Toxicity & Ecotoxicity

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Long Carbon Nanotubes Could Have Carcinogenic Effect Similar to Asbestos

Nanotechnology, the science of creating materials possessing very tiny fibers, is having an increasing influence on everyday life. Currently, researchers have demonstrated for [...]
The development of nanotechnologies has introduced important amounts of manufactured nanoparticles into the environment, including those in the ambient air and water.

Engineered nanomaterials represent a new and expanding class of chemicals whose environmental hazard is nowadays poorly determined. The peculiar behavior of nanomaterials makes them much more like new chemicals than to the corresponding bulk materials; this feature imposes reliable and standardized evaluation protocols for toxicity and ecotoxicity assessments.

The general scarcity of experimental data, their unequal distribution among the different nanomaterials and environmental conditions or the difficulties in manipulating nanomaterials are the cause of the results are not homogeneous.[1]

Nevertheless, to protect human health and wildlife from the potential adverse effects of a broad range of nanomaterials, an increasing number of studies have focused on the assessment of the toxicity of the nanoparticles commonly used in industry.[2]


Several studies exist on the toxicological properties of nanoparticles. Some of them have shown that few nanomaterials can pass through the various protective barriers of living organisms. Nanoparticles may enter into the human body via inhalation, dermal, oral and injection routes either unintentionally or deliberately.  The inhaled nanoparticles can end up in the bloodstream after passing through all the respiratory or gastrointestinal protective mechanisms. They are then distributed in the various organs and accumulate at specific sites, because of they are able to cross body membrane barriers effectively, affecting organs and tissues at cellular and molecular levels. They can travel along the olfactory nerves and penetrate directly into the brain, whether they can pass through cell barriers. The interactions of nanoparticles with biological milieu and resulted toxic effects are significantly associated with their small size distribution, large surface area to mass ratio, and surface characteristics.[3] Nanoparticles are so tiny that small quantities (expressed in terms of mass), could have major toxic effects, because of their large surface.[4] Soluble nanoparticles toxic effects are linked to their different components, regardless of the particle's initial size. However, the situation is totally different for insoluble or very low solubility nanoparticles.[5] [6]

The first feature of nanoparticles is their pulmonary deposition mode, whereby the particles will be deposited throughout the pulmonary system. It has been clearly shown that mucociliary clearance and phagocytosis are well-documented pulmonary clearance mechanisms for micrometric particles.5 6 Because of their size, nanometric particles can enter the extrapulmonary organs. This involves migration of solid particles through the epithelial layers and through the nerve endings, along the neuronal axons to the central nervous system. Thus, insoluble nanoparticles pass through the respiratory or gastrointestinal protective mechanisms and are distributed to the various organs throughout the body, including the brain. They are stored in the cells and end up in the bloodstream. These properties are currently being studied extensively in pharmacology, because nanoparticles could be used as vectors to transport drugs to targeted sites in the body.[7]

For instance, available research has indicated that ultrafine particles (UFPs) often induce mild yet significant pulmonary inflammatory responses as well as systemic effects.[8] Pulmonary exposure to NPs induces a greater inflammatory response compared to larger particles of conventional material at identical mass concentrations.[9] Following the exposure of human fibroblasts to ZnO and TiO2 NPs, cell viability was significantly reduced in a dose-dependent manner using the MTS Tetrazolium Salt assay.[10]  In vitro exposure of A549 cells to micro- and nanosized copper(II) oxide induced cell viability reduction in a dose-dependent manner; however, NPs reduced the cellular metabolic activity more severely.[11]

Currently, there is still a need for expanding efforts for addressing health and safety concerns in nanotechnology development and in nanotoxicology of engineered nanomaterials. The efforts include research to generate data for safety evaluation, toxicologic evaluation of potential human health effects, risk assessment to support risk-management decision-making, and regulations development to protect human health and the environment.[12]

Particle toxicity is associated with several parameters, mainly particle type, concentration and size distribution, frequency and duration of exposure, and pulmonary ventilation.[13] [14] The biological impacts of nanoparticles related to their physico-chemical properties are summarized in the following table3:

Table 1: Nanomaterial properties and possible biological effects.

Nanomaterial Properties Potential Biological Effects
Size/size distribution (aerodynamic, hydrodynamic) Crossing tissue and cell membranes
Cellular injury
Phagocytosis impairment, breakdown in defense mechanisms
Migration to other organs
Transportation of other environmental pollutants
Surface properties
Surface area/mass ratio
Increased reactivity
Increased toxicity
Chemical composition
Surface characteristics
ROS generation
Oxidative stress
Cytokine production
Glutathione depletion
Mitochondrial exhaustion
Cellular injury
Protein and DNA damage
Insolubility or low water solubility Bioaccumulation inside living systems such as human cells, tissues and lungs
Potential long-term effects
Agglomeration/aggregation Interruption of cellular processes
Cellular injury


Depending on the exposure profile and target cells, cellular responses can be minimal/reversible and can be recovered by the activation of adaptive responses, or they can be severe or irreversible and lead to a significant alteration of cellular structure and function as well as total cellular death or necrosis.

The nanoscale size distribution of NPs plays a significant role in their potential toxicity and their ability to cross tissue and cell membrane barriers and enter into individual cells.[15] [16] [17] Internalized NPs may interact with different subcellular compartments.[18] For example, particles smaller than 50 nm appear to enter cells and subcellular organelles such as mitochondria and the nucleus by passive diffusion.[19] [20] In addition, the very small size may lead to a direct cellular injury caused by particle-cell interactions.[21] Metal oxide NPs (e.g., Fe2O3, Y2O3and ZnO) have been internalized within human vascular endothelial cells in a dose-dependent manner proportional to the concentration in the culture medium.17

For the toxicology risk assessment, in vivo test methods would potentially be reduced to studies such as toxicokinetics, pulmonary inflammation, and potential models of fibrosis. A wide variety of in vitro assays are available to assess cellular toxicity.[22] The most frequently used in vitro assays to assess the cytotoxicity and biological responses of NPs have been reviewed.[23] [24] Researchers often tend to implement comparatively simple in vitro test systems that are relatively easy to perform, control, and interpret. However, there is a need to develop validated in vitro assay systems for toxicity testing of an expanding range of NPs.

In the field of respiratory toxicology, in vitro methods have been developed using human-based cellular systems such as airway cells, lung cells or tissues, and target-specific endpoints.[25]

Potential pulmonary toxicity of NPs can be revealed using human lung cells in vitro and relevant biological endpoints. Validated in vitro test systems can provide readily available toxicity information relevant to inhalational NM exposure. Such methods would bring many potential benefits for the risk assessment of NPs, providing simpler, faster, and less expensive toxicity screening tools.[26]

Regarding particle toxicology in vitro test methods using both human- and animal-based cellular systems can be employed. However, appropriate lung cell types need to be selected in order to represent significant features of its corresponding region in the respiratory tract. Physiologically relevant in vitro models that are able to replicate the natural cellular structure and tissue architecture of the proximal and distal parts of the respiratory tract are essential for nanotoxicology investigations.22 [27]



Nanoparticles once released into the environment organisms are likely to be exposed in different ways. Environmental issues concern air, as particulate matter dispersed in air will fall out by gravity once they are condensed or aggregated and thus reach a certain size. Both in this and other scenarios for spreading nanoparticles, water may serve as a transport medium and a temporary reservoir for nanoparticles. Yet, the ultimate recipients for any nonvolatile compound or particle spreading in the environment will be sediments and soils.[28]

Assessing the ecotoxicity of previously untested substances, as in the case of nanomaterials, is a challenging task. Nowadays, nanomaterials remain very poorly tested potential pollutants, in contrast with their large diffusion: main difficulties in assessing toxicity are a consequence of their colloidal nature and dynamics, as systems in which smaller or larger aggregates can form in poorly predictable ways, making it difficult to measure shape, size and concentration in the final sample. [29] [30] [31] [32] [33]

Measurements are conducted on different trophic levels including microorganisms, plants, invertebrates, and vertebrates, and test systems have been standardized for some organisms and for some exposure conditions. The standardized protocols specify some physical and physiological conditions (medium composition, age of organisms, etc.) as well as exposure times (e.g., for acute or chronic toxicity) and which endpoints to measure (growth, fecundity, activity of enzymes, expression of genes, etc.). Microorganisms (mainly bacteria, but also fungi, protozoa, and algae) have the advantage that they are ubiquitous and highly diverse (filling a range of habitats and functions), small (permitting miniaturized tests), and with short generation times (permitting rapid tests).28

Apart from microorganisms, there are many very useful organisms from different environments that may be used in ecotoxicity testing. It may be interesting to use organisms of different trophic levels (from different steps in a food chain), from primary producers to grazers and predators, as some environmental pollutants may accumulate in the food chain (biomagnification). In soil, organisms may also be selected based on specific modes of exposure (contact, ingestion, and prey preferences), specific habitats (surface, shallow, or deep sub-surface, aerated or anoxic environments, etc.), or specific functions (denitrification, bioperturbation, etc.). In either water or soil, organisms may also be chosen because they are important for carrying out an ecologically process, for example, related to biogeochemical cycling of elements.28 Ecotoxicity strictly means toxicity to environmental relevant organisms, while the term “bioassay” implies that toxicity or stress caused by a compound has been measured in an environmental matrix pertinent to the habitats where the organism lives in nature.[34]

The modification of nanoparticles after entering in contact with environmental matrix constituents, like ions, natural colloids, and other charged surfaces is likely not only to affect mobility, aggregation and so forth, but also to modify toxicity characteristics.[35] Therefore, ecotoxicology ENMs assessment may use a wide range of physiological and genetic endpoints, but in addition, ecotoxic organisms may be assayed on a functional level, which adds to the complexity of such investigations.


When considering the environmental risks of nanoparticles, a paradox arises when one understands that potentially dangerous nanoparticles also have the potential to produce more environmentally friendly processes, the so called green chemistry, and can be used to deal with environmental contaminants.[36] [37] An example of that is the use of engineered nanoparticles for water treatment and groundwater remediation.[38]

Finally, to assess nanoparticles risk in environment properly, in other means, to investigate whether a substance is dangerous or not, involves a determination of the material’s inherent toxicity, the manner of its intervention with living cells, and the effect of exposure time.38

Although much more research is still needed, general rules for assessing nanotoxicity and the state of the art are periodically published in reports by control agencies.

The use of approved or certified standards, a basic requirement for good practice in toxicology laboratories, seems far from fulfillment in the case of nanomaterials. In all naturally occurring systems (water, soil, air and their combinations), the organization of the dispersed nanophase depends equally from the physical-chemistry of the manufactured nanomaterials and from that of the environment, as well as from the modalities of suspension. Obtaining a gold standard for every case is far-fetched, redundant and expensive, while the use of a stable internal standard could attain a satisfactory level of laboratory practice. Furthermore, interlaboratory comparisons will improve the characterization and overcome the complexity of nanometrology.5

One feature characterizing the colloid stability is the zeta potential, which gives good information on nanomaterials mobility, aggregation rates and interactions with surfaces.[39] For magnetically charged particles, the dipole moment is a key feature in their characterization and appears to be related to the toxicity potential.[40]

The Critical Coagulation Concentration (CCC) of electrolytes (mol/l) is particularly interesting in evaluating the stability of colloid suspensions in hard and salt water.[41]

The recently released notes of the Organization for Economic Co-operation and Development (OECD, 2008) include these properties in the endpoints for phase one characterization of manufactured nanomaterials. In addition to them, water solubility and stability of dispersions, crystalline phase, dustiness, crystallite size, TEM picture(s), particle size distribution, surface chemistry, photocatalytic activity, pour density, porosity, octanol-water partition coefficient, redox potential, and radical formation potential are listed.

The second tool, i.e., a reliable measuring unit expressing toxicity, gave matter of discussion. Particle size, firstly suggested as a highly appropriate unit of measure related to toxicity, declined in popularity in recent years: the size of aggregate, not particles, seems to be more informative.[42] An alternative method, expressing nanomaterials toxicity based on mass or on surface area, seemed to be satisfactory.[43] [44] However, a revision of published data in an attempt to explain non-linear dose-response toxicity of some nanomaterials revealed that surface-to-mass area seemed to be preferred to surface-to-size, and the number of particles performed as well.[45] The problem, as claimed in many works, is probably more complex, and additional information is needed to obtain optimally informative metrics. [46] [47]

Another regrettable lacking matter is the systematic measure of elements released by dissolution. Dissolution is dependent from the chemical nature and size of the nanoparticle, as well as from environmental variables, such as pH and temperature.[48] Consequently, the measured effect and the eventually observed toxicity could be not a feature of the nanomaterial itself, but of its degrading products. However, dissolution has been very rarely documented.[49] [50] [51]

The common efforts of the worldwide control agencies and governmental organisms finally achieved their goal in coordinating the resources, and periodically updated guidelines for risk assessment of nanomaterials have been published.

The US EPA in its recently released Nanoscale Materials Stewardship Program (NMSP), elaborated an assessment of knowledge about nanomaterials: data scheduled in databases (Nanowerk Nanomaterials Database and Wilson Center Project on Emerging Nanotechnologies (PEN) Inventory of Nanomaterials in Consumer Products), or directly submitted to the NMSP were collected and compared. Sharing a part of commercially available products from those for research use only or under development, the engineering process standardization and description was the best characterized. The following best characterized point was the knowledge about professional risk factors in handling and manipulating nanomaterials. Nevertheless, experimental data about acute and chronic ecotoxicity and environmental fate score lower by far.

While natural systems represent the real target of these investigations, artificial laboratory conditions can be preferred for practical reasons. First, the steps of knowledge can speed up by using a combination of test media mimicking the natural environment, and test species adapted to the laboratory, representative of the field. Acute lethality should be conveniently studied first, long-term toxicity tests following as needed, focused on growth and reproduction as well as morphology and behavioural changes. Simplified or natural food webs should be tested to identify the levels and the risks for bioaccumulation and biomagnification[52], and modelling of exposure has been proposed.[53] Endpoints for phase one assessment of environmental toxicity of nanomaterials comprise a list of short and long-term effects on species inhabiting pelagic, sediment/benthic, soil and other terrestrial habitats.[54]

The Organisation for Economic Co-operation and Development (OECD) published in 2014 inside the Environment, Health and Safety Programme the “Ecotoxicology and Environmental Fate of Manufactured Nanomaterials: Test Guidelines”. This compilation results from expert meetings as part of its Programme on the Safety of Manufactured Nanomaterials.

Nonetheless, prevention of the escape of nanoparticles to the environment is, nowadays, the best approach under consideration. In this sense, the embedding of nanoparticles into organic or inorganic matrices reduces their mobility and prevents their appearance in the environment.[55] [56] The use of nanocomposites such as these might be the simplest way to increase the safety of nanoparticles. A complimentary approach to ensure the safety of nanoparticles is to use magnetic nanoparticles in their design[57], because of their facility to be recovered if leakage from the nanocomposites occurs by using simple magnetic traps.[58]

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