificity of the set of internal regulatory mechanisms that support the system in equilibrium, which should be considered in the toxicological effect extrapolation, as well as for assessment of ecosystem health and predicting the risk. Therefore, the models, adequately reflecting the responses of MBS in nature, require the knowledge of structure, function and the mechanisms for ensuring the existence and integrity of MBS and the behaviour of toxicants in the current climate. The main functions and features of MBS have been deeply commented by Lyubenova [5].

The main aim of chapter is to comment the contemporary knowledge and established practice in the usage of bioassays to study the environmental toxicity of ingredients. The acute, chronic, mono-species and multi-species tests are discussed. Moreover, the analytical and biochemical methods for determining the initial damage at the molecular level on acute and chronic exposure are commented, too. The molecular markers (biomarkers) or indicators are very important for the early diagnosis of damages and the interests for new developments are growing steadily.

#### **2. Ecotoxicological testing: contemporary knowledge and gaps**

The ecotoxicological effects of contaminants on bio systems and MBS are developed as sub lethal and lethal responses. The earliest toxicological responses (change in biological systems) are detected at the cellular level. Some of the most important effects are changes in the structural components of the cell membrane (e.g. breach links between proteins and lipids); suppression of certain enzymes (e.g. microsomal enzymes); damage to the whole or partial metabolic changes (e.g. the synthesis of carbohydrates) [6]; changes in DNA correlation, respectively mutations and modification of cell growth [7], etc. At the macro-bio system level, effects related with their structure and functioning can be observed: efficiency of energy utilization and transmission through the food chains [8]; bio-depressant effects (inhibition of growth and reproduction) [9] and bio-stimulant effects (e.g. eutrophication) on population or community [10]; changes in the nature of biological cycle—capacity (the amount of chemical elements involved in biomass per year), intensity (of productive processes, energy transformation and destructive processes), chemistry (determined by the leading elements in the cycle), openness (e.g. balance of import and export); bioaccumulation of toxicants (higher concentration in the biomass than in the surrounding medium) [11]; bio magnification (increasing the concentrations in each higher-trophic level) [12]; bio concentration (accumulation in separate organs or elements of bio-system), etc. In most cases, plants and animals are more exposed to the combined effects of many pollutants simultaneously [13]. The interaction between them may increase or decrease the toxicity of the mixture and hence alter the response of the biological system. The effects on biosystem exposure on two or more toxicants may result in the following combining toxicological responses: supplementary response (e.g. in simultaneous action of two organophosphate compounds) [14]; synergistic (reinforced) response (e.g. response of rat to concomitant ingestion of hepatic toxins – ethanol and carbon tetrachloride) and depressed (antagonistic, reduced) response, when the antagonistic reaction between toxicants exists: for example, chemical (e.g. the toxic effect of Se and Hg) [15], competitive (e.g. the toxic effect of CO), uncompetitive (e.g. the toxic effect of atropine and organophosphorus insecticides), functional (e.g. the toxic effect of barbiturate decreased vascular pressure) and predisposing (e.g. the reduction of organophosphorus insecticides toxicity with piperonyl bioksid by blocking the activity of cytochrome P450, responsible for the metabolism of organophosphates) antagonism. The interactions between toxicants and natural chemicals in the environment result in formation of new molecules or complexes, changing their expected utilization. When toxicity is unknown, the conducting tests use a wide range of concentrations and report of "all or none" response. The dose–response relationship is the tested biological effects to 4–5 toxicant concentrations that cause from 20 to 80% mortality. This value is only representative of acute exposure, not chronic one. The LD50 and ED50 variables are influenced by many factors: behaviour of animals [16], age [17], sex [18], temperature [19], water quality (hardness) [20], pH [21], etc. Nevertheless, the 50% response rate is used, because it is the most reproducible response and can be calculated with high reliability. There are three main types of systems for the contaminants exposure of aquatic organisms: *Flowing-through, Static and Renewal* [1 and others]. Flowing-through systems are preferred for the study of acute toxicity. They are recommended for toxicants with high volatility and pollutants that are unsustainable in water [22 and others]. The static systems are applied mainly in short-term tests (≤96 h with fixed or slowly degradable materials and at a low load (biomass/water volume) of the test organisms. Static systems are used at limited availability of studied pollutant critical load with residual water or receiving toxic effects at critical levels of the components of the test. The renewal systems are applicable in work with small organisms that may be lost in watercourse systems or that are very sensitive to streams. They are also used in the case of limited test material. A *recirculation test* is similar to a static test except that the test solutions and control water are pumped through an apparatus, such as filter, to maintain water quality but not reduce the concentration of test material. The water is returned to the test chamber. This type of test is not routinely used because of uncertainty about the effect of the apparatus (aerator, filter and sterilizer) on the test material [1].

The main aim of chapter is to comment the contemporary knowledge and established practice in the usage of bioassays to study the environmental toxicity of ingredients. The acute, chronic, mono-species and multi-species tests are discussed. Moreover, the analytical and biochemical methods for determining the initial damage at the molecular level on acute and chronic exposure are commented, too. The molecular markers (biomarkers) or indicators are very important for the early diagnosis of damages and the interests for new developments are

The ecotoxicological effects of contaminants on bio systems and MBS are developed as sub lethal and lethal responses. The earliest toxicological responses (change in biological systems) are detected at the cellular level. Some of the most important effects are changes in the structural components of the cell membrane (e.g. breach links between proteins and lipids); suppression of certain enzymes (e.g. microsomal enzymes); damage to the whole or partial metabolic changes (e.g. the synthesis of carbohydrates) [6]; changes in DNA correlation, respectively mutations and modification of cell growth [7], etc. At the macro-bio system level, effects related with their structure and functioning can be observed: efficiency of energy utilization and transmission through the food chains [8]; bio-depressant effects (inhibition of growth and reproduction) [9] and bio-stimulant effects (e.g. eutrophication) on population or community [10]; changes in the nature of biological cycle—capacity (the amount of chemical elements involved in biomass per year), intensity (of productive processes, energy transformation and destructive processes), chemistry (determined by the leading elements in the cycle), openness (e.g. balance of import and export); bioaccumulation of toxicants (higher concentration in the biomass than in the surrounding medium) [11]; bio magnification (increasing the concentrations in each higher-trophic level) [12]; bio concentration (accumulation in separate organs or elements of bio-system), etc. In most cases, plants and animals are more exposed to the combined effects of many pollutants simultaneously [13]. The interaction between them may increase or decrease the toxicity of the mixture and hence alter the response of the biological system. The effects on biosystem exposure on two or more toxicants may result in the following combining toxicological responses: supplementary response (e.g. in simultaneous action of two organophosphate compounds) [14]; synergistic (reinforced) response (e.g. response of rat to concomitant ingestion of hepatic toxins – ethanol and carbon tetrachloride) and depressed (antagonistic, reduced) response, when the antagonistic reaction between toxicants exists: for example, chemical (e.g. the toxic effect of Se and Hg) [15], competitive (e.g. the toxic effect of CO), uncompetitive (e.g. the toxic effect of atropine and organophosphorus insecticides), functional (e.g. the toxic effect of barbiturate decreased vascular pressure) and predisposing (e.g. the reduction of organophosphorus insecticides toxicity with piperonyl bioksid by blocking the activity of cytochrome P450, responsible for the metabolism of organophosphates) antagonism. The interactions between toxicants and natural chemicals in the environment result in formation of new molecules or complexes, changing their expected utilization. When toxicity is unknown, the conducting tests use a wide range of concentrations

**2. Ecotoxicological testing: contemporary knowledge and gaps**

growing steadily.

150 Toxicology - New Aspects to This Scientific Conundrum

**Figure 1.** Percentage distribution of ecotoxicological studies published based on random sampling.

In the random sample of 384 published studies, 535-conducted bio-tests were considered. For the studied period (2010–2016), an exponential increase of published ecotoxicological studies to 2012 can be observed—**Figure 1**. The level is kept in 2013 and the percentage sharp fell in 2014, while in 2015 the published ecotoxicological studies are closed to that in 2014. We do not have compete data for 2016, but it seems that this trend will likely keep. Furthermore, the scientific community is concerned about finding new environmentally acceptable agents and technologies in industry, agriculture and households, which is gradually becoming a priority in the new solutions. No less is the role of environmental legislation, the timely testing of new toxicants and the introduction of regulations and restrictions.

Among the reviewed studies, the tests for toxicity of aquatic environment prevail – 67%, of which these for the toxicity of freshwater are 35%, the terrestrial ones are about 20% and those concerning three environments – 13% (**Figure 2**).

**Figure 2.** Percentage distribution of ecotoxicological studies by media published in random sample.

During the years, the focus of researches has been on the toxicity of different environments, for example, in 2011, prevailed these for the aquatic environment; in 2012, for the terrestrial; in 2013, again for the aquatic; and in 2015, again for the terrestrial (**Figure 3**).

**Figure 3.** Percentage distribution of ecotoxicological studies by media and years published in random sample.

For the research period in the toxicological testing, 25 groups of biological systems have been used as most tests have been performed with crustaceans and fishes, 22% and 20%, respectively (**Figure 4**). Common test objects are also insects (9%), molluscs (9%), algae (8%) and the plants (6%). In the ecotoxicological studies, 61 crustacean species, 51 fish species, 27–17 insect species, molluscs, algae and higher plants have been used (**Figure 5**).

in the new solutions. No less is the role of environmental legislation, the timely testing of new

Among the reviewed studies, the tests for toxicity of aquatic environment prevail – 67%, of which these for the toxicity of freshwater are 35%, the terrestrial ones are about 20% and those

**Figure 2.** Percentage distribution of ecotoxicological studies by media published in random sample.

in 2013, again for the aquatic; and in 2015, again for the terrestrial (**Figure 3**).

**Figure 3.** Percentage distribution of ecotoxicological studies by media and years published in random sample.

During the years, the focus of researches has been on the toxicity of different environments, for example, in 2011, prevailed these for the aquatic environment; in 2012, for the terrestrial;

toxicants and the introduction of regulations and restrictions.

concerning three environments – 13% (**Figure 2**).

152 Toxicology - New Aspects to This Scientific Conundrum

**Figure 4.** Percentage participation of biological groups for ecotoxicological testing in random sample (2010–2016).

**Figure 5.** Participation of biological groups (number of species) for ecotoxicological testing in random sample (2010– 2016).

## **2.1. Ecotoxicological tests for short–term (acute) and continuous (long–term,6 chronic) toxicity**

The accepted types of toxicants impact on exposed biosystem are: acute (high doses and for a short time, typically 24 – 96 h); sub-acute (repeated exposure for one month or less at lower doses than those in the acute exposure); sub chronic (multiple exposure, for 1–3 months) and chronic (exposure for more than 3 months at doses representing about 1/100 to 1/1000 of the acute dose). The exposure intervals definition varies for different biological systems, media and toxicants.

For the period concerned, the studies of acute toxicity clearly prevailed over the chronic tests. In a number of studies for clarifying the actual toxicity, a series of both tests have been performed. The practical application of subacute and sub-chronic tests is low or negligible (**Figure 6**), but the trend of increasing the interest for these tests in 2014 and 2015 is noticeable.

**Figure 6.** Percentage participation of acute and chronic tests in random sample (2010–2016).

*Acute ecotoxicological testing* has two main applications in environmental risk assessment. One of the applications is in conducting the screen test, for example, to determine whether the toxicant is biologically active at test doses for used indicators. The second type of application is the determination of acute toxicity—measuring the dose-response function and determination of LC50/LE50 for a predetermined period. The acute toxicity tests is the first step for detecting the total toxic effects caused by toxicant. Many studies have been dedicated for searching to the most sensitive species to conduct acute tests. The practice shows that there are no universal species, that are the selection of species depends on the type of toxicant and the affected ecosystems. Virtually every hydrobiont is suitable for conducting the acute tests, but one of the most used are *Daphnia magna* [23–26], *Pseudokirchneriella subcapitata* [27, 28]; many mussel species like *Dreissena polymorpha* [7] and *Mytilus edulis* [29]. Fish species are also very often used as test system: *Danio rerio* [30, 31], *Gambusia holbrooki* [32], *Cyprinus carpio* [33, 34], *Oreochromis niloticus*s [35, 36]. The terrestrial test objects include bees [37, 38], *Mus musculus* [39], *Megascops asio* [40], *Podarcis sicula* [41] *Aquila adalberti*, soil invertebrates [42] and other. The bacterial species, especially *Vibrio fischeri*, are also used in many ecotoxicological tests [27, 28, 43] and others. Today the most used plant test objects are different species of algae [44] and duckweed (*Lemna* spp.) [45]. They have a high reproductive capacity and therefore the study includes several generations in a relatively short period. Intensity of photosynthesis [46] and growth [47] are measured at algae or growth [48] at duckweed. Reporting indicators for the photosynthesis intensity are: concentration and ratio of pigments in photosynthetic biomass [49], amount of released oxygen [50], assimilated 14CO2 [51], ATP production [52] and number of cells [53]. Short-term sublethal tests are used to evaluate the toxicity of effluents to aquatic organisms [54], but some authors use terrestrial organisms [38]. These methods are developed by the EPA, and only focus on the most sensitive life stages. The endpoints for these tests include changes in growth, reproduction and survival as NOECs, LOECs, LC50s and EC50s [25] and others. Acute toxicity resulted in abortion rate of eggs and embryonic stages [25], reduced offspring and egg production [55], reduction in hatching success [56], decrease in fecundity [57], decrease in fertility [57], failure of metamorphosis [58], delayed development [59] and abnormalities and deformities in fish larvae [60]. Despite the results cited above, [61] reported no significant effects of the insecticide indoxacarb on the eggs, young and old larval stages and the pupal stage of two species of Trichogramma [62] and observed no negative effects on *Daphnia magna* embryonic development or hatching rate to insecticide Carbaryl up to concentrations almost 1000 times of the median effect concentration (EC50) of neonate survival in acute tests. Furthermore, [63] suggested that adaptation to tolerate PCBs has altered the sensitivity of *Fundulus heteroclitus* to oxidative stress during embryonic development, demonstrating a cost of the PCB resistance adaptation and [64] reported resistance of *Fundulus heteroclitus* from the Atlantic Wood Superfund site on the Elizabeth River to the acute toxicity and teratogenesis caused by polycyclic aromatic hydrocarbons (PAHs) and others.

**2.1. Ecotoxicological tests for short–term (acute) and continuous (long–term,6 chronic)**

The accepted types of toxicants impact on exposed biosystem are: acute (high doses and for a short time, typically 24 – 96 h); sub-acute (repeated exposure for one month or less at lower doses than those in the acute exposure); sub chronic (multiple exposure, for 1–3 months) and chronic (exposure for more than 3 months at doses representing about 1/100 to 1/1000 of the acute dose). The exposure intervals definition varies for different biological systems, media

For the period concerned, the studies of acute toxicity clearly prevailed over the chronic tests. In a number of studies for clarifying the actual toxicity, a series of both tests have been performed. The practical application of subacute and sub-chronic tests is low or negligible (**Figure 6**), but the trend of increasing the interest for these tests in 2014 and 2015 is noticeable.

**Figure 6.** Percentage participation of acute and chronic tests in random sample (2010–2016).

*Acute ecotoxicological testing* has two main applications in environmental risk assessment. One of the applications is in conducting the screen test, for example, to determine whether the toxicant is biologically active at test doses for used indicators. The second type of application is the determination of acute toxicity—measuring the dose-response function and determination of LC50/LE50 for a predetermined period. The acute toxicity tests is the first step for detecting the total toxic effects caused by toxicant. Many studies have been dedicated for searching to the most sensitive species to conduct acute tests. The practice shows that there are no universal species, that are the selection of species depends on the type of toxicant and the affected ecosystems. Virtually every hydrobiont is suitable for conducting the acute tests, but one of the most used are *Daphnia magna* [23–26], *Pseudokirchneriella subcapitata* [27, 28]; many mussel species like *Dreissena polymorpha* [7] and *Mytilus edulis* [29]. Fish species are also very often used as test system: *Danio rerio* [30, 31], *Gambusia holbrooki* [32], *Cyprinus carpio* [33, 34], *Oreochromis niloticus*s [35, 36]. The terrestrial test objects include bees [37, 38], *Mus musculus* [39], *Megascops asio* [40], *Podarcis sicula* [41] *Aquila adalberti*, soil invertebrates [42] and other.

**toxicity**

154 Toxicology - New Aspects to This Scientific Conundrum

and toxicants.

*Chronic ecotoxicological testing* is subjected to determine whether the long-term toxicant exposure that is supposed to be present in the environment, can have a significant detrimental impact on ecosystems. The number of selected species that inhabit the ecosystem is tested to toxicant exposure. The threshold of chronic concentration complies with the reactions of the most sensitive species. In the practice, the following assumptions are made to the chronic toxicity determination: selected individuals are with respective sensitivity to toxicant corresponding to the representative natural groups; chronic threshold concentration set for the most sensitive species, is the starting chronic toxicity for the ecosystem; studied species are the most sensitive to the toxicant in the ecosystem. The chronic tests provide information allowing extrapolation of the effects at the community and ecosystem level [1]. In the chronic tests, the bio-reactions upon exposure to toxicants for a long time are examined [65], often as long as the entire life cycle. After running the assay, the established initial concentration causing chronic sensitivity of the ecosystem is compared with the expected concentration of toxicant in the environment. Effect of severe chronic toxicity can be expected at concentrations exceeding the established in the environment initial or threshold concentrations. For the predicting of toxicants chronic effects, commonly three categories of tests are used: including life cycle [66], including the most sensitive life stages [67] and functional. By the lifecycle tests, the contaminant impact of chronic exposure on reproduction, growth, survival and other indicators of several generations of test organisms are examined. The tests begin with eggs, larvae or juveniles and continue until the reproduction of test organisms. The used toxicant concentrations range from causing strong negative impact to at least one that has no influence on studied indicators (compared to controls). Most commonly aquatic organisms that can complete their life cycle in laboratory are used—algae, invertebrates and others. In these tests, the calculation of survival and fertility by age is conducted according to equations of Lotka or Yuler [1]. The isolated generations in the period of youth and the period of maturity may be also used for these tests. The toxicity tests on the life cycle require considerable time and costs, especially for vertebrates. The tests conducted on the most sensitive life stages are used for studying the contaminants impact of chronic exposure to the survival and growth of eggs [68] and larvae [69] of fish. The indicative tests have been developed with the early – eggs floating on the water surface or eggs laid on the bottom of rivers or estuaries, embryos or larvae. In surface water micro-layer are concentrated heavy metals, detergents, chlorinated oil hydrocarbons, etc. The sediments also accumulate a number of toxicants. The tests start with the exposure of groups of fertilized eggs or embryos through the system for supply of serial concentrations of tested toxicant. The range of concentrations should include substantial effects and lack of impact. The species inhabiting cold water, for example *Salmo gairdneri*, are usually exposed for a period of 330–570 days [1] while inhabitants of warm water, for example zebrafish, are exposed from 30 to 250 days [70]. The parameters of the measurement include survival, growth and teratogenesis. The benefits of testing embryos and larvae are: saving time and money; creating opportunities for the study of larger number of species compared with the life cycle tests; calculated thresholds for chronic toxicity can be extrapolated to many more species for a wide range of areas and trophic levels than the potential in the implementation of testing lifecycle; the needs to conduct these tests due to the insufficient data on the fish toxicity. The concentration of toxicant that causes chronic toxicity effects on eggs, embryos and larvae vary among the same species and among different species. It depends on the duration of the conducted test—stage of the life cycle or the whole life cycle. For example, the studies with small *Salmo gairdneri* fish has the highest degree of sensitivity to six toxicants, while the eggs are relatively resistant, because of the bio–absorption alteration. The early life-staged tests are not considered as valid, if mortality in the control sample is greater than 30% [1]. Some authors published results of conducting tests with eggs [71], fish embryos [28], larvae [72] and early stages of development [24], as through them the potential toxic effect is reflected.

By the *functional tests,* the effects of toxicants on various physiological functions of test objects are studied. The fishes and other aquatic organisms react physiologically and with behavioural changes on toxic exposures. For example, changes are observed in blood chemistry [73], enzymatic activity [74], histology [75], swimming behaviour [76], sensory perception and disease resistance. This testing has some disadvantages: the effect of adaptation to toxicant is absent and the reported effects differ from the real ones; the inability to capture all variation in functional parameter for MBS; the inability to extrapolate the results to MBS. In general, the information is about the relationship between functional individual bio-effects to toxicants and the survival, growth and reproductive capacity of the populations in community. The data for the discussed test categories are used to determine the threshold concentration of the toxicant causing chronic toxicity.

*Bioaccumulation tests* are toxicity tests that can be used for hydrophobic chemicals that may accumulate in the fatty tissue of organisms. The toxicants with low water solubility generally can be stored in the fatty tissue due to the high lipid content in this tissue. The storage of these toxicants within the organism may lead to cumulative toxicity. Some authors report results from bioaccumulation in different tissues and organs like liver, kidney, gills, embryo tissue and accessory glands [60, 77]. Bioaccumulation tests use bio concentration factors (BCF) to predict concentrations of hydrophobic contaminants in organisms [78] and others. The BCF is the ratio of the average concentration of test chemical accumulated in the tissue of the test organism (under steady state conditions) to the average measured concentration in the water.

*Tests with sediments.* At some point, most chemicals and elements originating from both anthropogenic [79] and natural sources [80] accumulate in sediments. For this reason, sediment toxicity can play a major role in the adverse biological effects seen in aquatic organisms, especially those inhabiting benthic habitats [81]. A recommended approach for sediment testing is to apply the Sediment Quality Triad (SQT), which involves simultaneously examining sediment chemistry, toxicity, and field alterations so that more complete information can be gathered [82]. Collection, handling and storage of sediment can have an effect on bioavailability and for this reason standard methods have been developed to suit this purpose [83]. Some ecotoxicological tests for assessing sediments quality are published [84]. The worms [42], clams [85], fish [86] and phytoplankton [87] are mostly used as test objects.

#### **2.2. Mono–species tests and multi–species tests**

juveniles and continue until the reproduction of test organisms. The used toxicant concentrations range from causing strong negative impact to at least one that has no influence on studied indicators (compared to controls). Most commonly aquatic organisms that can complete their life cycle in laboratory are used—algae, invertebrates and others. In these tests, the calculation of survival and fertility by age is conducted according to equations of Lotka or Yuler [1]. The isolated generations in the period of youth and the period of maturity may be also used for these tests. The toxicity tests on the life cycle require considerable time and costs, especially for vertebrates. The tests conducted on the most sensitive life stages are used for studying the contaminants impact of chronic exposure to the survival and growth of eggs [68] and larvae [69] of fish. The indicative tests have been developed with the early – eggs floating on the water surface or eggs laid on the bottom of rivers or estuaries, embryos or larvae. In surface water micro-layer are concentrated heavy metals, detergents, chlorinated oil hydrocarbons, etc. The sediments also accumulate a number of toxicants. The tests start with the exposure of groups of fertilized eggs or embryos through the system for supply of serial concentrations of tested toxicant. The range of concentrations should include substantial effects and lack of impact. The species inhabiting cold water, for example *Salmo gairdneri*, are usually exposed for a period of 330–570 days [1] while inhabitants of warm water, for example zebrafish, are exposed from 30 to 250 days [70]. The parameters of the measurement include survival, growth and teratogenesis. The benefits of testing embryos and larvae are: saving time and money; creating opportunities for the study of larger number of species compared with the life cycle tests; calculated thresholds for chronic toxicity can be extrapolated to many more species for a wide range of areas and trophic levels than the potential in the implementation of testing lifecycle; the needs to conduct these tests due to the insufficient data on the fish toxicity. The concentration of toxicant that causes chronic toxicity effects on eggs, embryos and larvae vary among the same species and among different species. It depends on the duration of the conducted test—stage of the life cycle or the whole life cycle. For example, the studies with small *Salmo gairdneri* fish has the highest degree of sensitivity to six toxicants, while the eggs are relatively resistant, because of the bio–absorption alteration. The early life-staged tests are not considered as valid, if mortality in the control sample is greater than 30% [1]. Some authors published results of conducting tests with eggs [71], fish embryos [28], larvae [72] and early stages of development

156 Toxicology - New Aspects to This Scientific Conundrum

[24], as through them the potential toxic effect is reflected.

causing chronic toxicity.

By the *functional tests,* the effects of toxicants on various physiological functions of test objects are studied. The fishes and other aquatic organisms react physiologically and with behavioural changes on toxic exposures. For example, changes are observed in blood chemistry [73], enzymatic activity [74], histology [75], swimming behaviour [76], sensory perception and disease resistance. This testing has some disadvantages: the effect of adaptation to toxicant is absent and the reported effects differ from the real ones; the inability to capture all variation in functional parameter for MBS; the inability to extrapolate the results to MBS. In general, the information is about the relationship between functional individual bio-effects to toxicants and the survival, growth and reproductive capacity of the populations in community. The data for the discussed test categories are used to determine the threshold concentration of the toxicant For the period concerned, the studies of acute toxicity clearly prevailed over the chronic tests. In a number of studies to clarify the actual toxicity the series of both tests have also been performed. The practical application of subacute and sub-chronic tests is low or negligible (**Figure 6**), but the noticeable trend of increasing the interest for these tests in 2014 and 2015 is observed (**Figure 7**). The analyses carried show that in 2012, 2014 and 2015, the tests with two species as test objects and multi-species ones were applied in most published studies. In 2013, the focus is mainly on tests with communities and multi-species ones.

*Mono-species tests* are appropriate in determining the toxicological effects on individual characteristics of species such as mortality [88], growth [89], reproductive capacity [41], behaviour [38] and other but have limited significance for the consequences on the entire ecosystem from the pollutants impact [36]. The disadvantages of mono-species testing are: the responses of individuals are often not sufficient to extrapolate responses of other (even very close) species and determine the real toxical effects in nature; the identifying of sensitive species or groups to the toxicant is expensive; the influence of indirect effects from intra-population and inter-population relationships on toxicity cannot be observed; the standardized laboratory conditions in conducting mono-species tests are different from the conditions in the biotope [90]. The influence of many additional abiotic and biotic factors is always present in the field effects changing significantly the eco–toxicological response. The mono–species tests have been used for years for the simulation of multi-species effects in ecosystem although the existing inadequacy. Therefore, the results obtained in mono-species tests are more often used in practice and have the same rank of importance to those of multispecies tests enable to assess indirect effects, such as the changes of structure and the functioning of ecosystem (changing in competition, predation, energy flow and circulation of substances, etc.).

**Figure 7.** Percentage participation of tests with different number of test objects used in random sample (2010–2016).

*Multi-species tests.* At every level of biological organization appear new features that could not be recognized even by the most intense and careful studies of low levels. The complexity of MBS makes unacceptable the assumption that the responses received in toxicity tests of various species are applicable for predicting the behaviour of system of integrated species [91]. Therefore, the biological effects within the natural system differ considerably from those in laboratory tests [1]. Only acute toxic effects can be accounted with available methods. Another way to resolve the testing problems is by the identification of sensitive or/and key species or groups of species for MBS, but it is very expensive approach. We also need to bear in mind that in multispecies tests many answers are skipped due to unification of indicators, individuality of responses and statistical unreliability of additional indicators. So, we are faced with the inability to reproduce the experimental results to MBS level due to the variability and specifics of relationships, self-organization and self-regulation of different MBS. In other words, the uniqueness of each MBS leads to the inability for the toxicant assessment unification. The toxic effects extrapolation to ecosystem level requires good description of the biotope conditions and availability of data on the structure and functional processes. The lack of classification and characterization of ecosystem types in relation to specific environmental factors and bio-system features hampers the ecotoxicological research interpretation. For example, the population generally shows a lower sensitivity to pollutants than the individuals do. It seems that environmentally more realistic approach includes monitoring the effects of exposure to toxic impact in natural ecosystems or man-made systems specially designed to resemble fully the characteristics of natural systems (multi-species tests with the key representatives of studied ecosystem – model ecosystems, microcosm, mesocosm, lake-coral systems and others). The most optimal combination of test objects for aquatic ecosystems includes species of algae, crustaceans and fish, reflecting their functional structure. The microecosystem is not an absolute analogue of the natural ecosystem, but a small system with sufficient complexity that could allow realistic ecological study of the typical characteristics of ecosystem in nature. Their application to environmental toxicology has been of interest, where fate and behaviour of contaminants markedly modify the exposure of biota to them and hence the environmental hazard [1]. In the aquatic micro-cosmology, there are two major achievements even in the last century: the ecosystem model and the food chain model. The ecosystem model is used to study the inherent ecosystem properties and functions in the level of integration—cycling of substances, energy flow and homeostasis. The food chains model is applied in studying the relationships predator-prey, environmental efficiency in the transmission of energy in trophic levels, etc. The experiments with food chains model is simpler and easier to manage compared to experiments with ecosystem model. The main problem is in selecting the appropriate microcosm types for different studies. Many species tests include a set of standard reproductive tests on daphnia, where the dose–response relationship is determined depending on the supply of nutrients [1]; large – (CEPEX, 1300 m3 ); medium – (10–150 m3 ) and small-sized (1.4 m3 ) bags or tanks is used for the isolation of communities living in the open sea. Usually the larger the system is, the longer it will operate and the environmental conditions, community structure and functioning will look more like natural. The artificial macro-bio-systems differ by the nature of communities; by the period of submission of the matter—long and short duration, also by using materially closed systems. The classical example is Taub test [1]. He performed to some degree a standardized toxicity test with 24 identical 3.6 1 containers that are "infected" with a total of 10 algal species, five animal species and unknown set of bacteria. The duration of the experiment was 60 days due to deterioration of the community in the absence of normal biological cycle. In Kersting classical test [1], the lack of mineral circle was overcome. He has developed a microcosm with 151 Compartments, in which primary production, secondary production and decomposition are separated to prevent overconsumption. The balance between production and decomposition in this type of microcosm proves its sustainability for months or even years in relatively stable conditions, which corresponds to the "ecological temporal periods". These types of models that are designed to resemble certain types of natural systems (isomorphic models) usually include sedimentary part and benthic invertebrates and operate under watercourse conditions. In most cases, it is expected the water, sediments and biota to be modelled from the site. There are designed different tanks in size (560 1 or 13 m3 ) with a variable depth to external structures that are best described as model flows, model channels and model lakes of various sizes. These test systems can combine the evaluation of biological effects with studies on transformation of pollutants in the environment. Test systems modelling the changes of interactions between populations under the chemical stress were also developed in the last century. The tests of Cairns and Lundgren [1], studying the interactions of algae and daphnia populations under chemical stress, are well known [1]. The usage of a battery of bioassays involving different species at different trophic levels is an efficient and essential tool for predicting environmental hazards to the aquatic ecosystem. For example [85], the adverse effects of wastewater treatment plants (WWTPs) on sediment quality at the Bay of Cádiz (SW, Spain) were investigated using a battery of acute bioassays and chemical contamination. The author concluded that the test may provide

indirect effects, such as the changes of structure and the functioning of ecosystem (changing

**Figure 7.** Percentage participation of tests with different number of test objects used in random sample (2010–2016).

*Multi-species tests.* At every level of biological organization appear new features that could not be recognized even by the most intense and careful studies of low levels. The complexity of MBS makes unacceptable the assumption that the responses received in toxicity tests of various species are applicable for predicting the behaviour of system of integrated species [91]. Therefore, the biological effects within the natural system differ considerably from those in laboratory tests [1]. Only acute toxic effects can be accounted with available methods. Another way to resolve the testing problems is by the identification of sensitive or/and key species or groups of species for MBS, but it is very expensive approach. We also need to bear in mind that in multispecies tests many answers are skipped due to unification of indicators, individuality of responses and statistical unreliability of additional indicators. So, we are faced with the inability to reproduce the experimental results to MBS level due to the variability and specifics of relationships, self-organization and self-regulation of different MBS. In other words, the uniqueness of each MBS leads to the inability for the toxicant assessment unification. The toxic effects extrapolation to ecosystem level requires good description of the biotope conditions and availability of data on the structure and functional processes. The lack of classification and characterization of ecosystem types in relation to specific environmental factors and bio-system features hampers the ecotoxicological research interpretation. For example, the population generally shows a lower sensitivity to pollutants than the individuals do. It seems that environmentally more realistic approach includes monitoring the effects of exposure to toxic impact in natural ecosystems or man-made systems specially designed to resemble fully the characteristics of natural systems (multi-species tests with the key representatives of studied ecosystem – model ecosystems, microcosm, mesocosm, lake-coral systems and others). The most optimal combination of test objects for aquatic ecosystems

in competition, predation, energy flow and circulation of substances, etc.).

158 Toxicology - New Aspects to This Scientific Conundrum

complementary information for diagnose of environmental factors that can impair aquatic communities. In other study [92], a battery of marine and freshwater species representing different trophic levels was used, and compared the bioassay of sensitivity levels to pharmaceutical residues of three antidepressants (fluoxetine, sertraline and clomipramine). The bioassays like the algal growth inhibition test (*Skeletonema marinoi* and *Pseudokirchneriella subcapitata*), the micro-crustacean immobilization test (*Artemia salina* and *Daphnia magna*), development and adult survival tests on *Hydra attenuata*, embryotoxicity and metamorphosis tests on *Crassostrea gigas*, and in vitro assays on primary cultures of *Haliotis tuberculata* hemocytes were selected. The importance of test battery usage showing the difference in sensitivity between bioassays hence high interspecies variability in EC50–values was underlined. The battery of bioassays and representative aquatic organisms (*Vibrio fischeri,* microalgae *Pseudokirchneriella subcapitata* and invertebrate *Daphnia magna*) for assessing the acute toxicity of water-extractable fractions of biochar-amended soil was published [93]. Based on the obtained results, the authors suggested that the battery of rapid and cost-effective aquatic bioassays that account for ecological representation can complement analytical characterization of biochar-amended soils and risk assessment approaches for surface and groundwater protection. The battery of bioassays was used [94] to assess the impact along a river due to a leak of effluent from an Installation of Cleansing and Uranium Recovery (Tricastin, France) and provided an estimation of exposure conditions that occurred along the river. The acute toxicity of the effluent was evaluated and compared to the toxicity of uranium nitrate in bioassays using *Chlamydomonas reinhardtii*, *Daphnia magna*, *Chironomus riparius* and *Danio rerio*.

The dysfunction occurring in plant communities and its effects on the plant populations structure and functioning is very well studied, especially for systems with poor species composition and simple structure. The pioneer studies have been published [1]. The imbalance is defined as a sudden change of the resource base that led to a clearly visible response. The hierarchical series of responses to these impacts can be predicted. The responses of the plant may influence the plant populations—reproduction, density spatial structure, rate of growth, mortality, body age, genetic variations, interspecies connections, etc. The different responses of plant populations can lead to major change of plant community as species composition, species richness, distribution of included genera, succession processes, etc. At the ecosystem level, these changes affect the primary productivity, the intensity of respiration, the intensity of mineralization and other functional processes. These responses depend on the type, frequency, intensity, duration and heterogeneity of dysfunctions. In some cases, we can evaluate the different obtained effects to the intensity and combination of impacts in models of vegetation structure and dynamics changes. By the comparison between the responses of exposed plant communities and the responses of untreated ones that grow on compatible soil types under similar topography and climate, the imbalance can be evaluated. Further, it would be possible to find a correlation of the results from laboratory tests, such as root growth, the growth of algae in soils or soil extracts, with actual plant data. There are some attempts to create models and study the complex toxicity on terrestrial ecosystems, but they are mostly with cultural ecosystems. For example, a model toxicological investigation of cultural plantsoil complex treated with wastewater have been published [95, 96]. Today, the conducting ecotoxicological studies with model ecosystems are common practice in the aquatic toxicology. While it can be considered that to some degree the problems associated with the study of the toxicological effects in the aquatic toxicology are resolved, this is not the case in terrestrial toxicology.

complementary information for diagnose of environmental factors that can impair aquatic communities. In other study [92], a battery of marine and freshwater species representing different trophic levels was used, and compared the bioassay of sensitivity levels to pharmaceutical residues of three antidepressants (fluoxetine, sertraline and clomipramine). The bioassays like the algal growth inhibition test (*Skeletonema marinoi* and *Pseudokirchneriella subcapitata*), the micro-crustacean immobilization test (*Artemia salina* and *Daphnia magna*), development and adult survival tests on *Hydra attenuata*, embryotoxicity and metamorphosis tests on *Crassostrea gigas*, and in vitro assays on primary cultures of *Haliotis tuberculata* hemocytes were selected. The importance of test battery usage showing the difference in sensitivity between bioassays hence high interspecies variability in EC50–values was underlined. The battery of bioassays and representative aquatic organisms (*Vibrio fischeri,* microalgae *Pseudokirchneriella subcapitata* and invertebrate *Daphnia magna*) for assessing the acute toxicity of water-extractable fractions of biochar-amended soil was published [93]. Based on the obtained results, the authors suggested that the battery of rapid and cost-effective aquatic bioassays that account for ecological representation can complement analytical characterization of biochar-amended soils and risk assessment approaches for surface and groundwater protection. The battery of bioassays was used [94] to assess the impact along a river due to a leak of effluent from an Installation of Cleansing and Uranium Recovery (Tricastin, France) and provided an estimation of exposure conditions that occurred along the river. The acute toxicity of the effluent was evaluated and compared to the toxicity of uranium nitrate in bioassays using *Chlamydomonas reinhardtii*, *Daphnia magna*, *Chironomus riparius* and *Danio rerio*.

160 Toxicology - New Aspects to This Scientific Conundrum

The dysfunction occurring in plant communities and its effects on the plant populations structure and functioning is very well studied, especially for systems with poor species composition and simple structure. The pioneer studies have been published [1]. The imbalance is defined as a sudden change of the resource base that led to a clearly visible response. The hierarchical series of responses to these impacts can be predicted. The responses of the plant may influence the plant populations—reproduction, density spatial structure, rate of growth, mortality, body age, genetic variations, interspecies connections, etc. The different responses of plant populations can lead to major change of plant community as species composition, species richness, distribution of included genera, succession processes, etc. At the ecosystem level, these changes affect the primary productivity, the intensity of respiration, the intensity of mineralization and other functional processes. These responses depend on the type, frequency, intensity, duration and heterogeneity of dysfunctions. In some cases, we can evaluate the different obtained effects to the intensity and combination of impacts in models of vegetation structure and dynamics changes. By the comparison between the responses of exposed plant communities and the responses of untreated ones that grow on compatible soil types under similar topography and climate, the imbalance can be evaluated. Further, it would be possible to find a correlation of the results from laboratory tests, such as root growth, the growth of algae in soils or soil extracts, with actual plant data. There are some attempts to create models and study the complex toxicity on terrestrial ecosystems, but they are mostly with cultural ecosystems. For example, a model toxicological investigation of cultural plantsoil complex treated with wastewater have been published [95, 96]. Today, the conducting ecotoxicological studies with model ecosystems are common practice in the aquatic toxicology. *Tests with nanoparticles* Nanoparticles represented by a group of toxicants as TiO2 [97], ZnO [44], carbon nanomaterials (CNMs) [98], core-shell copper oxide [99], silver (AgNPs) [49] and others are tested in ecotoxicology. Mainly, tests with aquatic organisms are conducted like algae [97– 99], plants and invertebrates [97].

*Biomarkers* The most used biomarkers is the activity of antioxidant enzymes like catalase (CAT) [100], superoxide dismutase (SOD) [101], glutathione S-transferase (GST) [102], glutathione peroxidase (GPX) [101] and glutathione reductase (GR) [101] or their gene expression. Many authors use Hsp70 (heat shock proteins) and their expression for determining the toxicity of a pollutant [102, 103]. When using fish as test object, the haematological parameters are often applied as biomarkers [104]. Behavioural biomarkers are applied for frog tadpoles [105], clams [106]; fish [31], cladocerans [107]. When using plants as test object, cell viability (mitochondrial activity) and plant physiology (chlorophyll) are used as biomarkers [108].

## **3. About the studies of ecosystem health (ecosystem diagnosis)**

The evaluation methods for ecosystems health assessment are usually based either on *risk assessment* or on *bio assessment* [1]. Most studies attach importance of *risk assessment*, but it is appropriate when the effect caused by known toxicant from one or more known sources with relatively high emissions and expected acute effect. The risk assessment focuses on the chemical composition, the impact of environmental toxicity and laboratory data. The acute tests with test object definitions as well as the available kits usually are used. The data of plants and soil invertebrates can be used to study the response of the short exposure, especially when impacts were made at regular intervals. The upper layer of the soil (5 cm) and plants (root, leaf and stem) are collected for the acute toxicity testing. It is essential also to measure physiological parameters—respiration, photosynthesis, pigments as well as microbial communities indexes. The short response of soil microbial community is also suitable indicator for this review—for example, the intensity of soil respiration of exposed and unexposed soil. The studies about the ecosystem's risk assessment using GIS, aerospace technologies and calculation of State Vector also were published [109–113 and others].

*Bio assessment* is applied for the complex effects of mixed toxicants or for low non-specific toxicity similar to chronic effects. The assessment focuses on ecosystem characteristics, factors causing stress and their importance and usage of measurements and models for chronic effects assessment. Bio assessment includes micro- and macro-research to perform controlled tests for ecosystem under impacts. It is necessary to know the characteristics of studied ecosystems and principles of their self-management and self-control. The bio-assessment of ecosystem state is based on the results of tests series with "critical" ecosystem components for chemical, physical and biological effects on ecosystems. There have been published 44 different characteristics important for the bio-assessment of ecosystem state and the eight of them are identified as critical ones of varying importance for different ecosystems. These ecosystem characteristics are as follows: (1) The features of the habitat to maintain biodiversity and reproduction of organisms; (2) The phenotypic and genotypic diversity of organisms; (3) The length of food chain, supported by biotope; (4) The determining biological production level of active and stored nutrients in biotope; (5) The features of biological turn-over for maintaining the ecosystem existence; (6) The energy flux to maintain the trophic structure; (7) The set of feedbacks for self-regulation and (8) The capacity to regulate the toxic effects, including capacity for biological absorption, metabolism and decomposition of toxicant, linking with the anthropogenic influences buffering. The importance of every critical characteristic depends on the ecosystem geographical location (i.e. eco region) and whether the system is aquatic or terrestrial. Some authors published bio assessments for different ecosystems, by selection of a set of indexes for the noticed critical characteristics [114–117]. The complex of 44 characteristics, even the complex of eight of them, the variation in the relative importance of each of them for the different ecosystems and the lack of standardized indices and methods for each of them, makes the representative assessment unlikely at each case. The representative assessment of ecosystem can only be done with a few "standard" test systems types. Today the scientific community makes efforts to resolve these methodological problems, not only in relation to ecosystem diagnosis, but also to assess the ecosystem capacity, assets and services [118].

The need of test systems classification leads to the publication of a set of investigations in the last century. For example, the classification [1] is based on the following criteria: environment (air, water and soil); time of exposure (long, medium, occasionally, etc.); concentration of toxicant (mg/l, mg/m3 ); used organisms (bacteria, fish, mammals, plants, etc.); type of exposure (through food, air, dermal, etc.); the effects on test objects (genetic, toxic, bio-accumulation, etc.); measuring methods; test type (common, standard, experienced, screening); requirements for variability, accuracy and precision of values and technical requirements for personnel and laboratory equipment. The developed protocols, however, require significant modifications depending on the type of ecosystem and environmental factors, the objectives of the study and more mentioned in the specific dynamic action analyses (SDA). To assess the possible effects on ecosystem level, the responses of dominant species are usually investigated on a set of sample plots. The mostly used indicators are grouped as indicators of plant community, plant chemistry (major cations, nitrogen, phosphorus, iron, zinc, etc.), aboveground and water insects (some populations of *Homeoptera* are extremely sensitive to changes in the chemical and species composition of communities), soil invertebrates, soil chemistry (indicators for mineralization potential of organic nitrogen and phosphorus, variable cations, pH, inorganic nitrogen, total nitrogen, organic and inorganic phosphorus), water chemistry, organic matter decomposition (the activity of heterotrophic microorganisms and species of class Arthropoda, etc. [119, 120 and others]. A lot of plant community indicators and indices are applied at ecosystem level as plant species composition and density (number/m2 ); plant biomass, separated into herbaceous and woody, above- and underground, live and dead; the average height of the stems; specific leaf mass (SLM); stalk weight; total dry weight (W); total leaf area (Le), etc. The relative indexes are also calculated as: the relative growth [Rw = (1/W)·(dW/dT]; relative leaf area [Re = (1/La)·(dLa/dT)]; leaf ratio [F = La/W]; full leaf evaluation [E = (1/La)· (dW/dT)] and others. Soil invertebrates as earthworms, spiders and nematodes are sufficiently sensitive to the quantity and quality of plant roots and plant detritus and can influence or be influenced by microbial populations. This group of consumers is identified as the potential regulator of the decomposition and production processes. The macro arthropods and earthworms are associated with the fragmentation of detritus, while the micro arthropods and nematodes are more important for the micro-organisms populations. Earthworms are a standard system for the bio assessment. The soil nematodes and the species of *Scarabaeidae* family are linked to net primary production and are sensitive to changes in plant chemistry. The number and trophic composition of soil invertebrates are measured together with the biomass of plant roots and rhizomes and dead biomass. The plant carbon distribution changes in stress are also a good indicator for toxicity. The data for a living and dead biomass in relation with the number and composition of soil invertebrates provides possibility for ecosystem state assessment. Usually the number of earthworms and large arthropods is measured in spring and autumn using soil samples of 0.1 m2 and 30 cm depth. The micro arthropods and nematodes are measured by mechanical separation of soil samples.

as critical ones of varying importance for different ecosystems. These ecosystem characteristics are as follows: (1) The features of the habitat to maintain biodiversity and reproduction of organisms; (2) The phenotypic and genotypic diversity of organisms; (3) The length of food chain, supported by biotope; (4) The determining biological production level of active and stored nutrients in biotope; (5) The features of biological turn-over for maintaining the ecosystem existence; (6) The energy flux to maintain the trophic structure; (7) The set of feedbacks for self-regulation and (8) The capacity to regulate the toxic effects, including capacity for biological absorption, metabolism and decomposition of toxicant, linking with the anthropogenic influences buffering. The importance of every critical characteristic depends on the ecosystem geographical location (i.e. eco region) and whether the system is aquatic or terrestrial. Some authors published bio assessments for different ecosystems, by selection of a set of indexes for the noticed critical characteristics [114–117]. The complex of 44 characteristics, even the complex of eight of them, the variation in the relative importance of each of them for the different ecosystems and the lack of standardized indices and methods for each of them, makes the representative assessment unlikely at each case. The representative assessment of ecosystem can only be done with a few "standard" test systems types. Today the scientific community makes efforts to resolve these methodological problems, not only in relation to ecosystem diagnosis, but also to assess the ecosystem capacity, assets and services [118].

The need of test systems classification leads to the publication of a set of investigations in the last century. For example, the classification [1] is based on the following criteria: environment (air, water and soil); time of exposure (long, medium, occasionally, etc.); concentration of

(through food, air, dermal, etc.); the effects on test objects (genetic, toxic, bio-accumulation, etc.); measuring methods; test type (common, standard, experienced, screening); requirements for variability, accuracy and precision of values and technical requirements for personnel and laboratory equipment. The developed protocols, however, require significant modifications depending on the type of ecosystem and environmental factors, the objectives of the study and more mentioned in the specific dynamic action analyses (SDA). To assess the possible effects on ecosystem level, the responses of dominant species are usually investigated on a set of sample plots. The mostly used indicators are grouped as indicators of plant community, plant chemistry (major cations, nitrogen, phosphorus, iron, zinc, etc.), aboveground and water insects (some populations of *Homeoptera* are extremely sensitive to changes in the chemical and species composition of communities), soil invertebrates, soil chemistry (indicators for mineralization potential of organic nitrogen and phosphorus, variable cations, pH, inorganic nitrogen, total nitrogen, organic and inorganic phosphorus), water chemistry, organic matter decomposition (the activity of heterotrophic microorganisms and species of class Arthropoda, etc. [119, 120 and others]. A lot of plant community indicators and indices are applied at

ecosystem level as plant species composition and density (number/m2

separated into herbaceous and woody, above- and underground, live and dead; the average height of the stems; specific leaf mass (SLM); stalk weight; total dry weight (W); total leaf area (Le), etc. The relative indexes are also calculated as: the relative growth [Rw = (1/W)·(dW/dT]; relative leaf area [Re = (1/La)·(dLa/dT)]; leaf ratio [F = La/W]; full leaf evaluation [E = (1/La)· (dW/dT)] and others. Soil invertebrates as earthworms, spiders and nematodes are sufficiently

); used organisms (bacteria, fish, mammals, plants, etc.); type of exposure

); plant biomass,

toxicant (mg/l, mg/m3

162 Toxicology - New Aspects to This Scientific Conundrum

For the forest ecosystem health assessment, the widespread indicator is defoliation that can do possible to calculate the ratio of damage of ecosystem: C = [∑(n·k)/NK]·100, where n is number of trees with respective scores of defoliation (first to fifth score); N—the total number of trees; K – maximum score on the scale. The forest ecosystems are considered to be damaged at C > 30% [121] Percentage of defoliation is determined by sight with guidance, where the habitus of crowns with different rates of defoliation for each tree species is given.

*Dendro-chronological analysis for ecosystem health assessment.* By the dendrochronological analysis, the impact of external factors (including contaminants) on the radial growth of stems may be determined. Depending on the change of these variables, the characteristic pattern of the tree ring series was formed. The pattern includes: successively alternating narrow and wide rings of lighter or darker wood; changes in the density of the tree rings; change in the ratio between early and late wood, changes in the chemical, cytological and histological characteristics of tree rings, etc. The year with changes in growth or with special annual ring, is named *different special year*. The different special years are very important in cross-dating and in identifying the age of trees, as well as the time of stressed events by climatic factors, pollution, disease, pests, etc. The samples are taken with Presler's driller on 1–1.5 m height of the stem and placed in special templates. They are measured by LINTAB™. Through the statistical processing of obtained series of values, the influence of considered stressors or bio assessment can be performed [121]. The main indicator for dendro-chronogical, especially dendrochemical analysis, is the growth index (It). It is the ratio between measured and calculated value for tree ring (Wt/Gt) by the best reflected to the course of tree stem growth trend (R2 > 0.45). Thus, the influence of age on the growth is eliminated and the environmental information in the rings is enhanced. The analysis of content of chemical elements in annual rings and its dynamics can provide valuable information about changes in the environmental toxicity. There is information for over 70 chemical elements that can be absorbed from the soil by root system, also through the bark or caught by the leaves and moved to the xylem. Many authors have found that the vertical transport of nutrients thousand times exceeds the horizontal one, that is, radial migration of elements in annual rings is minimal, because they form the insoluble complexes [1, 3, 111 and others]. Therefore, the content of chemical elements in the wood gives a general picture of the environmental factors influenced on tree species for their lifetime. The chemical memory of annual rings also can be used to estimate the changes in the environmental toxicity. Recently, the set of dendrochronological indicators have been developed for the forest state assessments: number of eustress periods; their duration, frequency and depth; eustress years (unfavourable climatic type of years), reactive tree functional types and eustress-climatic predictive patterns [122–124]. The authors perceive eustress as a repeating state of restricted radial growth rate of tree stems within a period of one or multiple years and caused by unfavourable environmental factors. This state encompasses the numerous other reactions of tree species. The level of radial stem growth (or tree ring width) is the main parameter that the developed holistic approach operates with, as well as the growth index, which is the main indicator for the statistical determination of low growth threshold (categorized as eustress). The study of the forest ecosystem state is based on the assessment of eustress depth (A) – = <sup>1</sup> ∑ = 1 (1 − ), duration (D) – the number of adjacent eustress years, and frequency (F) – the number of stress years for a period of 100 years, and the creation of eustress nomenclature by five-graded scales. The performance evaluation of eustress in particular localities allows the expression of reactive functional type of tree species. For example, functional type F4D5A4 means that in particular locality the typical for trees of that species are frequent, very long and deep eustresses and that "forest behaviour" puts the existence of the forest under some risk. For the analysis of eustress based on the periods with limited growth, SP-PAM 2.0 software has been developed [125]. Thus, these analyses can be applied for the forest ecosystem state assessment.

Nowadays, *the kits* are widely used for short-, long-term and risk ecotoxicological assessment, as well as for ecosystem health assessment and for quality monitoring of water and wastewater, because they are rapid, sensitive and cost-effective way [126–128]; determining the impact of bio-toxins produced by blue-green algae [129–131], chemicals for mutagenicity [132], chemicals and wastes released in aquatic, terrestrial environments and sediments [127]. Different model organisms are used like algae, aquatic invertebrates, bacteria and plants.

## **4. Conclusion**

New synthetic chemicals are recorded each year and the legislation in countries requires the immediate conduction of the both – toxicological and ecotoxicological testing. The scale of the potential ecological impacts on the environment and biota requires fast and accurate assessments of toxicological effects. The practical importance of ecotoxicology for the existence and functioning of the MBS is constantly growing. The toxicity may be different for different species in the ecosystem and for the same species in different ecosystems. Furthermore, toxicants do not only directly affect the biological system being evaluated, but may have an indirect negative effect on it, altering both abiotic and biotic parameters in the ecosystem. The various populations of the same species under different environmental conditions will respond differently to a given concentration of toxicant. In ecotoxicology practice, the number of species is used as test objects, and the results are extrapolated to all groups of organisms in the ecosystem. The variation in size, physiology, evolution, ontogeny and geographical distribution are some of the important parameters that usually do not fit exactly. However, some of the basic tests have demonstrated its great importance in the understanding of contaminants effects on the environment. The series of variables must be considered for the realistic assessment of environmental toxicity and MBS state. The reported sublethal effects often refer to changes in the structure of MBS that can lead to their degradation. A greater variation in the responses of individuals, populations and ecosystems observed in nature are compared with these reported under laboratory conditions, due to the mutual influence. This fact requires the more intensive usage of multi-testing systems—micro- and mesocosms and new developments. The analysis of situation and problems of ecotoxicological testing makes it possible to outline the directions in which to focus future efforts. They are related to the search of sensitive species for acute and risk testing, developing of new biomarkers and kits, especially for the study of terrestrial toxicity, formation of model systems (micro- and mesocosms) by key members of the ecosystem trophic network for multi-species testing and modelling the toxic effects at MBS level, which is especially true for the terrestrial ecotoxicology.

## **Author details**

complexes [1, 3, 111 and others]. Therefore, the content of chemical elements in the wood gives a general picture of the environmental factors influenced on tree species for their lifetime. The chemical memory of annual rings also can be used to estimate the changes in the environmental toxicity. Recently, the set of dendrochronological indicators have been developed for the forest state assessments: number of eustress periods; their duration, frequency and depth; eustress years (unfavourable climatic type of years), reactive tree functional types and eustress-climatic predictive patterns [122–124]. The authors perceive eustress as a repeating state of restricted radial growth rate of tree stems within a period of one or multiple years and caused by unfavourable environmental factors. This state encompasses the numerous other reactions of tree species. The level of radial stem growth (or tree ring width) is the main parameter that the developed holistic approach operates with, as well as the growth index, which is the main indicator for the statistical determination of low growth threshold (categorized as eustress). The study of the forest ecosystem state is based on the assessment of eustress depth (A) –

– the number of stress years for a period of 100 years, and the creation of eustress nomenclature by five-graded scales. The performance evaluation of eustress in particular localities allows the expression of reactive functional type of tree species. For example, functional type F4D5A4 means that in particular locality the typical for trees of that species are frequent, very long and deep eustresses and that "forest behaviour" puts the existence of the forest under some risk. For the analysis of eustress based on the periods with limited growth, SP-PAM 2.0 software has been developed [125]. Thus, these analyses can be applied for the forest ecosystem state

Nowadays, *the kits* are widely used for short-, long-term and risk ecotoxicological assessment, as well as for ecosystem health assessment and for quality monitoring of water and wastewater, because they are rapid, sensitive and cost-effective way [126–128]; determining the impact of bio-toxins produced by blue-green algae [129–131], chemicals for mutagenicity [132], chemicals and wastes released in aquatic, terrestrial environments and sediments [127]. Different

New synthetic chemicals are recorded each year and the legislation in countries requires the immediate conduction of the both – toxicological and ecotoxicological testing. The scale of the potential ecological impacts on the environment and biota requires fast and accurate assessments of toxicological effects. The practical importance of ecotoxicology for the existence and functioning of the MBS is constantly growing. The toxicity may be different for different species in the ecosystem and for the same species in different ecosystems. Furthermore, toxicants do not only directly affect the biological system being evaluated, but may have an indirect negative effect on it, altering both abiotic and biotic parameters in the ecosystem. The various populations of the same species under different environmental conditions will respond differently to a given concentration of toxicant. In ecotoxicology practice, the number of species is used as

model organisms are used like algae, aquatic invertebrates, bacteria and plants.

), duration (D) – the number of adjacent eustress years, and frequency (F)

= <sup>1</sup>

∑ = 1

assessment.

**4. Conclusion**

(1 −

164 Toxicology - New Aspects to This Scientific Conundrum

Mariyana Lyubenova\* and Silvena Boteva

\*Address all correspondence to: ryana\_l@yahoo.com and ryann@abv.bg

Department of Ecology and Environmental Protection, Faculty of Biology, Sofia University "St. Kliment Ohridski", Sofia, Bulgaria

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10.4172/2161-1009.1000e139


## **A Review of Cyanogenic Glycosides in Edible Plants A Review of Cyanogenic Glycosides in Edible Plants**

Islamiyat Folashade Bolarinwa, Moruf Olanrewaju Oke, Sulaiman Adebisi Olaniyan and Adeladun Stephen Ajala Islamiyat Folashade Bolarinwa, Moruf Olanrewaju Oke, Sulaiman Adebisi Olaniyan and Adeladun Stephen Ajala

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

http://dx.doi.org/10.5772/64886

#### **Abstract**

Cyanogenic glycosides are natural plant toxins that are present in several plants, most of which are consumed by humans. Cyanide is formed following the hydrolysis of cyanogenic glycosides that occur during crushing of the edible plant material either during consumption or during processing of the food crop. Exposure to cyanide from unintentional or intentional consumption of cyanogenic glycosides may lead to acute intoxications, characterized by growth retardation and neurological symptoms resulting from tissue damage in the central nervous system (CNS). Processing methods can detoxify cyanogenic glycosides and reduce the risk of cyanide poisoning. The efficiency of cyanide removal, however, depends on the processing technique employed and the extent of processing. Processing operations such as fermentation, boiling/ cooking, and drying, applied to process food‐containing cyanogenic glycosides have been reported to reduce cyanide content to acceptably safe levels. The present review discusses the level of cyanogenic glycosides in specific plant foods, health implications of consuming cyanogenic plants and effect of various processing method on cyanogenic glycosides with updated information gathered from the published reports on cyano‐ genic glycosides.

**Keywords:** cyanogenic glycosides, cyanide, toxins, processing, cyanide poisoning, food safety

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

## **1. Introduction**

Food plants contain many substances that can pose potential risks to consumers, and one of these types of substances is cyanogenic glycosides. Cyanogenic glycosides are chemical compounds contained in foods that release hydrogen cyanide when chewed or digested. The act of chewing or digestion leads to hydrolysis of the substances, causing cyanide to be released [1]. Cyanogenic glycosides are common in certain families such as the Fabaceae, Rosaceae, Leguminosae, Linaceae, and Compositae, and identification of their constituents is a useful tool for informative taxonomic markers [2]. There are approximately 25 known cyanogenic glycosides and these are generally found in the edible parts of plants, such as apples, apricots, cherries, peaches, plums, quinces, particularly in the seed of such fruits. The chemicals are also found in almonds, stone fruit, pome fruit, cassava, bamboo shoots, linseed/flaxseed, lima beans, coco yam, chick peas, cashews, and kirsch [3, 4]. Other food products that may contain cyanogenic glycosides include some food ingredients with flavoring properties such as ground almonds powder or paste, marzipan, stone fruit, and alcoholic drinks made from stone fruits. These foods therefore represent potential sources of hydrogen cyanide [5].

The toxicity of cyanogenic glycosides and their derivatives is dependent on the release of hydrogen cyanide. Toxicity may result in acute cyanide poisoning and has also been implicated in the etiology of several chronic diseases [6, 7]. Cyanide toxicity can occur in animal including humans at doses between 0.5 and 3.5 mg HCN per kilogram body weight. Symptoms of cyanide toxicity in humans have been reported to include vomiting, stomach ache, diarrhea, convulsion, and in severe cases death [5]. Children are particularly at risk because of their smaller body size [1].

The toxicity of cyanogenic glycosides is associated with their ability to be hydrolyzed either spontaneously or in the presence of enzyme to produce cyanide as end products of their hydrolysis. Thus, toxic levels of cyanogenic glycosides are estimated in terms of the quantity of free cyanide generated following hydrolysis. This makes it difficult to estimate total cyanogenic glycosides in diet. Hence, levels of amygdalin (the most common cyanogenic glycosides in fruits) reported in literature are inconsistent [8]. Although the level of cyanide up to 10ppm was reported by World Health Organization (WHO) to be safe for cassava flour [9]. Lack of quantitative toxicological test and epidemiological information makes it difficult to establish safe level of cyanogenic glycosides intake in many foods.

Cyanogenic glycosides are a group of nitrile‐containing plant secondary compounds that yields cyanide (cyanogenesis) following their enzymatic breakdown. Cyanogenic glycosides occur in at least 2000 plant species, of which a number of species are used as food. They are amino‐acid‐derived constituents of plants produced as secondary metabolites [5]. Despite great deal of structural diversity in cyanogenic glycosides, almost all of them are believed to be derived from only six different amino acids L‐valine, L‐isoleucine, L‐leucine, L‐phenylala‐ nine, or L‐tyrosine, and cyclopentenyl‐glycine (a nonprotein amino acid) [2, 10]. Cyanogenic glycosides play pivotal roles in organization of chemical defence system in plants and in plant‐ insect interactions [10].

Cyanogenic glycoside is not toxic on its own. However, when cell structures of plant are disrupted, cyanogenic glycoside will be brought together with the corresponding β‐glucosi‐ dase enzyme. Hydrolysis of cyanogenic glycosides usually occurs when cyanogenic plants are chewed by herbivores or when the plants are disintegrated during processes, such as grinding, pounding or in the presence of water for example during soaking or fermentation [11]. Hydrolysis is accomplished by the β‐glucosidase, producing sugars and a cyanohydrin that spontaneously decomposes to HCN and a ketone or aldehyde [11, 12]. Different kinds of cyanogenic glycosides may be found in different cyanogenic food plants, for example, taxiphyllin in bamboo shoots, linamarin in cassava [5]. Several commercial crop plants, such as sorghum (*Sorghum bicolor*), apple, apricot, almond kernels cassava (*Manihot esculenta*), and barley (*Hordeum vulgare*), are cyanogenic and accumulate significant amounts of cyanogenic glycosides [2, 13, 14]. Generally, the level of cyanogenic glycosides produced is dependent upon the age and the variety of the plant, as well as environmental factors [2].

## **2. Cyanogenic plant foods**

## **2.1. Cassava**

**1. Introduction**

180 Toxicology - New Aspects to This Scientific Conundrum

smaller body size [1].

insect interactions [10].

Food plants contain many substances that can pose potential risks to consumers, and one of these types of substances is cyanogenic glycosides. Cyanogenic glycosides are chemical compounds contained in foods that release hydrogen cyanide when chewed or digested. The act of chewing or digestion leads to hydrolysis of the substances, causing cyanide to be released [1]. Cyanogenic glycosides are common in certain families such as the Fabaceae, Rosaceae, Leguminosae, Linaceae, and Compositae, and identification of their constituents is a useful tool for informative taxonomic markers [2]. There are approximately 25 known cyanogenic glycosides and these are generally found in the edible parts of plants, such as apples, apricots, cherries, peaches, plums, quinces, particularly in the seed of such fruits. The chemicals are also found in almonds, stone fruit, pome fruit, cassava, bamboo shoots, linseed/flaxseed, lima beans, coco yam, chick peas, cashews, and kirsch [3, 4]. Other food products that may contain cyanogenic glycosides include some food ingredients with flavoring properties such as ground almonds powder or paste, marzipan, stone fruit, and alcoholic drinks made from stone fruits.

The toxicity of cyanogenic glycosides and their derivatives is dependent on the release of hydrogen cyanide. Toxicity may result in acute cyanide poisoning and has also been implicated in the etiology of several chronic diseases [6, 7]. Cyanide toxicity can occur in animal including humans at doses between 0.5 and 3.5 mg HCN per kilogram body weight. Symptoms of cyanide toxicity in humans have been reported to include vomiting, stomach ache, diarrhea, convulsion, and in severe cases death [5]. Children are particularly at risk because of their

The toxicity of cyanogenic glycosides is associated with their ability to be hydrolyzed either spontaneously or in the presence of enzyme to produce cyanide as end products of their hydrolysis. Thus, toxic levels of cyanogenic glycosides are estimated in terms of the quantity of free cyanide generated following hydrolysis. This makes it difficult to estimate total cyanogenic glycosides in diet. Hence, levels of amygdalin (the most common cyanogenic glycosides in fruits) reported in literature are inconsistent [8]. Although the level of cyanide up to 10ppm was reported by World Health Organization (WHO) to be safe for cassava flour [9]. Lack of quantitative toxicological test and epidemiological information makes it difficult

Cyanogenic glycosides are a group of nitrile‐containing plant secondary compounds that yields cyanide (cyanogenesis) following their enzymatic breakdown. Cyanogenic glycosides occur in at least 2000 plant species, of which a number of species are used as food. They are amino‐acid‐derived constituents of plants produced as secondary metabolites [5]. Despite great deal of structural diversity in cyanogenic glycosides, almost all of them are believed to be derived from only six different amino acids L‐valine, L‐isoleucine, L‐leucine, L‐phenylala‐ nine, or L‐tyrosine, and cyclopentenyl‐glycine (a nonprotein amino acid) [2, 10]. Cyanogenic glycosides play pivotal roles in organization of chemical defence system in plants and in plant‐

These foods therefore represent potential sources of hydrogen cyanide [5].

to establish safe level of cyanogenic glycosides intake in many foods.

High levels of cyanogenic glycosides have been reported to be present in many nutritious dense plants, thus limiting the use of some of the plants as food. For instance, cassava (*Manihot esculenta*) is one of such plant with high amount of cyanogenic glycosides. It is a crop of economic importance in Africa and a staple food in most African communities [15]. Cassava is also a valued crop in South America and Southeast Asia. The high carbohydrate content of the crop makes it a good source of calorie for feeding billions of people in the world [16]. According to Food and Agriculture organization, cassava is the third most important source of calories in the tropics, after rice and corn [1, 9]. Cassava leaves have higher protein content, contain vitamin C and vitamin A and provide some dietary fiber [17]. Much of the protein in the leaves is made up of linamarase, the enzyme that detoxifies the cyanogenic glycosides in cassava [18]. However, each parts of cassava plants (leaves, stem, root) contains high levels of cyanogenic glycosides; linamarin, lotaustralin, and amygdalin [14, 19], with linamarin been the most predominant cyanogen [19].

The cyanide level of cassava varies from about 75 to 350 ppm, but can be up to l000 ppm or more depending on the variety, plant age, soil condition, fertilizer application, weather, and other factors [20]. Studies have shown that the levels of cyanogenic glycosides in cassava roots are generally lower than that in the leaves and stems [21, 22]. Cassava roots have been reported to contain cyanide content of 10–500 mg/kg of dry matter [23] and the leaves were reported to contain 53–1300 cyanide equivalents/kg of dry matter [24].

Cassava root is processed into various forms before consumption. The root can be boiled and eaten as whole root, or processed into flour for the production of "thick gruel" commonly consumed in Africa. It can also be converted into chips for the production of flour, tapioca, and other foods. Cassava leaves are used in some African countries to produce soup or sauce, following processes such as scrubbing, fermentation, pounding, and boiling. Apart from human consumption, cassava roots and peels are also consumed by animal [1]. The major cyanogenic glycoside in cassava is linamarin, while small amount of lotaustralin (methyl linamarin) and amygdalin are also present, as well as an enzyme linamarase. Linamarin is rapidly hydrolyzed by linamarase to glucose, acetone cyanohydrin, and hydrogen cyanide (**Figure 1**).

**Figure 1.** Hydrolysis of linamarin to produce hydrogen cyanide.

Under neutral conditions, acetone cyanohydrin decomposes to acetone and hydrogen cyanide. Although processing methods can reduce linamarin and cyanide in food, improperly proc‐ essed cassava products would contain some amount of residual linamarin and hydrogen cyanide. This would results in the potential toxicity of the cassava products. Indeed, cases of cyanide toxicity from the consumption of inadequately processed cassava products have been reported [18, 25]. Recent studies have reported the presence of cyanogenic glycoside, amyg‐ dalin in cassava [14]. Amygdalin content of 8.84–48.33 mg/g has been reported to be present in cassava roots, depending on variety [14]. Bitter cassava varieties are more drought resistant and thus more readily available and cheaper but contain high level of cyanogenic glucosides. However, owing to food shortage in times of drought, less time is available for the additional processing required for cassava products. This leads to cassava products with high cyanogenic level with the potential of affecting consumers' health. Increasing the production of cassava breed with low cyanogen content [10] will improve healthy livelihoods of cassava producers, processors and consumers.

#### **2.2. Cocoyam**

Edible cocoyam is a nutrient dense tuber crop that belongs to Araceae family. Two major species of this family are Taro (*Colocasia esculenta L*) and Tannia (*Xanthosoma Sagittifolium* L). These species are commonly grown in the tropical region of Africa and are generally referred to as cocoyam [26]. Cocoyams are valued for its corms, cormels, and leaves. It can be converted into flour and used to make mashed meal or porridge; it can be consumed baked or boiled. It is a rich source of calorie for millions of people in the tropical and subtropical regions due to its high carbohydrate content [26, 27]. In addition to carbohydrate, cocoyam also contains other sources of nutrients such as protein, vitamins, carotenoids and minerals [9]. Apart from the nutrient composition of cocoyam tuber, the crops have been reported to contain low levels (2.10–17.13 mg/100g) of cyanogenic glycosides [28, 29]. In another study, cocoyam tuber was reported to contain 7.4 mg/100g equivalent cyanide [27].

## **2.3. Bamboo shoot**

human consumption, cassava roots and peels are also consumed by animal [1]. The major cyanogenic glycoside in cassava is linamarin, while small amount of lotaustralin (methyl linamarin) and amygdalin are also present, as well as an enzyme linamarase. Linamarin is rapidly hydrolyzed by linamarase to glucose, acetone cyanohydrin, and hydrogen cyanide

Under neutral conditions, acetone cyanohydrin decomposes to acetone and hydrogen cyanide. Although processing methods can reduce linamarin and cyanide in food, improperly proc‐ essed cassava products would contain some amount of residual linamarin and hydrogen cyanide. This would results in the potential toxicity of the cassava products. Indeed, cases of cyanide toxicity from the consumption of inadequately processed cassava products have been reported [18, 25]. Recent studies have reported the presence of cyanogenic glycoside, amyg‐ dalin in cassava [14]. Amygdalin content of 8.84–48.33 mg/g has been reported to be present in cassava roots, depending on variety [14]. Bitter cassava varieties are more drought resistant and thus more readily available and cheaper but contain high level of cyanogenic glucosides. However, owing to food shortage in times of drought, less time is available for the additional processing required for cassava products. This leads to cassava products with high cyanogenic level with the potential of affecting consumers' health. Increasing the production of cassava breed with low cyanogen content [10] will improve healthy livelihoods of cassava producers,

Edible cocoyam is a nutrient dense tuber crop that belongs to Araceae family. Two major species of this family are Taro (*Colocasia esculenta L*) and Tannia (*Xanthosoma Sagittifolium* L). These species are commonly grown in the tropical region of Africa and are generally referred to as cocoyam [26]. Cocoyams are valued for its corms, cormels, and leaves. It can be converted into flour and used to make mashed meal or porridge; it can be consumed baked or boiled. It is a rich source of calorie for millions of people in the tropical and subtropical regions due to its high carbohydrate content [26, 27]. In addition to carbohydrate, cocoyam also contains other sources of nutrients such as protein, vitamins, carotenoids and minerals [9]. Apart from the nutrient composition of cocoyam tuber, the crops have been reported to contain low levels (2.10–17.13 mg/100g) of cyanogenic glycosides [28, 29]. In another study, cocoyam tuber was

(**Figure 1**).

**Figure 1.** Hydrolysis of linamarin to produce hydrogen cyanide.

182 Toxicology - New Aspects to This Scientific Conundrum

reported to contain 7.4 mg/100g equivalent cyanide [27].

processors and consumers.

**2.2. Cocoyam**

Fresh immature bamboo shoots are consumed as vegetable in some Asian countries. Bamboo shoot contains appreciable quantities of vitamin C, carbohydrates, and protein [30]. Besides its nutritive value, bamboo shoots also contain lethal concentration of cyanogenic glycosides. The cyanogenic glycoside present in bamboo shoot is taxiphyllin, which is decomposed quickly in boiling water. Cyanide content of bamboo shoot ranged from 1000 to 8000 mg/kg hydrogen cyanide [5, 31]. Although cyanide content of bamboo shoot is much higher than that of cassava root, the cyanide content in bamboo shoots decreases substantially following harvesting and processing [32].

## **2.4. Sorghum (***Sorghum bicolor***)**

Dhurrin is the major cyanogenic glucoside in sorghum, representing 30% of the dry weight of shoot tips of seedlings, in addition to amygdalin [14]. In young sorghum leaves, dhurrin is localized in vacuoles of plants and the enzymes responsible for its hydrolysis to hydrogen cyanide is located in cytoplasm. In intact leaf, tissues are free from cyanide due to compart‐ mental separation of the enzyme and the substrate. Concentration of dhurin decreases as plant ages, immature sorghum leaves contains high concentration of dhurrin [33]. The cyanide content of sorghum increases rapidly during early growth stage, after which it declines with plant age [34]. The reports by [35, 36] confirmed the cyanogenic glucoside content in the tip of young seedlings of *Sorghum bicolor* that reaches 6% of the dry weight. Sorghum grains have been reported to contain 122.31 mg/g of amygdalin [14].

## **2.5. Fruits and fruits kernels**

The most common cyanogenic glycoside in fruits and fruit kernel is amygdalin. Amygdalin contents of the fruit seeds vary significantly from seed to seed [13, 14, 37].

## *2.5.1. Apple (Malus domestica)*

It is the most cherished fruit among the Rosaceae family. Apples can be consumed raw or processed into alcoholic (cider) or nonalcoholic beverage (apple juice) or apple sauce. Although apples are rich sources of vitamins and other nutrients, apple seeds contain high levels of cyanogenic glycosides. Amygdalin content of apple seeds ranged from 1 to 4 mg/g while that of apple juice was reported to be between 0.001 and 0.08 mg/ml [13, 37].

## *2.5.2. Apricot fruits (Prunus armeniaca)*

They are cultivated in the Middle Asia, Africa, America, and Europe. The fruits are consumed raw or used in dry form for confectioneries while the kernels are usually processed before consumption. Apricot kernels are of two varieties; bitter and sweet. Bitter apricot kernels contain high amount of the cyanogenic glycosides, amygdalin, which can cause cyanide toxicity problems at high dose and thus, unsafe for consumption. Sweet apricot kernels as well as apricot flesh are safe for human consumption because of their low level of cyanogens [13, 38]. The concentration of hydrogen cyanide in Apricot kernels varies widely (49–4000 mg/kg), depending on whether skin on or off varieties is surveyed. Cyanide level of raw or improperly processed apricot kernels can cause serious acute problems that could lead to death [4, 3]. Studies have shown that apricot kernel contain cyanide (CN) content of 1450 mg/kg, approx‐ imately 0.5 mg CN/kernel [39]. This value is similar to the toxic dose of cyanide (0.5 mg/kg body weight) reported by [5].

## **3. Health implications of cyanogenic glycosides**

The toxicity of cyanogenic glycosides and their derivatives is dependent on the release of hydrogen cyanide. Toxicity may result in acute cyanide poisoning and has also been implicated in the etiology of several chronic diseases [7]. Dietary exposure to elevated levels of some cyanogenic glycosides in food has the potential to cause acute cyanide poisoning or a debili‐ tating irreversible neurological condition in the long term.

#### **3.1. Konzo**

High and sustained cyanogens intake at sublethal concentrations from cassava or cassava flour in combination with a low intake of sulfur amino acids has been reported to cause Konzo in women and children [32]. Konzo is an upper motor neuron disease characterized by irrever‐ sible but nonprogressive symmetric spastic paraparesis that has an abrupt onset. It mostly affects children and women of childbearing age [40–42].

## **3.2. Tropical ataxic neuropathy (TAN)**

It is another health problem associated with continuous consumption of improperly processed cassava products. TAN is used to describe several neurological syndromes attributed to toxiconutritional causes. TAN has occurred mainly in Africa, particularly Nigeria [43] and is common among people of 40 years and above [32]. Dietary exposure to cyanide from the monotonous consumption of inadequately processed cassava products over years is respon‐ sible for the cause of the disease. Symptoms of TAN include sore tongue, optical atrophy, neuro sensory deafness, and sensory gait ataxia [43].

## **3.3. Goiter and cretinism**

They are common diseases in developing countries due to low intake of iodine (<100 μg/day). The disease is particularly common in Africa because of their over dependent on cassava as a staple food. Continuous exposure to dietary cyanide from insufficiently processed cassava products aggravate the disease [44] by the interferences of thiocyanate (the end products of cyanide detoxification in human system) with dietary iodine, thus leading to iodine deficiency. According to reference [44], populations with very low iodine intake and high thiocyanate levels from consumption of cassava, showed severe endemic goiter, which decreases with iodine supplementation. Study has shown that consumption of cyanogenic glycosides even at a very low concentration can also cause iodine deficiency leading to goiter [45].

## **3.4. Growth retardation**

depending on whether skin on or off varieties is surveyed. Cyanide level of raw or improperly processed apricot kernels can cause serious acute problems that could lead to death [4, 3]. Studies have shown that apricot kernel contain cyanide (CN) content of 1450 mg/kg, approx‐ imately 0.5 mg CN/kernel [39]. This value is similar to the toxic dose of cyanide (0.5 mg/kg

The toxicity of cyanogenic glycosides and their derivatives is dependent on the release of hydrogen cyanide. Toxicity may result in acute cyanide poisoning and has also been implicated in the etiology of several chronic diseases [7]. Dietary exposure to elevated levels of some cyanogenic glycosides in food has the potential to cause acute cyanide poisoning or a debili‐

High and sustained cyanogens intake at sublethal concentrations from cassava or cassava flour in combination with a low intake of sulfur amino acids has been reported to cause Konzo in women and children [32]. Konzo is an upper motor neuron disease characterized by irrever‐ sible but nonprogressive symmetric spastic paraparesis that has an abrupt onset. It mostly

It is another health problem associated with continuous consumption of improperly processed cassava products. TAN is used to describe several neurological syndromes attributed to toxiconutritional causes. TAN has occurred mainly in Africa, particularly Nigeria [43] and is common among people of 40 years and above [32]. Dietary exposure to cyanide from the monotonous consumption of inadequately processed cassava products over years is respon‐ sible for the cause of the disease. Symptoms of TAN include sore tongue, optical atrophy, neuro

They are common diseases in developing countries due to low intake of iodine (<100 μg/day). The disease is particularly common in Africa because of their over dependent on cassava as a staple food. Continuous exposure to dietary cyanide from insufficiently processed cassava products aggravate the disease [44] by the interferences of thiocyanate (the end products of cyanide detoxification in human system) with dietary iodine, thus leading to iodine deficiency. According to reference [44], populations with very low iodine intake and high thiocyanate levels from consumption of cassava, showed severe endemic goiter, which decreases with iodine supplementation. Study has shown that consumption of cyanogenic glycosides even at

a very low concentration can also cause iodine deficiency leading to goiter [45].

body weight) reported by [5].

184 Toxicology - New Aspects to This Scientific Conundrum

**3.1. Konzo**

**3. Health implications of cyanogenic glycosides**

tating irreversible neurological condition in the long term.

affects children and women of childbearing age [40–42].

**3.2. Tropical ataxic neuropathy (TAN)**

sensory deafness, and sensory gait ataxia [43].

**3.3. Goiter and cretinism**

It is a common health problem especially among children in developing countries. Exposure to cyanogenic glycosides has been a contributing factor to this health problem. Growth retardation is particularly a serious problem in populations consuming foods with inadequate proteins especially diets that are low in sulfur containing amino acids (methionine and cysteine). This is because detoxification of cyanide in human body requires sulfur donors from sulfur‐containing amino acids [46]. Thus, dietary exposure to cyanide is a contributing factor to growth retardation [47].

## **3.5. Cyanide poisoning**

Cyanide toxicity occurs when cytochrome oxidase a3 inhibits the terminal enzyme in the respiratory chain and halts electron transport and oxidative phosphorylation (which is essential to the synthesis of adenosine triphosphate (ATP) and the continuation of cellular respiration) [48, 49]. Cyanide poisoning occurs as a result of consumption of bitter cassava, almond kernels, or apricot kernels and their products without proper processing. Cases of cyanide poisoning after consumption of drink produced from the blends of apricot kernels and orange juice have been reported [50]. Clinical symptoms of cyanide toxicity are vomiting, nausea, dizziness, stomach pains, weakness, headache, diarrhea, and occasionally death [4, 51–54].

## **4. Effect of processing on cyanogenic glycosides**

Processing methods, such as peeling, drying, grinding, soaking, boiling or cooking, soaking and fermentation have been reported by several studies to cause significant reduction in the cyanogenic glycosides of processed foods. These processes have been applied to food crops such as roots, tubers, cereals, and leaves, to cause significant reduction in the cyanogen contents of the crops. Food‐processing methods generally disintegrate cyanogens contents of plants, and this leads to the production of cyanide. Since cyanide is volatile, further processing techniques, such as roasting and drying, will volatilize the remaining cyanide to low level.

## **4.1. Effect of soaking on cyanogenic glycosides**

Soaking of cassava root has been reported to decrease its total cyanide content by 13 52% after 24 h, 73–75% after 48 h and 90% after 72 h [55]. Ref. [56] reported that endogenous β‐glucosidase activity causes a significant degradation of amygdalin in ground apricot kernels soaked at 20°C.

## **4.2. Effect of fermentation on cyanogenic glycosides**

Fermentation of cassava pulp or dough for 4–5 days has been reported to decrease its total cyanide by 52–63% [55]. Soaking and fermentation of bitter apricot kernels decreased cyanogen levels by about 70% [57]. The cyanide content of cocoyam flour produced from fermented cocoyam was reported to reduce by 98.6% [27]. Prasad and Dhanya [58] reported a 84.6% reduction in the cyanide content of fermented sorghum leaves.

#### **4.3. Effect of storage on cyanogenic glycosides**

When foods produced from cyanogenic plants are stored at room temperature (35 ± 2°C), cyanide contents of the foods volatilize due to its low temperature (26°C). Ref [59] reported a decrease of 50–64% in the cyanide content of a cassava product (gari) stored for 4 weeks at room temperature.

#### **4.4. Effect of cooking on cyanogenic glycosides**

Cyanogenic glycosides are generally water soluble. During cooking, significant amount of cyanogens are leached into cooking water. Several studies have reported increased reduction of cyanide in cooked products. Steaming of a cassava product (akyeke) was reported to result in a 74–80% reduction in total cyanide levels. "Garification", a process whereby fermented and dried cassava mash is simultaneously cooked and dried in a shallow wok, resulted in a 90– 93% reduction in total cyanide content. Optimal cooking conditions for the reduction of cyanide levels in bamboo shoots (98–102°C for 148–180 min) resulted in a 97% reduction in cyanide [31]. Generally, traditional African processes typically decrease the cyanide content of cassava by 97–>99%. Also when the cooking method chosen is heating under dry, heat or at low moisture contents, the intake of the cyanogen is limited to only small amounts.

### **4.5. Effect of drying on cyanogenic glycosides**

The efficiency of cyanide removal during drying is dependent on moisture content of the roots, rate of moisture loss (which relates to drying conditions), and the extent of tissue disruption of the plant tissue [60]. Extending the period of drying with higher moisture levels would enhanced linamarin breakdown, thus explaining the fact that fast drying rates result in lower detoxification, while slower rates result in higher cyanogen removal. Famurewa and Emue‐ kele [61] reported that the higher moisture contents of the cassava root the greater the loss in cyanide content during drying. cutting of cassava tubers into small chips might create easy access for contact between the enzymes and cyanogenic glycosides resulting in higher hydrolysis. Reduction in cyanide content could also be as a result of variety, maturity and product sizes [62].

## **5. Cyanide detoxification in human**

Cyanide is detoxified in the body by the enzyme rhodanase with the help of sulfur‐containing amino acids to thiocyanate, which is excreted in the urine [63]. However, the detoxication mechanism of cyanide in the human body can only cope with low level of cyanide generated from consumption of small amount of cyanogenic plants.

## **6. Conclusion**

cocoyam was reported to reduce by 98.6% [27]. Prasad and Dhanya [58] reported a 84.6%

When foods produced from cyanogenic plants are stored at room temperature (35 ± 2°C), cyanide contents of the foods volatilize due to its low temperature (26°C). Ref [59] reported a decrease of 50–64% in the cyanide content of a cassava product (gari) stored for 4 weeks at

Cyanogenic glycosides are generally water soluble. During cooking, significant amount of cyanogens are leached into cooking water. Several studies have reported increased reduction of cyanide in cooked products. Steaming of a cassava product (akyeke) was reported to result in a 74–80% reduction in total cyanide levels. "Garification", a process whereby fermented and dried cassava mash is simultaneously cooked and dried in a shallow wok, resulted in a 90– 93% reduction in total cyanide content. Optimal cooking conditions for the reduction of cyanide levels in bamboo shoots (98–102°C for 148–180 min) resulted in a 97% reduction in cyanide [31]. Generally, traditional African processes typically decrease the cyanide content of cassava by 97–>99%. Also when the cooking method chosen is heating under dry, heat or at

low moisture contents, the intake of the cyanogen is limited to only small amounts.

The efficiency of cyanide removal during drying is dependent on moisture content of the roots, rate of moisture loss (which relates to drying conditions), and the extent of tissue disruption of the plant tissue [60]. Extending the period of drying with higher moisture levels would enhanced linamarin breakdown, thus explaining the fact that fast drying rates result in lower detoxification, while slower rates result in higher cyanogen removal. Famurewa and Emue‐ kele [61] reported that the higher moisture contents of the cassava root the greater the loss in cyanide content during drying. cutting of cassava tubers into small chips might create easy access for contact between the enzymes and cyanogenic glycosides resulting in higher hydrolysis. Reduction in cyanide content could also be as a result of variety, maturity and

Cyanide is detoxified in the body by the enzyme rhodanase with the help of sulfur‐containing amino acids to thiocyanate, which is excreted in the urine [63]. However, the detoxication mechanism of cyanide in the human body can only cope with low level of cyanide generated

reduction in the cyanide content of fermented sorghum leaves.

**4.3. Effect of storage on cyanogenic glycosides**

186 Toxicology - New Aspects to This Scientific Conundrum

**4.4. Effect of cooking on cyanogenic glycosides**

**4.5. Effect of drying on cyanogenic glycosides**

**5. Cyanide detoxification in human**

from consumption of small amount of cyanogenic plants.

room temperature.

product sizes [62].

Cyanogenic glycosides are abundant in edible plants. Consumption of improperly processed cyanogenic plants can lead to chronic and acute health problems. Understanding the appro‐ priate processing methods for specific cyanogenic plants will help in reducing the problem of unintentional cyanide toxicity. Similarly, to prevent adverse effects of cyanogenic glycosides in food plants, consumers should prepare foods properly before consumption. It is recom‐ mended that cyanogenic plants should be cut into smaller pieces and cook thoroughly to release toxic hydrogen cyanide before consumption in order to reduce the level of the toxin. Also, seeds of fruits to be processed into juice should be removed before crushing to avoid cyanide poisoning.

## **Author details**

Islamiyat Folashade Bolarinwa\* , Moruf Olanrewaju Oke, Sulaiman Adebisi Olaniyan and Adeladun Stephen Ajala

\*Address all correspondence to: ifbolarinwa@lautech.edu.ng, islamiyat202@yahoo.co.uk

Department of Food Science and Engineering, Ladoke Akintola University of Technology (LAUTECH), P.M.B. Ogbomoso, Oyo State, Nigeria

## **References**


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[7] FAO/WHO. WHO Food Additive Series: 65. Safety evaluation of certain food additives and contaminants. Prepared by the 74th Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). 2012. World Health Organization, Geneva.

[8] WHO: World Health Organization. Hydrogen cyanide and cyanides: Human health aspects. 2004. Concise International Chemical Assessment Document 61, Geneva.

[9] Food and Agricultural Organisation (FAO). Roots, tubers, plantain and bananas in human nutrition. Food and Agricultural Organisation of the United Nation. 1991; 7(1):

[10] Ganjewala D, Kuma S, Asha DS, Ambika K. Advances in cyanogenic glycosides biosynthesis and analyses in plants: a review. Acta Biol Szeged, 2010; 54: 1–14.

[11] CFS (Centre for Food Safety). Natural toxins in food plants. Risk assessment studies. Food and Environmental Hygiene Department, the Government of the Hong Kong

[12] Gruhnert C, Biehl B, Selmar D. Compartmentation of cyanogenic glucosides and their

[13] Bolarinwa IF, Orfila C, Morgan MRA. Determination of amygdalin in apple seeds, fresh

[14] Bolarinwa IF, Olaniyan SA, Olatunde SJ, Ayandokun FT, Olaifa IA. Effect of processing on amygdalin and cyanide contents of some Nigerian Foods. J Chem Pharm Res, 2016;

[15] Nhassico D, Muquingue H, Cliff J, Cumbana A, Bradbury JH. Rising African cassava production, diseases due to high cyanide intake and control measures. J Sci Food Agric,

[16] Nweke F. New challenges in the cassava transformation in Nigeria and Ghana. Environment and Production Technology Division, International Food Policy Research

[17] South Pacific Trade Commission. Exporting Fruit and Vegetables to New Zealand, A

[18] Bokanga M, Ekanayake IJ, Dixon AGO, Porto MCM. Genotype‐environment interac‐

[19] Lykkesfeldt J, Moller BL. Cyanogenic glycosides in cassava, Manihot esculenta Crantz.

[20] Gomez G, Valdivieso M. The effect of variety and plant age on cyanide content, chemical composition and quality of cassava roots. Nutr Rep Int 1983; 27(4): 857‐865.

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tions for cyanogenic potential in cassava. Acta Hort, 1994; 375: 131‐139.

apples and processed apple juices. Food Chem. 2015; 170: 437‐442.

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degrading enzymes. Planta, 1994; 195: 36‐42.

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[51] Lash EE, Shawa RE. Multiple cases of cyanide poisoning by apricot kernels in children from Gaza. Pediatrics, 1981; 68: 5‐7.

[36] Halkier BA, Møller BL. Biosynthesis of the cyanogenic glucoside dhurrin in seedlings of *Sorghum bicolor* (L.) Moench and partial purification of the enzyme system involved.

[37] Bolarinwa IF, Orfila C, Morgan MRA. Amygdalin content of seeds kernels and food products commercially‐available in the UK. Food Chem, 2014; 152: 1333‐139.

[38] Canadian Food Inspection Agency. Cyanides in bitter apricot kernels. 2009. Available from: http://www.hc‐sc.gc.ca/fn‐an/pubs/securit/2009‐apricots‐abricots/index‐

[39] Holzbecher MD, Moss MA, Ellenberger HA. The cyanide content of laetrile prepara‐

[40] Ministry of Health, Mozambique Mantakassa. An epidemic of spastic paraparesis associated with chronic cyanide intoxication in a cassava staple area Mozambique. Nutritional factors and hydrocyanic acid content of cassava products. Bull World

[41] Tylleskar T, Banae M, Bikangi N, Cooke RD, Poulter NH, Rosling H. Cassava cyanogens and konzo, an upper motorneuron disease found in Africa. Lancet, 1992; 339: 208‐211.

[42] Ernesto M, Cardoso AP, Nicala D, Mirione E, Massaza F, Cliff J, Hague MR, Bradbury JH. Persistent konzo and cyanogens toxicity from cassava in northern Mozambique.

[43] Oluwole OSA, Onabolu AO, Link H, Rosling H. Persistence of tropical ataxic neuro‐ pathy in a Nigerian community. J Neurol Neurosurg Psychiatry, 2000; 69: 96‐101.

[44] Rosling H. Cassava Toxicity and Food Security. Ed. Rosling. Tryck Kontakt, Uppsala,

[45] Odo CE, Enechi OC, Oburu CS. Concentrations of anti‐nutritional factors in raw edible

[46] Rosling H. Measuring effects in humans of dietary cyanide exposure from cassava. Acta

[47] Banea‐Mayambu JP, Tylleskar, T, Tylleskar K, Gebre‐Medhin M, Rosling H. Dietary cyanide from insufficiently processed cassava and growth retardation in children in the Democratic Republic of Congo (formerly Zaire). Ann Trop Pediatr, 2000; 20: 34‐40.

[48] Beasley DMG, Glass WI. Cyanide poisoning: pathophysiology and treatment recom‐

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eng.php


#### **Toxic Effects as a Result of Herbal Medicine Intake Toxic Effects as a Result of Herbal Medicine Intake**

Nudrat Fatima and Naira Nayeem Nudrat Fatima and Naira Nayeem

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

http://dx.doi.org/10.5772/64468

#### **Abstract**

Concurrent use of herbs with therapeutic drugs increases the potential of herb-drug interactions. The clinical importance of herb-drug interactions is associated with the particular herb, drug, and patient profile. Herbs are potentially potent as they affect body functions. The use herbal medicine and supplements can be risky as they are not subject to review by the FDA. In this chapter, we make an attempt to discuss the possible reasons for toxic effects, types of toxicities, some reported cases of toxicities involving the use of herbal medicine alone, and some herb-drug interactions. In addition to this, possible ways to reduce toxic effects of herbal medicines have also been discussed.

**Keywords:** herbal medicine, toxicity, reported cases, herb-drug interaction

## **1. Introduction**

Herbal medicines are advertised to be free from side effects, which is a myth. A large number of people still rely on herbal medicines, and some people take herbal medicines along with routine allopathic medicines especially in cases of diabetes, hypertension, thyroid disease, etc., where the patient is on long-term or lifelong treatment. Many commercial websites are available on Internet, which insist that herbal medicines have no side effects. In underdeveloped and developing countries, there are no specific laws for herbal practitioners and companies marketing herbal products. People are attracted by such companies and start using herbal medicines pertaining to be free from side effects. In this chapter, we will discuss about possible toxicities of some herbal medicines and their remedies.

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

## **2. Possible reasons for toxic effects of herbal medicines**

## **2.1. Self-treatment**

Herbal medicines are easily available in market and can be purchased without prescription. These products are advertised on media as a miracle treatment without any side effects to attract people that are fed up with side effects or lost hope for being cured. The patients who like to play safe game are attracted in a manner that they are allowed to continue their regular medicines along with herbal treatment. Even persons caring about their health start herbal treatment to remain healthy proving the proverb "Prevention is better than cure." As a result, a large number of people are attracted towards herbal medicines and they start self-treatment [1].

## **2.2. Unqualified practitioners**

In a large part of the world, unqualified practitioners are prescribing alternative therapies to patients of various diseases, apart from some countries where laws and regulations for herbal practitioners exist and implemented. Medical practitioners are provided vast knowledge about human body, drugs, mechanism of action, pharmacology, case studies, and then allowed to practice. Nowadays, Alternative Medicine Degree Course is available in certain universities with highly qualified and experienced faculty, which is a good source of herbal practitioners, but still 50% of herbal practitioners in the world are unqualified who acquire this profession after their forefathers such as a son of a farmer becomes a farmer, or after reading some books about herbal medicine, conducting 6-month online course about herbal medicine, etc., and start practicing. In underdeveloped countries, people are attracted towards these quacks due to economic reasons and start taking herbal medicine. These unqualified practitioners themselves are not aware of toxic effects of herbal medicines, and if the patient complains, they cannot rectify their mistake.

## **2.3. Sub-standard product**

There are many sub-standard herbal products available in the market. The reason is that these products are not tested accordingly for quality before marketing. Some contain less amount of active ingredient and some do not contain active ingredient at all as a result of incorrect identification of plant by the collector, using adulterant instead of original plant or due to improper storage of plant material, and it loses its efficacy. Sometimes the herbal products contain material not defined on label such as non-herb material, minerals, heavy metals, and addition of particular pharmaceutical product. Occasionally they may contain toxins and pesticides, which is much more dangerous and one of the major reasons of toxic effects after herbal medicine intake [2].

## **2.4. Improper intake**

Allopathic medicines are marketed after extensive testing and trials, and their dose is fixed according to age and weight of the patient. All possible adverse effects are listed in leaflet. But no such procedures are followed in case of herbal medicines. Some of herbal medicines are considered as dietary supplements, and proper dose is not mentioned. No measure cup or spoon provided with the medicine as in case of allopathic syrups. Usually, same dose is applied for persons of different age and weight. Companies selling these products misguide people and claim their product totally free from adverse effects. Even there is no period mentioned, some people continue for months or years, which in long term can be harmful for human health [3].

## **3. Types of toxicities**

## **3.1. Nephrotoxicity**

**2. Possible reasons for toxic effects of herbal medicines**

Herbal medicines are easily available in market and can be purchased without prescription. These products are advertised on media as a miracle treatment without any side effects to attract people that are fed up with side effects or lost hope for being cured. The patients who like to play safe game are attracted in a manner that they are allowed to continue their regular medicines along with herbal treatment. Even persons caring about their health start herbal treatment to remain healthy proving the proverb "Prevention is better than cure." As a result, a large number of people are attracted towards herbal medicines and they start self-treatment

In a large part of the world, unqualified practitioners are prescribing alternative therapies to patients of various diseases, apart from some countries where laws and regulations for herbal practitioners exist and implemented. Medical practitioners are provided vast knowledge about human body, drugs, mechanism of action, pharmacology, case studies, and then allowed to practice. Nowadays, Alternative Medicine Degree Course is available in certain universities with highly qualified and experienced faculty, which is a good source of herbal practitioners, but still 50% of herbal practitioners in the world are unqualified who acquire this profession after their forefathers such as a son of a farmer becomes a farmer, or after reading some books about herbal medicine, conducting 6-month online course about herbal medicine, etc., and start practicing. In underdeveloped countries, people are attracted towards these quacks due to economic reasons and start taking herbal medicine. These unqualified practitioners themselves are not aware of toxic effects of herbal medicines, and if the patient complains, they

There are many sub-standard herbal products available in the market. The reason is that these products are not tested accordingly for quality before marketing. Some contain less amount of active ingredient and some do not contain active ingredient at all as a result of incorrect identification of plant by the collector, using adulterant instead of original plant or due to improper storage of plant material, and it loses its efficacy. Sometimes the herbal products contain material not defined on label such as non-herb material, minerals, heavy metals, and addition of particular pharmaceutical product. Occasionally they may contain toxins and pesticides, which is much more dangerous and one of the major reasons of toxic effects after

Allopathic medicines are marketed after extensive testing and trials, and their dose is fixed according to age and weight of the patient. All possible adverse effects are listed in leaflet. But

**2.1. Self-treatment**

194 Toxicology - New Aspects to This Scientific Conundrum

**2.2. Unqualified practitioners**

cannot rectify their mistake.

**2.3. Sub-standard product**

herbal medicine intake [2].

**2.4. Improper intake**

[1].

Drug or toxin causing kidney damage upon exposure to a certain level and the kidneys are unable to pass excess urine, and waste product is the condition termed as nephrotoxicity. In this condition, there is an elevation in blood electrolytes such as potassium and magnesium. This condition starts temporarily but, if not detected earlier, could be severe. Nephrotoxicity can be detected by two simple tests of blood urea nitrogen (BUN) and creatinine levels in blood together termed as kidney function tests. Normal range values of BUN and creatinine are 10– 25 mg/dl and 0.7–1.4 mg/dl, respectively. These values may increase due to following factors:


Reason of nephrotoxicity after herbal medicine intake may be addition of toxins during careless preparation, addition of adulterants, heavy metals, and some pharmaceutical products intentionally to reduce cost or increase efficacy [4].

Herbs such as *Tripterygium wilfordii* Hook (thunder god vine) contain diterpenoid epoxide, which induces apoptosis causing kidney damage. *Averrhoa carambola* L. (star fruit) contains oxalate in high quantity, which can cause acute nephropathy. *Guaiacum officinale* L. (rough bark) and *Arctostaphylos uva-ursi* (cranberry) increase stone formation. *Aristolochia fangchi* causes well-known aristolochic acid nephropathy. *Callilepis laureola* DC (Impila) inhibits mitochondrial ATP synthesis. *Uncaria tomentosa* wild DC (Peruvian's Cat Claw) causes acute allergic interstitial nephritis. Studies are being conducted on *Salix alba* L. (willow bark) analgesic nephropathy induction. *Ephedra sinica* Stapf (Chinese ephedra) affects reninangiotensin-aldosterone system. *Glycyrrhiza glabra* L. (Licorice) and *Harpagophytum procumbens* DC (devil's claw) inhibit renal transport processes [5].

## **3.2. Hepatotoxicity**

Hepatotoxicity, termed after two Greek words *Hepar* and *Toxicon* meaning liver and poison, respectively, may be defined as liver damage due to chemical, drug, herb, or dietary supplement. The damage can be noticed by stomach pain, nausea, vomiting, change in urine and stool color, jaundice, rash, frequent tiredness, weakness, fatigue, and fever. Laboratory tests include some liver function tests, which are conducted on blood samples for the detection of hepatotoxicity. These tests are comprised of alanine transaminase test (normal range 7– 55 U/l), aspartate transaminase test (normal range 8–48 U/l), alkaline phosphatase test (normal range 45–115 U/l), albumin test (normal range 3.5–5.0 g/dl), and bilirubin test (normal range 0.1–1.2 mg/dl). Increased levels of ALT, AST, ALP and bilirubin and decreased level of albumin indicate liver damage. In pregnancy, ALP levels are also increased.

Causes of liver damage are both by hepatocellular and by extracellular mechanisms. Some of these mechanisms are mentioned below:

*Hepatocyte disruption:* When drug binds to intracellular proteins covalently, it may lower ATP level causing actin disruption, which in turn causes bleb and rupture of membrane.

*Transport protein disruption:* Drugs affecting transport proteins at canalicular membrane may disturb flow of bile juice. This interruption in certain processes and transport prevents bilirubin excretion leading to cholestasis.

*T-cell activation:* When a drug binds covalently to P-450 enzyme, it acts as immunogen and activates T cells and cytokines resulting in complicated immune response.

*Hepatocyte apoptosis:* Sometimes apoptotic pathway is activated due to tumor necrosis and αreceptor of F triggering flow of intercellular caspases, which leads to programmed cell death.

*Disruption of mitochondria:* Some drugs suppress mitochondrial function by effecting on βoxidation energy production and synthesis of nicotine amide adenine dinucleotide. A second effect is on flavin dinucleotide inhibiting ATP production.

*Injury of bile duct:* Some toxic metabolites from liver can injure epithelium of bile duct.

*Drug toxicity mechanisms:* Drugs are major cause of hepatotoxicity. Almost nine hundred drugs, toxins, and herbs are reported for hepatotoxicity. There are two kinds of drug reactions, first is reaction directly affecting the liver termed as intrinsic drug reactions, and other is reaction mediating to immune response termed as idiosyncratic drug reactions. In the first category, drug itself or its metabolite produces a dose-related injury, for example, paracetamol and carbon tetrachloride. In second category, hypersensitivity reactions, for example, phenytoin reaction, cause fever, rash, eosinophilia for a short period of time and immunoallergic or metabolic idiosyncratic reaction due to indirect drug reaction. The second type of reactions the response rate is variable, for example, halothane.

*Drug interaction mechanisms:* Some drugs when taken simultaneously react together and cause liver damage. For example, tylenol can be hepatotoxic in combination with INH, isotamine, laniazid, and nydrazid [6].

Discussing about hepatotoxicity caused by herbal medicine intake, mostly the incident rates are still to be reported. The severity of toxicity is widely variable between mild hepatitis to acute hepatic failure. The scoring system for allopathic medicines can be assessed but is not suitable for herbal medicines and needs validation. Many Ayurvedic and Chinese herbal medicines are reported to cause hepatotoxicity. Major hepatotoxic herbs are *Cimicifuga racemosa* (black cohosh), *Larrea tridentata* (chaparral), *Teucrium chamaedrys* (germander), *Scutellaria lateriflora* (American skullcap), and *Scutellaria baicalensis* (Chinese skullcap), etc.[7].

## **3.3. Cardiotoxicity**

Herbs such as *Tripterygium wilfordii* Hook (thunder god vine) contain diterpenoid epoxide, which induces apoptosis causing kidney damage. *Averrhoa carambola* L. (star fruit) contains oxalate in high quantity, which can cause acute nephropathy. *Guaiacum officinale* L. (rough bark) and *Arctostaphylos uva-ursi* (cranberry) increase stone formation. *Aristolochia fangchi* causes well-known aristolochic acid nephropathy. *Callilepis laureola* DC (Impila) inhibits mitochondrial ATP synthesis. *Uncaria tomentosa* wild DC (Peruvian's Cat Claw) causes acute allergic interstitial nephritis. Studies are being conducted on *Salix alba* L. (willow bark) analgesic nephropathy induction. *Ephedra sinica* Stapf (Chinese ephedra) affects reninangiotensin-aldosterone system. *Glycyrrhiza glabra* L. (Licorice) and *Harpagophytum procumbens*

Hepatotoxicity, termed after two Greek words *Hepar* and *Toxicon* meaning liver and poison, respectively, may be defined as liver damage due to chemical, drug, herb, or dietary supplement. The damage can be noticed by stomach pain, nausea, vomiting, change in urine and stool color, jaundice, rash, frequent tiredness, weakness, fatigue, and fever. Laboratory tests include some liver function tests, which are conducted on blood samples for the detection of hepatotoxicity. These tests are comprised of alanine transaminase test (normal range 7– 55 U/l), aspartate transaminase test (normal range 8–48 U/l), alkaline phosphatase test (normal range 45–115 U/l), albumin test (normal range 3.5–5.0 g/dl), and bilirubin test (normal range 0.1–1.2 mg/dl). Increased levels of ALT, AST, ALP and bilirubin and decreased level of albumin

Causes of liver damage are both by hepatocellular and by extracellular mechanisms. Some of

*Hepatocyte disruption:* When drug binds to intracellular proteins covalently, it may lower ATP

*Transport protein disruption:* Drugs affecting transport proteins at canalicular membrane may disturb flow of bile juice. This interruption in certain processes and transport prevents bilirubin

*T-cell activation:* When a drug binds covalently to P-450 enzyme, it acts as immunogen and

*Hepatocyte apoptosis:* Sometimes apoptotic pathway is activated due to tumor necrosis and αreceptor of F triggering flow of intercellular caspases, which leads to programmed cell death.

*Disruption of mitochondria:* Some drugs suppress mitochondrial function by effecting on βoxidation energy production and synthesis of nicotine amide adenine dinucleotide. A second

*Drug toxicity mechanisms:* Drugs are major cause of hepatotoxicity. Almost nine hundred drugs, toxins, and herbs are reported for hepatotoxicity. There are two kinds of drug reactions, first

*Injury of bile duct:* Some toxic metabolites from liver can injure epithelium of bile duct.

level causing actin disruption, which in turn causes bleb and rupture of membrane.

activates T cells and cytokines resulting in complicated immune response.

effect is on flavin dinucleotide inhibiting ATP production.

DC (devil's claw) inhibit renal transport processes [5].

196 Toxicology - New Aspects to This Scientific Conundrum

indicate liver damage. In pregnancy, ALP levels are also increased.

these mechanisms are mentioned below:

excretion leading to cholestasis.

**3.2. Hepatotoxicity**

Cardiotoxicity is a term used for damage to heart or altering heart functions. It is a state in which there is alteration in electrophysiological function of heart or cardiac muscle damage, which weakens the heart causing inefficient pumping and circulation of blood. This can be detected by symptoms such as dry, non-productive cough, inflammation of ankles, hand, feet, and neck veins; irregular heartbeat; tachycardia; cardiomegaly; weakness; vertigo, etc.

Some common tests for finding cardiotoxicity include physical examination of heart through stethoscope to check the sounds of heartbeat, chest X-ray to check the size of the heart, echocardiogram imaging test using ultrasound, electrocardiogram (ECG) to measure the electrical activity of heart, multi-gated acquisition scan (MUGA) by injecting a radiotracer into veins to check pumping and function of blood vessels to heart, and troponin blood tests. Troponins are proteins of heart muscles released by dying heart cells into blood stream.

These tests may show results positive for cardiotoxicity due to a number of cardiac events including changes in blood pressure, thrombosis, arrhythmias, inflammation of myocardium, and pericardium leading to cardiac arrest or failure. Cardiotoxic agents include chemotherapeutic drugs of anthracycline class, alkylating agents such as cyclophosphamide, cisplatin, chlormethine, mitomycin, etc. Some other agents such as paclitaxel, etoposide, fluorouracil, asparaginase, tretinoin, pentostatin may cause cardiotoxicity. This may be increased due to cumulative dose, rate, and schedule of administration, history of preexisting cardiovascular problems, and disturbed balance of cardiac electrolytes [8].

Herbal medicines having direct effect on heart include medicine prepared from plants such as *Digitalis purpurea* (digitalis), *Catharanthus roseus* (vinca)*, Aconitum napellus* (monk's hood), *Atropa belladonna* (deadly nightshade), *Ephedra distachya* (sea grape), *Mandragora officinarum* (mandrake), *Glycyrrhiza glabra* (licorice), etc. [9].

## **3.4. Neurotoxicity**

Neurotoxicity is a term used for a state in which there is a physical brain damage due to exposure to neurotoxin, a substance that disrupts or kills neurons, and in turn alters the activity of nervous system. Signs and symptoms of this type of toxicity are anxiety, depression, limb weakness and numbness, impaired vision, headache, sexual dysfunction and behavioral changes. The reasons may be chemotherapy, radiation therapy, drug abuse, organ transplants, exposure to heavy metals, some food additives, pesticides, cosmetics, cleaning solvents and naturally occurring substances.

The nervous system comprises of central nervous system (CNS) and peripheral nervous system (PNS). CNS consists of brain and spinal cord, and cerebellum is a part of brain primarily affected by neurotoxic substances. Cerebellum is responsible for processing information to conduct muscle activities and maintain body posture. The damaged cerebellum produces altered reflexes, unsteady walk, loss of body control, and confusion. PNS is a network of cranial and spinal nerves emerging from CNS to all parts of body. The system consists of myelinated neurons with layers of Schwann cells, which act as electrical insulator. By the damage of these nerves, the electrical signals are interrupted. Another part of PNS is autonomic nervous system (ANS), which functions without conscious effort. The movements such as cardiovascular, respiratory, gastrointestinal, and endocrine systems are under the control of this system. The toxicity of this system is most dangerous and it results in loss of activities function, retention of urine and stool, impotence, paralysis and impotence [10].

Some common plants used as herbal medicines have potential neurotoxic effects. Among them are *Papaver somniferum* (opium), *Catharanthus roseus* (vinca), *Datura stramonium* (thorn apple), *Atropa belladonna* (deadly nightshade), *Hyoscyamus niger* (henbane), *Cannabis indica* (marijuana), *Conium maculatum* (hemlock), *Coscinium fenestratum* (yellow vine) [11], and *Brugmansia* species (angel's trumpet) [12].

#### **3.5. Skin toxicity**

Cutaneous toxicity is a term used for an evident adverse effect such as skin irritation, inflammation, or rashes of epidermal growth factor receptor caused by exposure to a plant, chemical, or environmental factor.

Skin is the largest body organ and a protective barrier comprising of a layer of dead cells and several layers of living cells. When an irritating substance reaches these living cells, these sensitive cells respond by inflammation or dermatitis. Inflammation has four parts, which include redness, pain, heat, and swelling. The skin toxicity is easiest to detect as the reaction is immediately observed.

The most common test for detecting cutaneous toxicity is patch test. In this, the skin is exposed to a small amount of diluted substance in patches and observing the reaction. The most common source of skin toxicity is food and cosmetics, and others are medicated lotions, balms, creams, inhalers and essential oils. A variety of herbal material is available in all of the abovementioned cosmetics and medicated products. Types of skin sensitization reactions include the following:

*Atropa belladonna* (deadly nightshade), *Ephedra distachya* (sea grape), *Mandragora officinarum*

Neurotoxicity is a term used for a state in which there is a physical brain damage due to exposure to neurotoxin, a substance that disrupts or kills neurons, and in turn alters the activity of nervous system. Signs and symptoms of this type of toxicity are anxiety, depression, limb weakness and numbness, impaired vision, headache, sexual dysfunction and behavioral changes. The reasons may be chemotherapy, radiation therapy, drug abuse, organ transplants, exposure to heavy metals, some food additives, pesticides, cosmetics, cleaning solvents and

The nervous system comprises of central nervous system (CNS) and peripheral nervous system (PNS). CNS consists of brain and spinal cord, and cerebellum is a part of brain primarily affected by neurotoxic substances. Cerebellum is responsible for processing information to conduct muscle activities and maintain body posture. The damaged cerebellum produces altered reflexes, unsteady walk, loss of body control, and confusion. PNS is a network of cranial and spinal nerves emerging from CNS to all parts of body. The system consists of myelinated neurons with layers of Schwann cells, which act as electrical insulator. By the damage of these nerves, the electrical signals are interrupted. Another part of PNS is autonomic nervous system (ANS), which functions without conscious effort. The movements such as cardiovascular, respiratory, gastrointestinal, and endocrine systems are under the control of this system. The toxicity of this system is most dangerous and it results in loss of activities function, retention

Some common plants used as herbal medicines have potential neurotoxic effects. Among them are *Papaver somniferum* (opium), *Catharanthus roseus* (vinca), *Datura stramonium* (thorn apple), *Atropa belladonna* (deadly nightshade), *Hyoscyamus niger* (henbane), *Cannabis indica* (marijuana), *Conium maculatum* (hemlock), *Coscinium fenestratum* (yellow vine) [11], and *Brugmansia*

Cutaneous toxicity is a term used for an evident adverse effect such as skin irritation, inflammation, or rashes of epidermal growth factor receptor caused by exposure to a plant, chemical,

Skin is the largest body organ and a protective barrier comprising of a layer of dead cells and several layers of living cells. When an irritating substance reaches these living cells, these sensitive cells respond by inflammation or dermatitis. Inflammation has four parts, which include redness, pain, heat, and swelling. The skin toxicity is easiest to detect as the reaction

The most common test for detecting cutaneous toxicity is patch test. In this, the skin is exposed to a small amount of diluted substance in patches and observing the reaction. The most

(mandrake), *Glycyrrhiza glabra* (licorice), etc. [9].

198 Toxicology - New Aspects to This Scientific Conundrum

of urine and stool, impotence, paralysis and impotence [10].

**3.4. Neurotoxicity**

naturally occurring substances.

species (angel's trumpet) [12].

**3.5. Skin toxicity**

or environmental factor.

is immediately observed.

*Primary irritant dermatitis:* It is a direct irritation of skin with symptoms such as redness, itching, pain, blusters, peeling, or open wounds. Primary irritant dermatitis may be caused by plants such as *Cannabis sativa* (weed oil), *Dieffenbachia amoena* (dumb canes), *Asclepias syriaca* (milk weed), *Narcissus pseudonarcissus* (daffodils), *Digitalis purpurea* (foxglove), *Ricinus communis* (castor bean), *Tulipa gesneriana* (tulip bulb), *Primula veris* (cowslip), *Hevea brasiliensis* (rubber tree), *Ficus carica* (fig tree sap), *Ranunculus acris* (butter cup), etc. Common foods such as *Pastinaca sativa* (parsnip), *Solanum lycopersicum* (tomatoes), *Daucus carota* (carrot), *Cucumis sativus* (cucumber), *Brassica rapa* (turnip), *Petroselinum crispum* (parsley), *Apium graveolens* (celery) and *Agaricus bisporus* (mushrooms) also can cause primary irritant dermatitis.

*Allergic contact dermatitis:* It is a true allergic response and is varied from individual to individual. *Toxicodendron diversilobum* (poison oak) and *Toxicodendron rydbergii* (poison ivy) are the most common plants producing allergic contact dermatitis. Others include *Hedera helix* (English ivy), *Toxicodendron vernix* (poison sumac), *Dendranthema grandiflorum* (chrysanthemum), *Narcissus pseudonarcissus* (daffodils), *Tulipa gesneriana* (tulip bulb), *Marchantiophyta species* (liverwort), *Primula vulgaris* (prime rose), *Flavoparmelia caperata* (lichens), *Pinus sabiniana* (pine), *Cedrus deodara* (cedar), *Anacardium occidentale* (cashew), *Apium graveolens* (celery), *Allium cepa* (onions), and *Allium sativum* (garlic).

*Photosensitization dermatitis:* It is cutaneous toxic response caused by exposure to sunlight when a photosensitizer (compound sensitive to sunlight) is present in body and can be detected by sunburn-like reactions in non-pigmented areas. Plants such as *Tetradymia species* (horse brushes), *Hypericum species* (St John's wort), *Tribulus terrestris* (goats head), *Agave lechuguilla* (lechuguilla), *Bassia scoparia* (kochia), and *Lantana camara* (lantana) cause photosensitization dermatitis.

There is another type of phototoxic photosensitization caused by contact of some plants. When a photoactive chemical produced by plants comes in skin contact, absorbed and activated by sunlight, this type of reaction occurs. The intensity varies depending upon time and amount of exposure. Plants such as *Ficus carica* (figs), *Anethum graveolens* (dill), *Brassica alba* (mustard), *Petroselinum crispum* (parsley), *Citrus aurantifolia* (lime), *Daucus carota* (carrots), *Ranunculus acris* (butter cup), *Hypericum perforatum* (Klamath weed), and *Apium graveolens* (celery) with pink rot are reported to produce contact photosensitization [13].

## **4. Reported cases of toxicity using herbal medicine solely**

Although a large number of herbal toxicity cases are not reported, still there are many reported cases. Some of such cases are listed in **Table 1**.


**Table 1.** Some of the documented herbal toxicity cases.

## **5. Herb-drug interactions**

Natural products are a mixture of phyto-constituents unlike the conventional drugs. Usually, the quantity and quality of the bioactive substance from the herbs vary depending on the part of the plant used, environmental factors, method of collection and storage conditions. The use of herbs as medicine is getting popular; hence, there is a need to address and review the interaction between the herb and the drug. Certain herbal supplements may cause dangerous side effects when taken with prescription drugs. Furthermore, the complex nature of a natural product adds to the complexity of determination of herb-drug interactions. There are no standards for herbal products prescribed/regulated by the FDA [26]. Currently, there are limited reports of adverse herb-drug reactions. The available reports are either individual case reports giving the details of the specific case studied or the suspected interaction or clinical trials, in which the drugs and herbs are combined and are closely monitored. Data concerning the drug-herb interaction are usually unavailable as there is lack of information about nature of the herbal product and their complex reactions. Sometimes the literature available can be confusing due to lack of clarity or even contradictory. This is due to the way these adverse interactions are reported. The data received by experimentation and pharmacodynamics studies may give indications of potential interactions.

Herb-drug interactions can be either pharmacodynamic or pharmacokinetic in nature. When the constituents have synergistic or antagonist activity in relation to the drug, it is termed as pharmacodynamics, which results in concentration-dependent activity, while alteration in ADME of the drug by herbal products results in pharmacokinetic interactions [27].

The Med Watch system is used by the FDA to report adverse reactions for conventional drugs as well as dietary supplements. The complex nature of the herbs creates complications while determining the herb-drug interactions.

A method for the evaluation of herb-drug interactions has been developed for determining the reliability of the case reports on drug-herb interactions. A 10-point scale has been used for detecting the probability of drug-herb interaction. This method consists of ten items and each item being allotted 01 point. The interactions are interpreted as 8–10 points' likely, 4–7 points possible, or 0–3 points unevaluable. A total of 320,860 adverse events were reported to the system in 2002 [28].

The following are the evaluating parameters for determining the probability of herb-drug interactions (1 point is allotted per item) [29] Some herb-drug interactions are listed in (**Table 2**):

**a.** Adequate patient history.

**No. Herb Toxicity reported Indication Reference**

Hepatic failure Hepatorenal syndrome encephalopathy

Tachycardia, difficulty in respiration, insomnia

Acute hepatitis Insomnia [14]

Acute drug-induced liver injury Arthritis [16]

Nephrolithiasis Dietary supplement [18]

Ventricular arrhythmia Pain [21]

Renal and cardiotoxicity Arthritis [22]

Acute hepatitis Menopausal symptoms [23]

Acute liver failure Tranquillizer [24]

Liver toxicity, neurotoxicity Sedative [25]

Chronic renal failure Anorexia and

Renal dysfunction Analgesic

Progressive renal failure and

Natural products are a mixture of phyto-constituents unlike the conventional drugs. Usually, the quantity and quality of the bioactive substance from the herbs vary depending on the part of the plant used, environmental factors, method of collection and storage conditions. The use of herbs as medicine is getting popular; hence, there is a need to address and review the interaction between the herb and the drug. Certain herbal supplements may cause dangerous side effects when taken with prescription drugs. Furthermore, the complex nature of a natural

proteinuria

Liver detoxification [14]

Nutritional supplement [15]

[17]

[19]

hypoproteinemia

anti-rheumatic

Male impotence [20]

1. *Stephaniae sinica*

*Bu Huan*

2. *Larrea tridentata*

3. *Ephedra sinica*

4. *Scutellaria baicalensis*

Yangrong Tang

6. *Vaccinium macrocarpon*

7. *Salix daphnoides*

8. *Pausinystalia johimbe*

9. *Aconitum napellus*

11. *Cimicifuga racemosa*

12. *Piper methysticum*

hood

Huang Qin

5. *Panax ginseng*

*Vernacular* name: Anshu Ling J*in*

200 Toxicology - New Aspects to This Scientific Conundrum

*Vernacular* name: Chaparral

*Vernacular* name: Ma Huang

*Vernacular* name: Renshen

Common name: Cranberry

*Vernacular* name: Willow bark

*Vernacular* name: Aconite, monk's

*Vernacular* name: Thunder god vine

**Table 1.** Some of the documented herbal toxicity cases.

*Vernacular* name: Black cohosh

*Vernacular* name: Kava kava

**5. Herb-drug interactions**

13. *Valeriana officinalis Vernacular* name: Valerian

*Vernacular* name: johimbe

10. *Tripterygium wilfordii* Hook F

*Vernacular* name: Chinese skull cup;



**Table 2.** Some of the documented herb-drug interactions.

Although one or two reports may not guarantee an absolute contraindication to combinations of herbal and prescription therapies, certain precautions have to be taken while collecting the medical history of patients during counseling sessions so as to obtain this information regarding the use of drugs and herbs in combination by the patient. Herbal drugs should be prescribed with caution in case of elderly patients, pregnant women, patients suffering from liver and kidney impairment and patients who have undergone organ transplant. The healthcare professionals can monitor the use of herbal medicines, especially when the patients are taking them along with the prescribed medicine. The patient has to be counseled with enough information about signs and symptoms of herb-drug interactions such that they are able to recognize any adverse reaction whenever it occurs. The patient should be advised to have a gap of 1–2 h or several hours between the intake of herb and drug.

Evidence-based research should be encouraged to document the data regarding the positive and/or negative effects of the use of herb and drugs in combination. Furthermore, it would be of help to if an internationally accessible database documenting the herb-drug interaction would be available.

## **6. Possible ways to reduce toxic effects of herbal medicines**

Natural substances are the best healers, but according to Paracelsus (1493–1541), all substances are poison and that's only the correct dose, which make them a remedy. There are some rules mentioned in the literature, which can be summarized as follows:


## **Author details**

**No. Herb Allopathic**

4. *Ginkgo biloba* Sodium

1. *Hypericum perforatum*

6. *Zingiber officinalis*

7. *Cimicifuga racemosa* 

11. *Glycyrrhiza glabra*

14. *Evolvulus alsinoides*

15. *Banisteriopsis caapi* 

**drug** 

202 Toxicology - New Aspects to This Scientific Conundrum

valproate

Antihypertensives

**Table 2.** Some of the documented herb-drug interactions.

Alprazolam A twofold decrease in the AUC for

creatine kinase.

5. *Panax ginseng* Phenelzine Headache, insomnia, tremulousness. Case study [34]

clearance of metronidazole.

Atorvastatin It may potently inhibit human cytochrome

enzymes.

decreased to 71%.

12. *Papain* Warfarin Skin, urinary, GIT. bleeding. Case study [41] 13. *Betula alba* Warfarin GI bleeding and a doubled prothrombin time. Case study [42]

Fluoxetine Tremors, shivering, sweating, severe nausea,

Although one or two reports may not guarantee an absolute contraindication to combinations of herbal and prescription therapies, certain precautions have to be taken while collecting the medical history of patients during counseling sessions so as to obtain this information regarding the use of drugs and herbs in combination by the patient. Herbal drugs should be prescribed with caution in case of elderly patients, pregnant women, patients suffering from

and vomiting.

10. *Allium sativum* Saquinavir Reduced plasma. saquinavir concentration. Clinical

8. *Scutellariae radix* Losartan The metabolic activities of losartan were

9. *Camellia sinensis* Folic acid Results in decreased bioavailability of folic acid.

Metronidazole Absorption and plasma half-life were

orally. administered ampicillin.

muscle pain, dark urine, and elevated

significantly increased, significantly decreased the elimination rate constant and

(CYP) 3A4, which may result in increase of atorvastatin levels, causing an elevation of liver

Patients with essential HT are more sensitive to the inhibition of beta-HSD by liquorice. Symptoms were more in women than men.

Phenytoin Loss of seizure control. Case study [43]

life.

2. *Catha edulis* Ampicillin Significantly reduced the bioavailability of

3. *Piper Methysticum* Caffeine Myoglobinuria, rhabdomyolysis, severe

alprazolam plasma concentration vs time and a twofold increase in alprazolam clearance. Shortening of alprazolam. elimination half-

**Indications Model Reference** 

3–4 seizures within 2 weeks. Case study [33]

Clinical study

Clinical study

Animal study

Clinical study

Clinical studies

study

Clinical study

Case study [44]

Case study [32]

Case study [36]

[30]

[31]

[35]

[37]

[38]

[39]

[40]

Nudrat Fatima\* and Naira Nayeem

\*Address all correspondence to: drnudrat\_ekq@yahoo.com

College of Pharmacy, Northern Border University, Rafha, Saudi Arabia

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*Edited by Sonia Soloneski and Marcelo L. Larramendy*

This edited book, Toxicology - New Aspects to This Scientific Conundrum, is intended to provide an overview on the different xenobiotics employed every day in our anthropogenic activities. We hope that this book will continue to meet the expectations and needs of all interested in the implications for the living species of known and new toxicants and to guide them in the future investigations.

Toxicology - New Aspects to This Scientific Conundrum

Toxicology

New Aspects to This Scientific Conundrum

*Edited by Sonia Soloneski* 

*and Marcelo L. Larramendy*

Photo by Haawie / iStock