Impact of nanomaterials on ecosystems: Mechanistic aspects in vivo

Journal Pre-proof Impact of nanomaterials on ecosystems: Mechanistic aspects in vivo Mandeep Singh Bakshi PII:

S0013-9351(19)30895-3

DOI:

https://doi.org/10.1016/j.envres.2019.109099

Reference:

YENRS 109099

To appear in:

Environmental Research

Received Date: 3 October 2019 Revised Date:

27 December 2019

Accepted Date: 27 December 2019

Please cite this article as: Bakshi, M.S., Impact of nanomaterials on ecosystems: Mechanistic aspects in vivo, Environmental Research (2020), doi: https://doi.org/10.1016/j.envres.2019.109099. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

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Impact of Nanomaterials on Ecosystems: Mechanistic Aspects In Vivo Mandeep Singh Bakshi Department of Natural and Applied Sciences, University of Wisconsin - Green Bay, 2420 Nicolet Drive, Green Bay, WI 54311-7001, USA E mail: [email protected]

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Abstract Nanotechnologies are becoming increasingly popular in modern era of human development in every aspect of life. Their impact on our ecosystem in air, soil, and water is largely unknown because of the limited amount of information available, and hence, they require considerable attention. This account highlights the important routes of nanomaterials toxicity in air, soil, and water, their possible impact on the ecosystem and aquatic life. The mechanistic aspects have been focused on the size, shape, and surface modifications of nanomaterials. The preventive measures and future directions along with appropriate designs and implementation of nanotechnologies have been proposed so as to minimize the interactions of nanomaterials with terrestrial flora and aquatic life. Specifically, the focus largely remains on the toxicity of metallic nanoparticles such as gold (Au) and silver (Ag) because of their applications in diverse fields. The account lists some prominent mechanistic routes of nanotocxicity along with in vivo experimental results based on the fundamental understanding that how nanometallic surfaces interact with plant as well as animal biological systems. The appropriate modifications of the nanometallic surfaces with biocompatible molecules are considered to be the most effective preventive measures to reduce the nanotoxicity.

Keywords: Nanotoxicity, metallic nanoparticles, zebra fish, mechanistic aspects, nanometallic surface coating.

1. Introduction 1.1 Nanotechnology With growing population and decreasing natural resources, sustainability has become the necessity of modern times and its dependence on advanced technologies such as nanotechnology. While nanotechnology has proved its vast potential in all walks of life, it has brought significant unexplored risk factors which require considerable attention. Among various possible modes of sustainability, engineered nanomaterials are an emerging class of new materials1-4 with enormous potential in environmental remedifications and nanomedicine. Some of the prominent

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applications include surface treatments of fabrics, nanoscale films on eyeglasses, windows, and other surfaces as water repellent, antifog, antimicrobial agents, catalysts for chemical reactions, environmental sensors, air purifiers, and filters etc. Synthesis, characterization, and applications of suitable materials at nano-scale provide several advantages in terms of large surface area as well as ability of such materials to carry deliverables.5,6 However, toxicity7-10 of such materials is an important issue and needs to be addressed simultaneously to evaluate their impact on the biological sustainability of terrestrial and aquatic environments containing vast natural resources. Frequent use of metallic (most common noble metals), oxide, and carboneous nanomaterials increases their concentration in air, water, and soil on which biological sustainability of both terrestrial as well as aquatic environments depends. It ultimately affects the growth of ecosystem and life cycle of aquatic species in different ways that in turn could affect the human health.1,4 In fact, numerous applications that utilize nanotechnology result in the production of nano-waste that contains engineered nanomaterials. The nano-pollutants thus produced are hard to monitor due to their nanoscale dimensions, and hence, require their disposal in appropriate ways so as to minimize the adverse environmental and health impacts. Thus, the aim of the present account is to bring forward the risk factors involved in diverse applications of nanomaterials as far as different forms of life is concerned on this planet. An effective solution for the risk factors depends on our fundamental understanding of their mechanistic aspects which include their molecular origin, routes, and end results. In vivo studies in complex biological systems are considered to be the most realistic ways in tracing the fundamental basis of the mechanistic routes involved in the risk factors. Therefore, this account attempts to bring forward the potential parameters of risk factors by following the mechanistic steps through in vivo studies, effective preventive measures, and future directions. 1.2 Nanometallic particulates Among the noble metal nanoparticles (NPs), Au and Ag are the most common NPs frequently used for different applications because they produce quite stable aqueous suspensions consist of NPs of same size and shape essentially required for a successful application. They can be easily synthesized in nano-dimensions at room temperature through aqueous phase synthesis by using weak reducing agents to convert metal ions into their respective atomic states.11,12 Different forms of complex biological fluids such as plant extracts are frequently used to

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synthesize Au and Ag NPs so as to produce sustainable nanomaterials with high affinity to interact with the biological systems.13-16 Plant extracts usually contain weak reducing ingredients in the form of water soluble proteins, carbohydrates, or other biomolecules with enough reduction potential to convert the metal ions into nucleating centres without the use of any external reducing or stabilizing agents. Apart from inducing reduction of metal ions, biomolecules also possess the ability to simultaneously provide the colloidal stability to the growing nucleating centres thus produce. The freshly produced lattice planes of growing nucleating centres act as active sites for the simultaneous adsorption of biomolecules through both specific as well as non-specific interactions.5 This process allows the biomolecules to produce a strong coating which is fully capable of perusing inherent biological functionalities. This kind of unique three components (biomolecule + gold salt + water) green chemistry approach of producing nanometallic NPs is completely carried out in aqueous Biomolecules + Au3+(aq) + 3 e
Auo + oxidizing species environment and without the use of any organic solvent. NPs thus produced are considered sustainable nanomaterials because they usually carry surface adsorbed bio-conjugated species that can be used for different sustainable applications.13,18-20 Such bioconjugated nanomaterials when become the part of food supply lines of aquatic and terrestrial flora due to their biocompatibility with aqueous environment, soil nutrients, and air, they may induce inherent toxicity. 1.3 Nano-oxides and carboneous particulates Other sources of nanotoxicity may be contributed by nano-oxides and carboneous particulates which find their way to ecosystem through (i) artificial soil (that is becoming increasingly popular to achieve plant growth in quickest ways); (ii) pesticides made from inorganic and organic compounds as well as from colloidal sulphur and lime, and (iii) recycled water.21-24 Dependence of agricultural sustainability on nanotechnologies has allowed potential exposure of such NPs to plant living cells which is an essential source of human consumption. These particulate matters encounters enough opportunities to interact with both plant and animal cells. Although nano-oxides (Al2O3, SiO2, Ti2O3, ZnO etc.) and carboneous particulates usually exist in large dimensions, they are considerably smaller in size than that of the living cells. In

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fact, nanotoxicity in our environment is related to the nanoscale size of NPs that provides a large size disparity between the NPs and the living cells in agricultural soil. Cells are usually of several microns in size and much bigger in dimensions than the NPs. Such a huge size disparity allows live cells to be an easy target of nano-contaminations because several NPs can simultaneously interact with a single live cell.5 A large surface area of small NPs provides plenty of opportunities for NPs to interact with the cell membranes though different modes of interactions with little detection and far reaching consequences with little information to date. 1.4 Bioavailability of nanoparticulates The nanoparticulate matter of a wide range of sizes and different surface chemistries are bioavailable to plants through cell wall pores of plant leaves, the roots, and through water channels.20 Naked or bioconjugated NPs are also easily available to plants hydroponically. Hydroponic environment in turn provides plenty of opportunities for different kinds of biomolecules to adsorb on the free nanometallic surfaces and hence, metallic NPs in disguise of nutrients sneak into the food supply of ecosystem.13 Several model studies show the in situ synthesis of NPs carried out by different biomolecules or bioextracts that result in bioconjugation of growing nucleating centres and eventually lead to the formation of bioconjugated NPs.13-16 A systemic mechanism of in situ reduction of metal ions, colloidal stabilization, and crystal growth controlled in a step wise manner is depicted in Figure 1.25-28 Such bioconjugated NPs can be easily taken up by plants from different soil, water, and aerial sources, and hence, capable of inducing nanotoxicity to a considerable extent.1,3,4 This account highlights different characteristic features of nanomaterials and their toxicity toward terrestrial and aquatic biological systems at nano-scale. In other words, the objective is to develop a correlation between the shape, size, composition, and surface modifications of nanomaterials, and their environmental nanotoxicity.

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Figure 1. Schematic representation of the various steps involved in the synthesis of Au microplates (a−e) and nanowires (f−j) in the presence of dextran and carboxymethyl cellulose, respectively. Steps (a) and (f) represent the reduction reaction, (b) and (g) nucleation, and (c−e) and (h − j) growth processes of respective cases. (Reproduced with permission from ref. 27)

2. Aerial channel Diversified uses of nanotechnologies in every aspect of human life release plenty of NPs in air. Nano-dimension sizes of NPs allow them to easily air-born and carry away by the air currents to make terrestrial flora a primary target (excluding animal respiratory system in this account). Bioavailability of air born NPs to terrestrial flora happens in a restricted manner because the supply channels are available only at the surfaces of plant leaves where the probability of penetration depends on several factors such as size and shape of the NPs and their

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concentration in air.20 Even their high aerial concentrations do not induce significant nanotoxicity especially in the vicinity of iron ore mines where there is no shortage of metallic NPs of different shapes and sizes. This is probably in line with the fact that the depletion of ecosystem particularly in the areas close to the iron ore mines are not blamed for the air-born particulates of different shapes and sizes rather contaminated soil around the mines that becomes the source of primary supply route of metal particulates. A systematic research suggests that supply of nano-particulates to wheat leaf shows the presence of much less Au concentration than the estimated detection limit20 as determined by scanning X-ray fluorescence. Similarly, wheat leaf tissues reveal no evidence of accumulation of Au NPs in the aerial portions of the plants.20 In contrast, the nature of chemical root exudation of plant species is considered to play a dominant role in the uptake of NPs. However, some recent studies indicated the foliar supply route of NPs and tried to determine the size, shape, and concentration effect of NPs on their penetration rate.29,30 Usually NPs of size limit of 50 nm show penetration while bigger NPs are filtered out (Figure 2).30 Figure 2 is a schematic representation and referring to the shape and size of the NPs which can pass through stomatas or cell pores on the basis of size. A size of 50 nm is still considered to be much larger than the pore size of plant cell wall or root hairs which is close to 4 nm. The permeation properties differ between cuticles on the epidermal cells and trichomes on stomata31 because of the different chemical compositions and functionalities. NPs enter through stomatas are deposited on the cell wall of substomatal cavity.32 They travel through the cell wall in order to enter into protoplast. Only NPs less than 5 nm size are expected to traverse the cell wall of undamaged cells efficiently but they too are expected to face obstructions from the globular protein of ~ 30 kDa with approximately 3 nm in size. Thus, small roughly spherical polyhedral NPs are considered to be more toxic than the other shapes such as plates, rods, or wires because they cannot easily

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Figure 2. Schematic representation of modes of penetration of NPs of different shapes through stomatas and possible penetration through the nuclear pores depending on the size and shape of NPs.

penetrate through the pores. Interestingly, the low concentration of NPs is more toxic than the higher concentration. The issue is more complex to understand because it is related to the interparticle interactions which become prominent at high concentration when NPs are present on foliage surfaces in close vicinity of each other.32 The stability and inter-particles interactions of colloidal particles are best explained on the basis of DLVO (Derjaguin, Landau, Verwey, Overbeek) theory33,34. The potential energy of interacting colloidal particles is the sum of potential energy due to electrostatic (VR) and van der Waals (VA) interactions. The inter-particle interactions among the naked and coated NPs are governed by the mutual balance between the VR and VA. The magnitude of VA is higher than that of VR for the uncoated NPs while reverse is true for the coated NPs. Higher concentration of naked NPs allow them to interact with each other through predominant VA. It leads to the formation of large clusters which are unable to penetrate through the pores, thus making their higher concentration less toxic than the low concentration. Overall, it seems that the air nanotoxicity does not significantly affect the growth of foliage density of plants because the possible routes of supply lines such as stomatas and pores are not easily accessible to the NPs for the penetration. 3. Soil channel

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3.1 Bioavailability and nanotoxicity of metal NPs, ions, and combined affect Soil route of environmental nanotoxicity is an important channel that impact the growth cycle of territorial flora. The rapid industrialization and its dependence on the nanomaterials spill plentiful nano-particulates in soil that eventually affect the growth cycle of terrestrial flora in many different ways. Both NPs as well as metal ions induce toxicity independently as well as in a collective manner to form metallic alloys. Synthesis of nanometallic alloys is usually triggered by the surface catalysis of metal NPs when metal ions undergo auto-reduction on free metallic surfaces.11 Apart from this, the presence of different types of NPs in soil induces their mutual NPs – NPs interactions with each other in addition to their inherent toxic effect. The amount of nanomaterials taken up by the plants through soil is further affected by the presence of other metal NPs as well as ions, and their affinity to form metal alloy. This is quite common in nanotoxicity induced by Au NPs through soil supply route which is also contaminated with Ag

Figure 3. Scanning electron microphotographs of high-density metal structures in the plant ash prepared for each treatment. All images were obtained using a dual segment back scatter collector that shows high-density particles as bright objects. The chemical identity of the particles in these images was confirmed using X-ray microanalysis. (A) control, (B) silver-only, (C) copper-only, (D) gold-only, (E) copper and silver, (F) silver and gold, (G) copper and gold, and (H) copper, silver and gold. (Reproduced with permission from ref. 35)

and Cu ions. Cu and Ag contaminations around Au mines directly affect the bioavailability of Au.35 During the phytomining of Au,36,37 Cu and Ag possess high affinity to complex Au to form bimetallic alloy in the form of nano or microstructures. This process is facilitated by the weak reducing ability of plant biomass that converts Au(III) and Ag(I) into their respective metallic states in comparison to that of Cu(II). It predominantly produces Au – Ag bimetallic nanoalloys

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in comparison to either Au – Cu or Ag – Cu alloys because of the higher positive potential of Au(III) than Ag(I) and Cu(II). During this process, the uptake concentration of Au is significantly reduced when an elevated concentration of Cu and/or Ag are also present in the soil (Figure 3).35 The order of this effect is Cu < Cu/Ag < Ag, and the Au concentration is significantly lower in the event of Au-Ag bimetallic alloy formation rather than that of Au-Cu bimetallic alloy. The bimetallic NPs produced in this way are much larger in dimensions because

Figure 4. Schematic diagram showing the mechanisms behind the biogenic synthesis of metallic nanoparticles. (Reproduced with permission from ref. 39) their growth is driven by the seed-growth method11,12 in the soil where small Au NPs act as seeds which invite Ag and Cu ions for further catalytic reduction. The higher reduction potential of Au also facilitates its reduction through biogenic processes to produce small NPs in the first place that act as seeds and are involved in the growth process to produce much bigger bimetallic NPs. Biogenic processes are mainly contributed by various physiological and biochemical pathways38 present in biological systems that result in the reduction of metal ions into metal NPs. A detailed compilation can be found elsewhere (Figure 4).39 Specifically, proteins and secondary metabolites in water soluble fractions of geranium leaves or terpenoids act as efficient reducing agents for silver ions.40 Flavonoid and terpenoid derivatives of neem leaf broth help in the

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stabilization of NPs41 while aldehydes and ketones play an important role in the shape control synthesis of Au NPs. The biomolecule stabilized NPs thus produced do not induce a direct toxicity by incorporating themselves in the plant-growth cycle rather they are involved in the indirect process of impeding the nodulation formation which is the main source of food supply line.

Figure 5. Nodulation frequency (number of nodules per plant). Treatments with the same letter are not significantly different from one another at α = 0.05. Error bars = standard error. ENM = engineered nanomaterial. (Reproduced with permission from ref. 42)

(Tables 1 and 2 are reproduced with permission from ref. 42)

3.2 The mechanism 3.2.1 Role of metallic NPs The mechanism of nanotoxicity through soil supply route starts with the reduction of nodulation.42 Nodulation is the process by which roots associate with symbiotic nitrogenfixing bacteria to perform symbiotic relationships. Bioavailability of metal NPs in soil provides

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ample opportunities for the nanometallic surfaces to interact with symbiotic bacteria available in large amounts for nodulation. This could happen in many different ways.43 Biomolecules coated NPs can interact with the negatively charged cell wall of nitrogen fixing bacteria like rhizobia and bradyrhizobium through their electropositive functional groups. This association causes membrane damages and oxidative stress44-46 with concentrations higher than 10 mg·L−1. The nodulation rate in the plants that are grown in the presence of engineered nanomaterials significantly lowers than that observed in the control (Figure 5).42 Even the shoot length and shoot biomass show significant retarded growth in comparison to the control (Table 1). A simple comparison between the bioavailability of metal from the Ag, TiO2, and ZnO engineered nanomaterials and that in the dissolved media indicates the fact that metal in the NPs pose greater inhibition than that present in the bulk and the effect is more pronounced on fungi and gram negative bacteria (Table 2).42 Germination and seeding growth of faba bean plants is reduced by 40 % when they are exposed to Ag NPs at concentration 800 µg kg−1 soil.47 Ag NPs considerably retard the process of nodulation, nitrogenase activity, mycorrhizal colonization, and glomalin content. High concentration of Ag NPs results in detectable alterations including the intracellular deterioration of cytoplasmic components by means of autophagy and disintegration of bacteroids. In fact, all algae, bacteria, and fungi have the capacity to biosynthesize Au and Ag NPs following dissolved metal uptake (Figure 6).48-51 This is another important topic in terms

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Figure 6. Scanning electron microscopic images of whole cell (first row) and transmission electron microscopic cross-sectional images (second row) were collected for P. subcapitata incubated with 0−0.5 mM HAuCl4 for 72 h, filtered through a 0.2 µm filter, and fixed with 2.5 % glutaraldehyde. Electron-dense gold is displayed as white in ESEM images and black in TEM images. The third row summarizes the characteristics of the Au NPs for each condition. (Reproduced with permission from ref. 48)

of growing interest in the optimization of gold recovery following the biogenic processes.52 It happens when negatively charged cell wall electrostatically attracts gold ions where peptidoglycan or enzymes reduce them to Au(0). This results in the nucleation and subsequent growth of NPs which eventually transport through the cell wall.53,54 Alternate pathways for Au(III) to Au(0) bio-reduction by bacteria and fungi take place through thylakoid membranes, reductase enzymes, and cytochromes.48,53 Pronounced toxicity exhibited by Cu and Ag NPs has allowed Au NPs as safer and more biocompatible antimicrobials55-57 though Au NPs are generally regarded as non-bactericidal.58 They can be made strong antimicrobial agents simply by surface modification which enhances their ability even against multidrug-resistant bacteria59 by disrupting the bacterial wall or by generating the reactive oxygen species as well as by choosing appropriate NPs shapes.60-63 3.2.2 Bioaccumulation of metals In addition to nanotoxicity towards nitrogen fixing bacteria, decrease in nodulation is also caused by down regulation of several genes related to nodulation and up regulation of genes related to flavonoid biosynthesis and oxidative stress and metal tolerance.64 Bioaccumulation of Zn changes gene expression that causes suppression of nodulation.42 M. truncatula nodulation is particularly sensitive to Zn exposure.65,66 Recent studies show the transformation of CeO2 NPs in plants by releasing Ce3+ ions and transforming into CePO4 or Ce carboxylates.68 They accumulate in plants in various forms rather than as only CeO2.69 CeO2 NPs are highly popular in agricultural and biomedical applications because of their unique redox cycling between oxidation states of Ce(III) and Ce(IV).68 Transformation of the rod shaped CeO2 NPs happens much faster with greater chemical reactivity in comparison to other shapes with nearly 40 % of Ce in the form of Ce(III) species in roots (CePO4) and shoots (Ce carboxylates). Smaller CeO2 NPs release more Ce3+ ions while functionalized CeO2 NPs releases less amount.70

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3.2.3 Role of carbonaceous nanomaterials Another source of nanotoxicity is the carbonaceous nanomaterials that include multiwalled carbon nanotubes, graphene nanoplatelets, and carbon black apart from naturally occurring carbon particulates.71 Rapid use of coal, hydrocarbons, and carbon alloys produce nanosized carbonaceous nanomaterials in different forms. Activated carbon, carbon black, graphene, graphite, and carbon nanotubes with significant industrial applications are spilling into soil. The annual global production of carbon nanotubes and graphene has increased tremendously72,73 that eventually end up in the soils.74,75 Apart from this, carbon based materials are frequently used in agricultural sands for their modification and to fertilize and protect crops.76 Their hazardous effects on terrestrial plants have already been discussed to some extent77-79 while the effect of modern carbonaceous nanomaterials such as carbon nanotubes and

Figure 7. (left) Relative comparison of inverse dose response of different nanomaterials on N2 fixation potential. (right) Transmission electron microscopy images of soybean root nodules at the final harvest from either (A, B) the controls (Ctrl), or the low exposures (0.1 mg kg−1) of either (C, D) carbon black (CB), (E, F) multiwalled carbon nanotubes (MWCNTs), or (G, H) graphene nanoplatelets (GNPs). Scale bars are indicated in each image. Dense accumulations of bacteroids are evident in the Ctrl images at (A) high and (B) low magnifications. Within some bacteroids (e.g., Ctrl in part A), electron transparent (white-appearing) features (indicated by white arrows) are inferred to be poly(β-hydroxybutyrate) inclusions. White double arrows point to apparent nanomaterials inside root nodule cells in the images for the CB (C) and GNP (G, H) treatments, only. The center double arrow in the CB image C indicates where nanomaterials appear associated with a bacteroid. Densely packed bacteroids are evident in one image (H) for the GNP treatment. Accumulations of putative starch granules (indicated by white arrowheads, external to bacteroids) are observed in the images for the CB (D), MWCNT (F), and GNP (G) treatments. (Reproduced with permission from ref. 71)

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graphenes is worth discussing. Low amounts of carbon nanotubes (~ 0.1 mg kg−1 dry soil) lead to significantly shorter growth than the control (Figure 7, left panel). They also show slower leaf cover expansion with much lower final total leaf area than the control.71 Similar effect is observed when graphene (500 − 2000 mg L−1) is used. It also inhibits the shoot length, leaf count, and leaf area of tomato, cabbage, and red spinach.80 Low amount of carbon nanotubes (Figure 7E, F) makes nodules to appear comparatively empty with large vacuoles81 and the bacteroid density inside nodules was significantly lower than the control71 (Figure 7C, D). Thus, multiwalled carbon nanotubes, graphene nanoplatelets, and carbon black just like the noble metal and metal oxide nanomaterials significantly affect the plant growth particularly the symbioses of N2 fixing bacteria with greater impact of lower concentrations of carbonaceous nanomaterials (~ 0.1 mg kg−1 dry soil) than the higher concentrations (~ 1000 mg kg−1 dry soil).71 The higher concentration effect in fact reduces the bioavailability of carbonaceous nanomaterials due to extensive agglomeration. These findings contribute to a better understanding of the potential implications of carbonaceous nanomaterials in agricultural systems. 4. Aquatic channel 4.1 Zebra fish as a “in vivo” model system

(i)

(ii)

(iii)

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Figure 8 (i). Characterization of Ag NPs embedded in embryos. (A) Representative (a) color image and (b) LSPR spectra of single Ag NPs embedded in chorion layers, showing that single Ag NPs with multiple colors (blue, green, red) are present inside chorion layers, and some NPs are overlapped with the chorion pore canals (note an array of chorion pore canals, highlighted by a triangle). Scale bar 1 µm. (B) Representative images of individual Ag NPs embedded in the chorion layers, illustrating those Ag nanoparticles (as indicated by a circle) trapped in the chorion pore canals, outlined by ellipses. (ii). Histograms of distribution of normally developed (green) and dead (red) zebrafish, (A) versus concentration of Ag NPs and (B) versus concentration of supernatants resulting from washing Ag NPs (negative control). (C) Histogram of distribution of deformed zebrafish (120 hpf) versus concentration of Ag NPs. (D) Histogram of distribution of five representative types of deformities of the zebrafish versus concentration of Ag nanoparticles: finfold abnormality (purple), tail and spinal cord flexure and truncation (black), cardiac malformation (pink), yolk sac edema (light green), head edema (brown), and eye abnormality (orange). (iii). Dependence of types of deformed zebrafish on Ag NP concentration. (A) Optical images of deformed zebrafish show (a,b) finfold abnormalities; (c,d) tail/spinal cord flexure; (e) cardiac malformation and yolk sac edema; and (f,g) eye abnormality. (B) Histograms of distributions of embryos that developed into deformed zebrafish with five distinctive types of deformities at NP concentrations of 0.02, 0.05, and 0.10 nM. The scale bar is 500 µm for all images in A. (Reproduced with permission from ref. 85, 86)

After air and soil, “aquatic resources” is another vast area which could expose to nanotoxicity and hence, may affect the aquatic life. Aquatic life has close relationship to human health. Apart from the contaminated sea food, it is worth studying the influence of nanotoxicity in vivo on fish growth that resembles in many ways to the human physiological system. The toxicity in sea food is considered to be a gold standard for ecotoxicology. It has been successfully addressed by taking zebra fish as a model system. Zebra fish is a derived member of family Cyprinidae and its name originates from the five uniform pigmented, horizontal, blue stripes on its body. It can grow maximum 6.4 cm in a lifespan of around two to three years.82,83 Biologists choose it as a model system because its genome is fully sequenced, well-understood, and its embryonic development is very rapid and transparent.84 Because of its transparent body texture, it proves to be an excellent model in vivo to observe the nanometallic toxicity on various stages of embryonic development in a real-time study of transport and biocompatibility of single nanoparticle. It could provide new insights into molecular transport mechanisms.85 The surface plasmon resonance (SPR) of noble metal NPs depends on the shape and size of the NPs. It allows them to easily traceable in a transparent biological medium. Rayleigh scattering of 2 nm Ag NPs is about 104 times higher than that of a single fluorescent rhodamine molecule which helps to locate them in complex biological fluids as of zebra fish embryo.85 Unlike fluorescent probes,

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noble metal NPs do not suffer photodecomposition and hence, can be used as probes to continuously monitor dynamic events on the growth process of zebra fish. When the zebra fish embryo encounters the toxicity of NPs, the chorion layers try to restrict the diffusion of NPs through the chorion pore canals (Figure 8i). However, considering the much smaller dimensions of NPs, majority of NPs freely diffuse into embryos while some single nanoparticles stay in chorion pore canals for an extended period of time.85 These NPs serve as nucleation sites and aggregate with the incoming ones to form larger particles (Figure 8iB) that in turn clog chorion pore canals and hence, affect embryo’s transport system. A concentration higher than 0.19 nM leads to dead and deformed zebra fish indicating a critical concentration of Ag NPs for the development of zebra fish embryos (Figure 8ii). Deformities include finfold abnormality (which occurs at the highest rate), tail/spinal cord flexure, cardiac malformation, yolk sac edema, head edema, and eye deformity. NPs accumulated inside embryos alter the charge, diffusion, and interact with biomolecules in a dose-dependent manner that in turn induces interference or malfunctioning of signalling processes.85 4.1.1 NPs morphology and concentration effects NPs size is another important factor in embryonic development where large Ag NPs of ~40 nm are more toxic than the smaller ones and induce severe deformities such as cardiac abnormalities, yolk sac edema, and eye/head abnormalities.86 They can induce abnormalities with a concentration as low as 0.02 nM (Figure 8iii) or equivalent to 5 µg/ml which is considered to be quite high elemental concentration. The transition metal NPs such as of Au or Ag are quite stable in aqueous state and the possibility of degradation of NPs into ionic state is usually very low. Thus, the reported toxicity is mainly induced by the nanometallic form of Ag NPs rather than the elemental concentration usually considered in the environmental pollution from different industrial sources. As far as the embryonic development stages are concerned,87 the cleavage stage is highly sensitive to the toxic effect of NPs followed by gastrula stage, early, and late segmentation stage, while hatching stage is the most resistant one. In fact, cleavage and gastrula stages result in the deformed zebra fish with different abnormalities. Among different organs, the fish gill88 is a most vital organ that is the primary target of pollutants89,90 and particulate matter. Particulate matter induce morphological abnormalities in the epithelial layer of gill.91,92 Ag nanoplates demonstrate high toxicity at low concentration in cell death of about 70

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% of rainbow trout gill fish cell line, while the Ag nanospheres require relatively higher concentration (25 µg/mL) in order to induce cytotoxicity with much more superoxide formation.88 It suggests that the surface reactivity is the driving force towards the cyctoxicity which can very well be controlled by the surface modifications that suppresses the superoxide production. This is most prominent in the case of nanoplates rather than in the case of other morphologies because nanoplates can provide a large surface area of nanometallic pollutants that can come in contact with the cell membrane. Apart from this, single crystal surfaces are highly potent to interact with the biological membranes because they possess a uniform surface to interact with surface active biomolecules.93-95 Surface adsorption of biomolecules reduces the surface energy and adsorption is further related to the structural compatibility of biomacromolecules to the uniform crystal lattice planes of single crystal. Kinks and steps referred as crystal defects are the active sites which significantly promote these interactions and

Figure 9. Characterization of SERS NPs embedded inside a fully developed (48 hpf) zebrafish using SERS measurement. (A) Optical image of a normally developed zebrafish. The red markers highlight representative areas: (I) forebrain, (II) pharyngeal skeleton, (III) hindbrain, (IV) heart, (V) yolk, and (VI) somite. (B) SERS spectra obtained from those tissue sections outlined in panel A. (Reproduced with permission from ref. 98)

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lead to the disruption of biomolecules or cellular structures.96 Single crystal nanoplates are usually only few nm thick, bound with {111} crystal planes. Thin nanoplates are usually prone to the crystal defects27 and hence, possess several active sites for the interaction and adsorption of amphiphilic biomolecules constituting the biological membranes. Nanowires on the other hand can provide much less surface area to be in contact with the cell membranes and are considered to induce relatively much lower toxicity. However, long aspect ratio nanorods show significant toxicity97 by damaging the microvilli that result in the digestive malfunction, nutritional deficiency, retarded growth, and abnormal physical development. It is not easy for the zebra fish embryo or larvae to uptake long nanowires but nanorods are equally up taken as much as polyhedral nanomaterials.

4.1.2 SERS analysis Surface enhanced Raman spectroscopy (SERS) is a powerful tool to quantify the interactions of NPs with biological systems. SERS can quantify the internalization of NPs in embryonic zebra fish. It can monitor multiple sites simultaneously during embryonic development with single molecule sensitivity (Figure 9).98 SERS can be applied to understand the nanotoxicity under in vivo conditions. Au NPs of 40 nm size yield higher enhancement in live embryos. During the embryonic development and cell division, the number of SERS NPs in each cell decreases to produce a weaker signal. In vivo imaging shows98 that the NPs spread throughout the cytoplasmic bridges which connect all zebra fish blastomeres. The observed distribution of NPs in various tissues of the embryo happens during the cleavage stage of the development and hence, NPs eventually travel into different organs and tissue progenitor cells which ultimately develop into zebrafish.99 Stronger SERS signals inside the embryo are caused by the self-aggregation of NPs in small cluster.100 They may also be triggered by the NPs – NPs fusion or nucleation. Biological medium is a complex mixture of several different kinds of bioactive molecules which may complex with NPs and thus, promote their self-aggregation or nucleation. SERS is a fine technique to apply for observing the nanotoxicity of NPs under in vivo conditions while monitoring the locations of NPs at different stages of the embryonic development. 5. Nanotoxicity in relation to the shape, size, and surface modifications of nanoparticulates

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Among the shape and size of the NPs, size effect is considered to be more important in comparison to the shape effect. Several studies highlighted the importance of size effect where smaller size NPs i.e. less than 40 nm, are considered to be more toxic than the larger NPs.85,86 Different aerial, soil, and aquatic routes are the ideal channels for the nanometallic toxicity where smaller NPs pose greater challenges in comparison to the bigger ones. Although stomatas are quite wide enough (several microns) to allow the passage of large NPs (Figure 2), cell pores only allow the passage of small NPs. Thus, small NPs are potentially hazardous air as well as soil pollutants because they affect the plant growth mainly by penetrating through the pores of the plant cells. Small NPs are also potent antimicrobial agents and hence, they effectively reduce the nodulation formation.47-51 Interactions between the plants or bacterial cells and the NPs are further related to the surface area in contact with the cell membrane. This aspect is mainly related to the shape of the NP rather than the size, thus, it makes the shape an important criterion for the nanotoxicity as well. The shapes which provide greater surface area in contact with the cell membrane, possess higher cell membrane disrupting potential. Surface contact of NP may induce strong local membrane deformation or may result in the cell internalization depending on the shape. In both cases, this association becomes energetically favorable where the amount of energy released from the binding of NPs is overcome by the amount of free energy required to bend the membrane (Figure 10A). The former energy is associated with the external surface area while the latter is proportional to the curvature or inversely proportional to the square of the radius (r) of the particle.101-103 A large surface area thus provides a greater binding energy for pulling the membrane to the particle surface and hence, lower bending energy is required to wrap the larger NP in comparison to the smaller one.102 This makes the membrane wrapping and engulfment of NPs thermodynamically favorable. Among the various shapes, rice shape of NP

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(A)

(B)

(C) Figure 10. (A) Schematic illustration of the size- and surface dependent interaction with blood cell membrane. NP with radius r can be wrapped around or engulfed by the blood cell if the energy (Ei) released from the blood cell-NP interaction is greater than the energy (Eb) required for membrane bending. (B) Analysis of high-charge-density rice NP translocation through the lipid bilayer. (a−d) Coarse-grained molecular dynamics simulation snapshots of rice NP (orange) approaching the lipid bilayer (green, magenta, and gray) and undergoing reorientation during translocation. (C) Post-translocation, 3D contour plot of changes in the cell−membrane thickness (nm) (e). (f) PMF (kJ/mol) profile as a function of center-of-mass separation (nm) of the NP from the center of the lipid bilayer. The background image of the plot is provided as a visual aid to track the reorientation of the NP along the PMF curve. (Reproduced with permission from ref. 102, 104)

is highly potent. Adhesion of rice NPs orient parallel to the oppositely charged lipid membrane (Figure 10B).104 The parallel orientation maximizes adhesion caused by the attractive Coulombic interactions that overcome entropic orientation diffusion effects (Figure 10Ba−d) resulting in a substantial disruption of the lipid bilayer self-assembly (Figure 10C). It causes a bilayer thickness decrease from its usual value of ~5 nm to as low as 1 nm at the highest impact points and is appropriately explained by the potential of mean force (PMF) curve (Figure 10C).104

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However, a rice NP with low positive surface charge density does not undergo reorientation because of diminished Coulombic interactions. This makes the surface charge on the NP surface another important criterion which determines the mode of cell membrane bending and wrapping (Figure 11).105 The neutral NP simply stays at the embedded pore of the lipid bilayer due to the

Figure 11. (a) Schematic diagram of a lipid bilayer membrane with bound neutral and randomly charged nanoparticles. (b−e) Representative trajectories and distributions of turning angles for the nanoparticles with randomly (b, c) and uniformly (d, e) distributed surface charges. Color bar denotes the time lapse of each trajectory. (Reproduced with permission from ref. 105) hydrophobic surface of the NP106 while the membrane concaves up to wrap a negatively charged NP and it concaves down for a positively charged NP. This mechanism is further related to the tilt angle. A charged NP can alter the tilt angle of the head of zwitterionic lipids which acts as a dipole.107 The positively charged NP interacts preferentially with the negative end, reducing the tilt angle while negatively charged NP raises the angle of the dipole. Simulations studies indicate that NP with negatively charged functionalization showed no preference for translocation across the lipid bilayer108,109 because they are electrostatically repelled by the negatively charged lipid bilayer and energetically less favourable, while a change in the surface charge density demonstrate pronounced effect on translocation for the positively charged NP. NP adhesion disrupts the equilibrium charge distribution of the cell membrane that eventually permits NP internalization.110 Especially in aquatic environments, NP behaviour also depends on the physiochemical aspects of the surrounding medium111,112 which are further related to the pH,

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ionic strength, ionic composition, and hydrodynamic conditions.113 These factors are important in determining whether NPs exist in self-assembled state that may alter the mechanism of their interactions with cell membranes and which in turn may significantly alter transport mechanism as well as nanomaterial toxicity.111,113 The physiochemical properties may also facilitate the interactions of NPs with organic matter, humic substances, and dissolved molecules such as phosphates and sulfates.113 Thus, aggregation and deposition are two closely related processes which may have marked effect on the nanomaterials toxicity because larger aggregates up taken by the organisms may find their way into food chains.114 On the other hand, a carefully chosen zeta potential of colloidal NPs can reduce their impact on the cell membrane disruption effects. The negatively charged NPs neither demonstrate hemolytic activity nor cytotoxicity.115 In addition, cytotoxicity studies indicated that TiO2 and Fe2O3 caused decreasing cellular viability over 48 h while Al2O3, ZnO, and CeO2 cellular viability remained similar to control.116 Thus, zeta potential may be used as a predictive measure of nanotoxicity. Although, there is no clear evidence how nanomaterials of different shapes, size, and surface modifications affect the genotoxicity, a great diversity of test systems and methods have been used to assess the genotoxicity of nanomaterials.117,118 The detailed mechanisms of NPs induced genotoxicity are not completely understood. However, sustainable NPs are usually designed to perform certain specific applications and hence, they are coated or functionalized.119-124 The functionalization provides charge to the NP surface unless they are completely hydrophobic and designed to be used in the non-polar environments. Thus, the toxicity of charged or functionalized NPs depends on the nature of polarity to repel or attract the lipid bilayer of cell membrane, and accordingly indulge in the membrane disruption activity.105 Biocompatible functionalization of sustainable NPs reduces toxicity to a large extent.5,8,63 For instant, serum albumin coating of NPs makes them excellent drug delivery vehicles for their uses in the systemic circulation because they do not induce hemolysis while travelling in the blood stream.8 Modified NP surface strongly reduce NP adhesion in comparison to what is observed for the bare material.125 The lowered adhesion in the presence of proteins decreases the NP uptake efficiency. This happens due to the reduction in the interactions between the bare material and the cell membrane because of the presence of protein coating in-between. Sustainable NPs meant for the industrial and environmental applications can be designed to have minimum cell disrupting ability and hence, minimum toxicity.

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6. Future directions and preventive measures Future directions would require to keep sustainability ahead of nanometallic toxicity by designing nanomaterials of minimum toxicity. Nanotoxicity in air, soil, and water resources is quite difficult to locate because of the nanometer size in comparison to the much bigger conventional pollutant particulates. Even their nanomolar concentrations have proved to be toxic for the aquatic life with largely unknown consequences for the spontaneous growth cycle of organisms. Therefore, while identifying the routes of nanotoxicity in air, soil, and water, it is possible to design sustainable nanomaterials which could have minimum interactions with the biological systems and especially with the cell membrane. The most viable solution for this problem seems to be an appropriate coating of biocompatible layer on free NPs surfaces that could coexist with biological medium. It proves to be an effective measure against the cell membrane disruption and/or oxidative stress. There are plenty of biological low cost molecules which can be easily used to generate cost effective nanomaterials. The applicability is chosen in such a manner that the biological molecules should help in achieving the applicability. In addition to this, widespread industrial applicability of engineered nanomaterials requires appropriate standards for their disposability just like that of conventional chemicals where special precautions are taken for halogenated/non-halogenated organic solvents and nuclear/radioactive wastes. While designing efficient sustainable nanomaterials for replacing conventional technologies, it is also required to study their long term consequences on ecosystem and water resources.126 Keeping this in consideration, the preventive measures should be taken on the basis of the shape, size, and surface functionalities to minimize their nanotoxicity before using them for efficient sustainable technologies. For example, NPs based metallic spray paint should consist of metallic NPs which could have minimum air born lifespan by increasing their flocculation and coagulation behaviors through surface modifications. Similarly, nanomaterials used for water purification in terms of heavy metal ions, humic substances, or bacterial contaminations, must have surface passivation with minimum impact on aquatic life. The nanoporous and magnetic nanomaterials are the better options because they can entrap the pollutants specifically and magnetically, respectively, but they still need surface passivation to minimize their impact on the cell membrane. Finally, this account highlights the major environmental challenges of diverse nanotechnologies and paves the way for possible solutions to minimize the environmental nanotoxicity in air, soil, and water.

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Acknowledgment: These studies were supported by the financial assistance from UWGB, NAS, Green Bay.

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Impact of Nanomaterials on Ecosystems: Mechanistic Aspects In Vivo Mandeep Singh Bakshi

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: