The biophysicochemical interactions at the interfaces between nanoparticles and aquatic organisms: adsorption and internalization

Abstract

Nano–bio interfacial interactions that can likely regulate the potential toxicity of nanoparticles (NPs) toward aquatic organisms are receiving increasing research interest worldwide and warrant more investigation. This review presents an overview of already-known nano–bio interactions and some speculations on the interfaces between NPs and aquatic organisms, in order to gain a new insight into the biological effects of NPs in the aquatic environment. The fundamental interfaces between NPs and organism cells and the main biophysicochemical interactions that occur at the nano–bio interfaces are described. The interfacial interactions, focused on adsorption and internalization, during the contact of NPs with microorganisms, hydrophytes, invertebrates and fish were reviewed. The effects of NP properties and suspending states as well as environmental conditions including pH, ionic strength, natural organic matter and other factors on the interfacial interactions were elucidated. Furthermore, the analytical methods employed in the interfacial interaction investigations were also briefly introduced. Future research directions of nano–bio interactions were prospected.

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Daohui Lin

Dr Daohui Lin is Professor of Environmental Chemistry (since 2009) at Zhejiang University, where he has been involved in teaching and research since 1998. He received his Ph.D. from Zhejiang University (2005), M.Sc. degree from Beijing Normal University (1998) and B.Sc. degree from Hangzhou University (1995). He was a visiting scholar (2006–2008) at the University of Massachusetts at Amherst and was selected as a New Century Excellent Talent in University by Ministry of Education of China in 2010. His research interests focus on environmental behavior and risk of contaminants, especially of engineered nanomaterials. He has published about 50 peer-reviewed articles.

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Environmental impact

As an increasing number of nanoparticles (NPs) are being released into the aquatic environment, their nanotoxicity to aquatic organisms has aroused research interest, but studies on the interfacial interactions from a micro perspective are really limited. In this review, we provide an overview of the biophysicochemical interactions between NPs and cells, and then demonstrate the nano–bio interactions at the interfaces when NPs meet microorganisms, hydrophytes, invertebrates and fish. Besides, the influencing factors (including the NP properties and the environmental conditions) affecting the interfacial interactions were stated. Analytical methods employed to study the nano–bio interfaces were briefly introduced, and the perspectives were also put forward. We believe this review will shed light on the ecological effect of NPs.

1 Introduction

Nowadays, it is known that nanoparticles (NPs) will be increasingly released into the aquatic environment and their behaviors greatly depend on the environmental conditions, such as natural organic matter, pH, and ionic strength.^1,2 As long as NPs are released into the aquatic environment, they may inevitably interact with microorganisms, hydrophytes, invertebrates, and/or fish, and thereby exhibit potential toxicity to these aquatic organisms and consequently human beings through food chains. Increasing research has therefore been performed to investigate the nanotoxicity of NPs to aquatic organisms;³ however, the nano–bio interfacial interactions that can likely determine the potential nanotoxicity are still waiting to be fully understood.

Unlike other contaminants, with unique physicochemical properties, NPs exhibit quite different toxicities when exposed to aquatic organisms during their existence in the aquatic environment. Oxidative stress, released metal ions, physical damage, and shading effects are the four mainly addressed toxic mechanisms of NPs toward aquatic organisms, especially microorganisms.³ Increasing evidence indicates that NPs can generate reactive oxygen species (ROS), and consequently induce oxidative injury, cell damage, and even apoptosis.^4–8 Toxic metal ions released from metal NPs⁹ and carbon nanomaterials¹⁰ can cause toxicity toward aquatic organisms. Physical contact, including adsorption and internalization, is another mechanism contributing to the nanotoxicity.^11,12 Shading and agglomeration can play an important role in the nanotoxicity toward phototrophic organisms.^13–15 Each mechanism is not supposed to work alone. As to larger aquatic organisms, such as invertebrates and fish that contain complex tissues and organs, accumulation and distribution of the NPs inside the organisms, as well as the nano–bio interactions at the outer-surfaces of the organisms are of vital importance; and the toxic mechanisms may become much more sophisticated than that between unicellular animals and NPs. Besides the release of toxic metal ions, the other toxic mechanisms all involve a crucial precondition of close contact between NPs and aquatic organisms.¹⁶

It is worth noting that there are various microcosmic interfaces where biophysicochemical interactions occur between NPs and aquatic organisms, and in most cases the interactions can cause subsequent toxicity towards the organisms. A few researchers have already focused on nano–bio interfaces in order to fully understand the interactions between NPs and organisms.¹⁷ The main forces acting at the nano–bio interfaces include hydrodynamic, electrodynamic, electrostatic, solvent, steric and polymer bridging interactions.¹⁷ NPs may be adsorbed and thereupon be internalized by the aquatic organisms due to these interactions. However, there are many questions regarding the nano–bio interfaces and interactions which remain to be answered: How do NPs attach to organism surfaces and be internalized? What interfaces and interactions between NPs and aquatic organisms should be of importance? What factors will influence these interfaces and interactions? Therefore, this review is aimed at providing an overview of details from documented studies and introducing available knowledge on the interfacial interactions between NPs and aquatic organisms. The interfaces between cells and NPs as well as the interfacial interactions are described, and then the specific details of interfaces between NPs and microorganisms, hydrophytes, invertebrates and fish are elaborated. Furthermore, the influencing factors governing the interfacial interactions and some analytical methods were delineated and several future research needs are put forward.

2 Biophysicochemical interactions at NP–cell interfaces

2.1 NP–cell interfaces

In the aquatic environment, there are complex food webs supporting the whole ecosystem (Fig. 1). Micro algae and hydrophytes are regarded as the first trophic level as they are the primary producers. Bacterium is the decomposer, but also becomes the beginning of the food chain together with algae and hydrophytes after being eaten by plankton. Invertebrates, both zooplankton and zoobenthos, form the second trophic level as they feed on microorganisms, especially algae. Fish that eat invertebrates are in the higher trophic level. The released NPs may be suspended in the water phase or deposited in the sediment. The suspended NPs can likely interact with zooplankton, such as daphnia, while the NPs in the sediment may react with zoobenthos, including oyster, for instance.

Fig. 1 The food web in the aquatic environment.

When NPs come into contact with aquatic organisms, the interface between these two objects refers to the micro region existing on the surfaces of both of them. The most important and fundamental interface is between the NPs and cells. Alga, bacterium, and hydrophyte cells generally have cell walls, which are the outermost layers of the cells when meeting NPs. Cellulose and pectin are the main compositions of the cell walls of algae and plants, while for bacteria the major component is peptidoglycan. In spite of this slight difference, the tough cell walls of these organisms can play a protective role in supporting the cell structure and resisting potential harmful substances including NPs. To date, however, research aimed at the interactions between NPs and cell walls is really scarce. Animal cells have no cell wall, so the membrane consisting of a phospholipid bilayer is the concerned interface for invertebrates and fish in the aquatic environment. Generally, there is a hydrated sheath on the interfaces in water, the thickness of which depends on various factors including the surface properties of the two contacting objects and the environmental conditions.

2.2 Interfacial interactions

Once NPs come into contact with cell surfaces, they may be adsorbed on the cell walls or membranes by multiple forces, and then it is likely that they will enter the cells through various routes. The biophysicochemical interactions at the interfaces between the NPs and cells, in conclusion, mainly include adsorption and internalization.

Fig. 2 schematically illustrates the main adsorption mechanisms at the NP–cell interfaces. NPs with bio interfaces can be combined by van der Waals forces, a common attracting force. This force was confirmed to determine the interactions of SiO₂ NPs with a dioleoyl phosphatidylcholine monolayer.¹⁸ NPs with hydrophobic surfaces would be adsorbed on the hydrophobic surface zones of the cells through hydrophobic forces. Electrostatic attraction, as another important and general adsorption mechanism, that can cause the charged NPs to become adsorbed on the cell surfaces with opposite charges, which has been documented in many studies.¹⁹ Moreover, some specific interactions exist, e.g. hydrogen bonding and receptor–ligand interactions, depending on the surface properties of both NPs and cells.¹⁷

Fig. 2 The interactions between NPs and a cell membrane. NPs can be adsorbed on the membrane (phospholipid bilayer) through receptor–ligand interactions, hydrophobic interactions, electrostatic attractions and hydrogen bonds. When membrane fusion occurs, the NPs can cross the membrane and enter the cells directly. Metal ions released from the NPs can be transported into the cells via certain membrane channels. Endocytosis occurs directly across the membrane and the NPs are wrapped inside.

The cell-adsorbed NPs may be internalized via different routes. Rigid NPs with sharp shapes may directly penetrate through cell wall and membrane and then enter into cells. For cells without cell walls, NPs would interact directly with the membranes (Fig. 2). NPs may disrupt the cell membrane and directly enter the cells, e.g. cationic NPs²⁰ and polycationic polymers²¹ could induce the formation of nanoscale holes, membrane permeability alterations, membrane thinning, and/or membrane erosion in the lipid bilayers of the cells and thus be internalized. On most occasions, internalization can not occur independently. A study has shown that the internalization of polymer iron oxide NPs into rat pheochromocytoma cells involves three distinct steps: plasma membrane attachment, endocytosis, and gradual accumulation in the membrane-bound intracellular vesicles.²² Endocytosis is the most accepted route for NP uptake. It was demonstrated that single-walled carbon nanotubes (SWCNTs) could enter cells by both penetration and endocytosis.²³ PAA-coated Au nanospheres were also implied to enter cells via receptor-mediated endocytosis or membrane fusion.¹⁹ Interestingly, in contrast, CdTe nanocrystals capped by thiolated methoxypolyethyleneglycol were shown to penetrate through the lipid bilayer of giant unilamellar vesicles and giant plasma membrane vesicles which constitute basic endocytosis-free model membrane systems.²⁴ Obviously, for different NPs and cells, endocytosis plays a different role. In addition, internalization of NPs could occur without overt membrane disruption, e.g., cationic nano-objects were observed to pass through cell membranes by generating transient holes.²⁵ Besides, metal ions released from the NPs could pass through the membrane via specific channels and enter the cells.²⁶ Hence, under different circumstances, the mechanisms of internalization may be quite different or even be contradictory – the specific reasons of which remain unknown and deserve further and systematic research. Most of the recorded studies concerning NP–cell interactions were focused on mammalian or model cells. We suppose that the internalization would be quite specific when NPs meet aquatic organisms as mentioned later.

Besides the interactions between NPs and membranes as well as cell walls, from a more detailed perspective, the association between NPs and biomolecules should be taken into consideration. Proteins, lipids, and polysaccharides are common components existing on the inner or outer interfaces of the organisms, thus they would inevitably come into contact with NPs at the nano–bio interfaces. The interactions between NPs and biomolecules have therefore received increasing research attention.^27,28 It was indicated that proteins can associate with NPs as a “corona” and thereupon bind the NPs and cells together.^29–32 Hydrogen bonding, ionic interactions, and dehydration of polar groups may be the main forces connecting NPs and polysaccharides.³³ Both the specific and nonspecific NP–biomolecule interactions may facilitate cell internalization of the NPs. It was indicated that nonspecific adsorption of serum proteins mediated the uptake of gold NPs into mammalian cells,³⁴ whereas strong specific ligand–receptor interactions, between folate on silicon nanowires and folate receptors on the cell membranes, were suggested to expedite the internalization of the nanowires.³⁵

3 Biophysicochemical interactions at the interfaces between NPs and microorganisms

3.1 Adsorption of NPs on microorganisms

Table 1 shows the selected research results at the nano–bio interactions between NPs and microorganisms. Alga and bacterium are the two common target aquatic microorganisms in nanotoxicity studies. In general, microorganisms have negative charges on their surfaces, thus adsorption may occur as soon as they meet NPs with positive charges. Positively charged CeO₂ NPs⁵¹ and alumina coated SiO₂ NPs⁵² were observed to be adsorbed (driven by electrostatic attraction) on the algal cell surfaces. This kind of purely physical process was also reported between positively charged Fe⁰ NPs and negatively charged cyanobacterial cells,³⁹ and between oxide NPs [Al₂O₃ (ref. 43) and CeO₂ (ref. 12)] and bacterial cells. NPs may not directly attach to the cell walls of microorganisms, but could interact with the extracellular substances such as proteins and polysaccharides. It was confirmed that Ag NPs could bind bacterial extracellular proteins through electrostatic attraction.⁵³

Table 1 The nano–bio interactions between NPs and microorganisms^a

NPs	Microorganisms	Toxicity	Adsorption		Internalization		Ref.
			Yes/no	Mechanism	Yes/no	Mechanism
a Note: “yes” refers to the occurrence of the interaction; “no” refers to the absence of the interaction; “/” refers to the fact that the interaction and the mechanism were not addressed in the references.
Si	Pseudokirchneriella subcapitata	Growth inhibition	Yes	/	No	/	36
Ag	Chlorella vulgaris, Dunaliella tertiolecta	Oxidative stress	Yes	Electrostatic attraction	No	/	37
	Pseudomonas putida biofilms	Growth inhibition	Yes	/	Yes	/	38
Fe^0	Cyanobacteria	Oxidative stress, shading effect	Yes	Electrostatic attraction	/	/	39
CdTe/CdS QDs	Chlamydomonas reinhardtii	Oxidative stress, genotoxicity	Yes	/	Yes	Ion released	9
NiO	Chlorella vulgaris	Growth inhibition, morphological alteration	Yes	/	Yes	Ion released	14
CuO	Chlamydomonas reinhardtii	Oxidative stress	Yes	/	Yes	Penetration	40
	Microcystis aeruginosa	Oxidative stress, genotoxicity	Yes	/	Yes	Endocytosis, ion released	41
ZnO	Chlorella sp.	Shading, growth inhibition, ion released	Yes	/	Yes	Ion released, cell surface damaged	42
	Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens	Physical damage, ion released	Yes	Electrostatic attraction, receptor–ligand interaction	/	/	43
	Escherichia coli	Ion released, growth inhibition	Yes	/	Yes	/	44
TiO2	Chlorella sp., Scenedesmus sp.	Cell damage, growth inhibition	Yes	Specific interactions between functional groups	No	/	45
	Anabaena variabilis	Oxidative stress	Yes	/	Yes	Membrane damage	46
	Salmonella typhimurium	Genotoxicity	Yes	/	Yes	Non-specific membrane damage and specific uptake	47
SiO2	Scenedesmus obliquus	Shading, photosynthetic activity inhibition	Yes	/	No	/	48
CeO2	Pseudokirchneriella subcapitata	Oxidative stress, physical damage	Yes	/	Yes	Membrane damage	49
	Escherichia coli	Oxidative stress, growth inhibition	Yes	Electrostatic attraction	/	/	12
Al2O3	Cupriavidus Metallidurans, Escherichia coli	Oxidative stress	Yes	/	Yes	Transfer across peptidoglycan layer	50
MWCNTs	Chlorella vulgaris, Pseudokirchneriella subcapitata	Shading effect, growth inhibition	Yes	Hydrogen bonds	No	/	13
	Chlorella sp.	Oxidative stress, shading, physical damage	Yes	/	Yes	Physical penetration	16

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Specific interactions may occur between surface functional groups on the NPs and microorganisms. Carboxyl, amide, phosphate and hydroxyl functional groups on the bacterial cell walls and of the extracellular polymer substances may provide active sites for molecular-scale interactions with the oxide NPs.⁵⁴ Schwab et al.¹³ suggested that hydrogen bonds between the algal cell surfaces and oxygen defects of carbon nanotubes (CNTs) could form to coagulate them. Furthermore, the majority of metal NPs can likely release metal ions that may bind with cationic and anionic exchange sites on the algal cell surfaces and thus provide binding sites for the NPs.⁵³ The lipopolysaccharide chain molecules that form on the outer membrane of a Gram-negative bacterium, together with phospholipids, peptidoglycan, and exopolyscaccharide layers have been indicated to contain a large number of metal-complexing sites.⁵⁵

From a kinetics point of view, the generation of adsorption largely depends on the interaction energy. Zhang et al.⁵⁶ explored the adsorption pattern between hematite NPs and Escherichia coli using a series of adsorption experiments and data analysis. It was indicated that the interaction energy is rather complex because the interactions may concurrently include van der Waals forces, electrostatic interactions, acid–base forces, and specific interactions.^56,57 The force distribution varies under different circumstances, which can result in a change of the interaction energy of the interfaces and subsequently influence the adsorption.

The adsorption of NPs on microorganisms could result in a series of subsequent toxicity, including physical damage and biochemical injury.^12,48,58,59 Based on the observation of SiO₂ NPs adsorbed on algal cells and no evidence of uptake into the cells, Hoecke et al.³⁶ concluded that the apparent toxicity of SiO₂ NPs occurred through the surface interactions. Algal surface accumulated CNTs could inhibit the algal growth by enhanced shading and agglomeration effects.¹³ Microorganism-adsorbed NPs were also observed to result in toxicity by pitting the cell walls,⁵⁸ inducing the intracellular leakage via damaged cell membranes,^46,59 and disturbing the permeability and respiration functions of the cells.⁶⁰ The physical characteristics of microorganism cells, including adhesiveness and hardness or elasticity, would change due to NP-adsorption.⁶¹ The adsorbed NPs may also block the nutrient uptake and influence the cell metabolism. It was suggested in a research study that the direct interaction of Ag NPs with bacteria could lead to impaired metabolism, because the protein on the bacterial surface that binds to the NP surfaces alters the conformation or shields the active sites of the essential enzymes on the surfaces of the bacterial cells.⁶² Furthermore, oxidative injury is another highly concerning toxicity which follows the adsorption of NPs.⁶³ It is known that cellular uptake and intracellular ROS production requires close contact with the NPs.^64,65

3.2 Internalization of NPs into microorganisms

An increasing amount of evidence has been provided, that shows that many different kinds of NPs are capable of entering microorganism cells,^46,47,50,59 however, the specific mechanism remains to be explored. For most algae and bacteria, the cell wall is a protective barrier that forbids NPs to enter the cells by blocking the possible entrances, such as endocytosis.⁴⁸ For example, the physical properties of the bacterial peptidoglycan layer could resist the access of NPs into the cells.⁵⁰ The most common ingress is the natural pores on the cell walls, the size of which decides the size of NPs that can enter the cells directly.^48,66 However, sometimes internalization immediately occurs after adsorption of the NPs on the cell surfaces, because, as mentioned above, adsorption could induce cell wall pitting or membrane damage. For example, Ag NPs could be transported into microorganism cells because of the impaired cell walls and increase in cell membrane permeability.^67,68 There are other possible specific internalizations of NPs. A specific uptake of ZnO and TiO₂ NPs through porins existing on the surface of the bacterium S. typhimurium has been suggested.⁴⁷ Under some circumstances, it is the metal ions that are released from NPs, rather than the NPs themselves, that enter the microorganism cells.^55,68

After entering the cells, the NPs will disperse inside the cells and locate in the cytoplasm and organelles, such as vacuole, chloroplast (for algal cells) and nuclear body. In this way, NPs would become accumulated in the cells. As a consequence, the intracellular interactions between NPs and these cellular substances may lead to nanotoxicity.⁴⁰

4 Biophysicochemical interactions at the interfaces between NPs and hydrophytes

Hydrophytes, including emergent plants, floating-leaved plants and submerged hydrophytes, can likely meet the increasing number of NPs being released into the aquatic environment. There have been plenty of studies focusing on the phytotoxicity of NPs towards terrestrial plants,^69,70 however, those toward hydrophytes are really limited. Despite the difference between terrestrial and aquatic plants, the biophysicochemical interactions between NPs and hydrophytes are still mainly reviewed based on phytotoxicity studies on the terrestrial plants, since a majority of these studies were performed in hydroponic culture systems. There is usually a layer of ceraceous substances or cuticles on the surfaces of leaves; the adsorption of NPs would therefore be difficult but could still occur. As pointed out in a review, small and lipophilic NPs could be incorporated into the apolar zones of the cuticles through apolar and/or polar pathways.⁶⁹ The root is vulnerable when exposed to NPs. It has been widely observed that plant roots can accumulate and internalize various NPs such as ZnO NPs,^71,72 CuO NPs,⁷¹ water-soluble fullerenes,⁷³ and CdSe/ZnS QDs.⁷⁴ The irreversible adsorption of fullerenes on the aquatic plant Ceratophyllum has been reported, and it was pointed out that C₆₀ molecules could bind to cellulose in the plant structure via hydrogen bonds.⁷⁵ After being adsorbed on the plant surfaces, NPs can likely be internalized into the plant tissues. Compared with terrestrial plants, aquatic plants generally have much thinner cuticle layers and their surfaces are much softer; furthermore, there are more pores on the stems and leaves of aquatic plants, which provide more uptake routes of NPs, namely allowing NPs with specific sizes to enter into the cells.⁷⁶ It was revealed that multi-walled carbon nanotubes (MWCNTs) could penetrate the cell wall and enter the cytoplasm after uptake through the roots.⁷⁷ It was indicated that CNTs could enter living walled plant cells through cellulose-induced nanoholes.⁷⁸ Once uptake occurs, the NPs would be transported and translocated to various tissues of the plants, such as shoots, epidermis, vacuoles, plasmodesmos, mesophylls, and gas spaces.⁶⁹ Considering the important position of hydrophytes as the primary producer in the aquatic environment, the adsorption and internalization of NPs may affect the aquatic biosphere through the trophic levels, and even influence the entire aquatic ecosystem owing to the potential impact on photosynthesis.

5 Biophysicochemical interactions at the interfaces between NPs and invertebrates

Invertebrates, including zooplankton and zoobenthos, are regarded as the largest community in water environments. Zooplankton, such as daphnia, shrimp and some snails, feed on micro algae, and would undoubtedly ingest NPs because of the adsorption of NPs on the algal cells. Even though some zooplanktons do not feed on algae, it is quite easy for the suspending NPs to enter the organisms via the water phase through certain dietary pathways. On the other hand, zoobenthos, like oysters and mussels, would obviously come into contact with the settled NPs. Recent research results on the nano–bio interactions between NPs and invertebrates are summarized in Table 2.

Table 2 The nano–bio interactions between NPs and invertebrates^a

NPs	Invertebrates	Toxicity	Adsorption		Internalization		Involved organs (parts)	Ref.
			Yes/no	Mechanism	Yes/no	Mechanism
a Note: “yes” refers to the occurrence of the interaction; “no” refers to the absence of the interaction; “/” refers to the fact that the interaction and the mechanism were not addressed in the references.
Ag	Daphnia magna	Chronic toxicity, bioaccumulation	Yes	/	Yes	Endocytosis	Branchia, guts	79
Au	Mytilus edulis	Oxidative stress	/	/	Yes	Digestion and endocytosis	Guts, digestive gland	4
CuO	Mytilus galloprovincialis	Oxidative stress	Yes	/	Yes	Endocytosis, ion released	Gill	80
ZnO	Daphnia magna	Molting inhibition, oxidative stress, genotoxicity	/	/	Yes	Endocytosis	/	81
TiO2	Daphnia magna	Molting inhibition, physical damage	Yes	/	Yes	/	Shell, exoskeleton	11
C60	Crassostrea virginica	Lysosomal destabilization, oxidative stress	/	/	Yes	Endocytosis, phagocytosis	Hepatopancreas	82
CNTs	Daphnia magna	Movement inhibition	Yes	/	Yes	/	Guts	83
MWCNTs	Ceriodaphnia dubia	Bioaccumulation	Yes	Specific connection by functional groups	/	/	Digestive tract, brood chamber	84
Carbon black	Bivalve mollusc mytilus	Inflammation, oxidative stress, genotoxicity	/	/	Yes	Endocytosis	Gills and digestive gland, the immune cells, the hemocytes	85
Polystyrene NPs	Mytilus edulis, Crassostrea virginica	Bioaccumulation	/	/	Yes	Endocytosis	Guts, digestive gland	86

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Adsorption of NPs on the surfaces of aquatic invertebrates has been documented. It should be noted that there are setae on the surfaces of some invertebrates such as daphnia; NPs can likely be adsorbed onto these setae. It was observed that NPs could gather on the surfaces of daphnia probably due to the adhesion to the exoskeleton of daphnia.^11,87 Direct adherence of TiO₂ aggregates to the outer envelope (chorion) of abalone embryos was also observed.⁸⁸ As a matter of fact, the interface between invertebrates and NPs is not as simple as just a layer or flat surface. It was pointed out that oysters could produce an organic–inorganic adhesive displaying a cross-linked organic matrix, and elevated protein content.⁸⁹ Thus, when considering the interfacial interactions, nano–protein interactions should be taken into account. It was demonstrated that Au NPs were able to bind thiol-containing proteins in the digestive glands of mussels.⁴ For invertebrates with shells, such as mussels and oysters, the adhesion of NPs on the shell surfaces deserves attention, and indeed it has already aroused research interest. It was confirmed that Ag NPs could be incorporated into the surfaces of the nacreous layer of the marine bivalves Mytilus edulis, and were transported to the mollusk extrapallial fluid (EPF) between the soft tissues and shell of blue mussels.⁹⁰ In addition, due to their filter feeding habits, the bivalves gill epithelium was the first contact interface between the organism and NPs.⁸⁰ For some invertebrates, besides the gills, mantle and labial palps were also the exposure organs contributing to the internalization of NPs,⁸⁷ though they were not the main entrance compared to the ingestion route.

As mentioned above, NPs can enter invertebrates, and interact with the internal organs, therefore the interfaces formed when NPs meet invertebrates not only include the outer surfaces but also contain the inner interfaces of the organisms (Fig. 3). Different entrances and routes for the uptake of NPs can lead to various distributions of the NPs inside the invertebrates. For instance, it was suggested that CdSe/ZnS quantum dots (QDs) were observed in the lower intestine of daphnia because such crustacea were capable of pumping water through the anus into the intestine.⁹¹ However, since ingestion is the most common uptake route of NPs, the digestive system likely becomes the vulnerable target inside the invertebrates. The guts are the most concerned organs during the localization and distribution of the NPs. Heinlaan et al.⁹² have paid special attention to the interaction between CuO NPs and the midgut epithelial cells of daphnia, and concluded that the CuO NPs could be located between the midgut epithelium microvillies, indicating the potential internalization. Actually, during the ingestion, the state of some NP aggregates may be altered by the action of cilia on the gills and labial palps,⁸⁷ which would facilitate the dispersion and distribution of the NPs inside the organisms. Moreover, the interaction between NPs and hemocytes cannot be ignored, because NPs could be transferred from the digestive system to the hemolymph and circulating hemocytes.⁸⁷

Fig. 3 Uptake routes and distributions of NPs in daphnia. The figure shows both the outer nano–bio interface and inner distribution of NPs. The NPs could be adsorbed on the exoskeleton and setae. The NPs can enter the daphnia through dietary routes or through the anus via water phase – they then mainly accumulate in the digestive system. The NP aggregates could be dispersed by the action of cilia on the gills when entering the organism.

Endocytosis could be the main internalization mechanism for NPs to cross the membrane and finally reach the inner part of animal cells.⁹³ Polystyrene NPs were observed entering the tubules of the digestive glands and being internalized by the digestive cells of oysters via endocytosis.⁹⁴ Fullerene particles could be readily accumulated inside the hepatopancreas tissues of Crassostrea virginica, which have rich lysosomes cells due to endocytosis.⁸² Interestingly, on the contrary, it was reported that the internalization of NPs did not occur inside the midgut of daphnia, even though the NPs were observed around the microvilli of the midgut epithelium cells.⁹⁵ Therefore, more research is required for the actual internalization mechanisms.

These interactions that occur in the microworld between NPs and invertebrates can potentially cause either acute or chronic toxicity. The adsorption of NP clusters on the outer surfaces may affect the movement of zooplankton and thus have an impact on their predation, and subsequently influences the whole food chain performance.⁹⁶ Exposure of daphnia to CuO NPs could lead to ultrastructural changes of the midgut epithelial cells.⁹² For molluscs with a protective shell, interfacial interactions with NPs can probably alter the nacre surface micromorphology.⁹⁷ After entering the digestive system, NPs may impact the inner organs, like the gut, through inducing lysosome perturbations and oxidative injury.⁹⁸

6 Nano–bio interactions at the interfaces during the exposure of fish to NPs

Being in the higher trophic level of the aquatic ecosystem, fish are vulnerable to contaminants in their surroundings. NPs can not only come into contact with fish directly, but can also enter the body via the fish predation of NP-exposed microorganisms and invertebrates. Nano–bio interactions at the interfaces during the exposure of fish to NPs have been concerned in some studies on the toxicity of NPs toward fish (Table 3).

Table 3 The nano–bio interactions between NPs and fish^a

NPs	Fish	Toxicity	Adsorption		Internalization		Involved organs (parts)	Ref.
			Yes/no	Mechanism	Yes/no	Mechanism
a Note: “yes” refers to the occurrence of the interaction; “no” refers to the absence of the interaction; “/” refers to the fact that the interaction and the mechanism were not addressed in the references.
Ag	Sheepshead minnows (Cyprinodon variegatus)	Ion released, chronic toxicity	Yes	Receptor–ligand interaction	Yes	/	Gill	99
	Medaka embryo	Oxidative stress, genotoxicity	Yes	/	Yes	Penetration	Chorion	100
Al	Zebrafish (Danio rerio)	Genotoxicity	Yes	/	/	/	Gills	101
ZnO	Trout hepatocytes	Oxidative stress	/	/	Yes	/	Liver	102
TiO2	Rainbow trout (Oncorhyncusmykiss) gonadal tissue (RTG-2 cells)	Oxidative stress, genotoxicity	Yes	/	Yes	Membrane damage	Gonad	103
	Larval zebrafish (Danio rerio)	Histopathological injuries and oxidative stress	Yes	/	No	/	Embryo	104
	Juvenile rainbow trout (Oncorhynchus mykiss)	Oxidative	/	/	Yes	/	Gill, gut, liver, brain and spleen	105
CuO	Juvenile carp (Cyprinus carpio)	Neurotoxicity	Yes	Interaction with mucus and protein	Yes	Ion released, across mouth and gill	Skin, scale, gill, intestine, liver, brain, muscle	106
CeO2	Carp (Cyprius carpio)	Cytotoxicity	/	/	Yes	/	Gastrointestinal tract, hepatocytes	107
C60	Carp (Cyprinus carpio)	Oxidative stress	Yes	Protein–NPs binding	Yes	/	Gill, brain	108
SWCNTs	Rainbow trout	Respiratory toxicity, oxidative stress, organ pathologies	Yes	Trapped by gill mucus	Yes	/	Gill, intestine, brain, liver	109

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Fig. 4 schematically shows the possible adsorption/accumulation sites and internalization pathways of NPs for fish. Generally, the skin is the outermost layer of the surface of fish, containing an epidermis and dermis. The epidermis secretes a mucus that covers the fish’s body, which may embed NPs. It has been found that NPs can penetrate into the fish’s mucous layer by peri-kinetic forces, and are subsequently electrostatically attracted to and bind with the mucoproteins; NPs with a non-spherical shape would be more likely to tangle with strands of mucus.¹¹⁰ In fact, the epidermis of fish consists of multilayers of epithelial cells. Accordingly, the adsorption of NPs on these epithelial cells plays an important role in the interfaces that form when fish meet NPs. Besides the skin, fins and scales are also present on the surfaces of fish, which can also become adsorption sites attracting NPs. Cu was detected in the skin and scales of juvenile carp, and the mechanism was ascribed to the adsorption of CuO NPs and binding of Cu ions.¹⁰⁶ Fish have gills as their respiratory organ which provides an access route for NPs, as water and air would pass through this organ. There are also protective covers, like a mucus layer on the gills of some species of fish, in which NPs may be trapped,¹¹¹ as in the case of the association of SWCNTs with the gill mucus in trout.¹⁰⁹

Fig. 4 Adsorption of NPs on the outer surfaces and distribution in the main internal organs of fish. The involved outer interfaces contain scales, mucus layers, fins, and skin. NPs inside the fish may group in some of the organs, including gill, stomach, intestine, gallbladder, liver, kidney, spleen, heart, and brain.

Owing to their much larger body, compared to microorganisms or invertebrates, fish are more likely to accumulate NPs in the body. It has been observed that Cu_xTiO_y and pure TiO₂ NPs were able to penetrate into the embryo cells as well as skin, nerve and yolk sac epithelium cells of zebrafish larvae as aggregated particles.¹¹² Ag NPs were observed to be adsorbed on and transported through zebrafish embryos¹¹³ as well as fathead minnow (Pimephales promelas) embryos,¹¹⁴ penetrating the chorion via pore canals, with uptake kinetics characteristic of diffusion instead of active transport. Ag NPs were also found adhered to the surfaces and insides of the chorion of medaka fish.¹⁰⁰ As stated above, the NPs on the boundary between fish and the surrounding water may be trapped in the mucus layer formed around the gill epithelial cells, and will then probably enter the cells via endocytosis.¹¹⁰ The gill is regarded as the first organ to be affected by NPs. NPs can enter a fish through respiration (via gills) and access through the mouth with dietary exposure, as a result, the concentrations of NPs in the gills and intestine were the highest.¹⁰⁶ Limited uptake of NPs can occur directly from water and across the epithelial membrane in the gill.¹¹⁵ After entering the gastrointestinal system, the NPs can be translocated to internal organs, including the gut, liver, spleen, kidneys, gallbladder, and even brain.^105,107 The accumulation of NPs inside the fish body causes inevitable interactions with various fish cells. Once the NPs enter the cells, they are likely to be stored in some organelles, such as lysosomes, endoplasmatic reticulum, or Golgi apparatus.¹¹⁶ The internalized NPs would also interact with the biomacromolecules in the cells and thereupon affect the cell functions. Griffitt et al.¹⁰¹ indicated that the internalization of Al NPs could result in a concentration-dependent decrease in sodium potassium ATPase activity in the freshwater fish.

The accumulated NPs could be excreted from or transformed in the fish. Desorption of Ce NPs from the gill surfaces and excretion from the alimentary tract occurred through respiration and digestion activity.¹¹⁷ Macrophages in vertebrates may phagocytose NPs.¹¹⁸ NPs in the blood of fish may form aggregates with thrombocyte, which can also be phagocytized by macrophages.¹¹⁹ After entering a biological fluid (such as blood and plasma), the NPs will become coated with proteins that may undergo conformational changes, leading to the exposure of new epitopes and the formation of protein corona at the nano–bio interface.¹⁷

The nano–bio interactions stated above can certainly cause diverse toxicity toward fishes. The adsorbed NPs will increase the living burden and stress of fish, inhibiting the movement and predation. The interactions that occur in the internal organs may result in various adverse effects. Edema, fusion and hyperplasia in the gill lamellae and filaments were observed when TiO₂ NPs were accumulated in the gills; no matter whether the interaction was via uptake or just adhesion.¹²⁰ TiO₂ NPs in the kidney of a fathead minnow caused a reduction in neutrophil function, which is probably due to the potential interactions with neutrophils in the kidney.¹¹⁹ Furthermore, liver pathologies of juvenile carp, including necrotic and apoptosis hepatocytes, occurred due to the accumulation of TiO₂ NPs.¹²⁰ Overall, NPs on the surfaces or in various internal organs of the fish, would induce sub-lethal toxicity due to many micro interfacial interactions that are still lacking thorough research.

7 Influencing factors of the interfacial interactions

7.1 Properties of NPs and their state in water

The size and shape of NPs are important factors that can likely regulate the nano–bio interactions. From an adsorption kinetics point-of-view, smaller NPs may have a faster adsorption rate than larger NPs, which may be attributed to their faster particle mobility and lower energy barriers to the target organism surfaces.⁵⁶ Nano-sized TiO₂ particles were confirmed to be adsorbed much more easily on the daphnia exoskeleton, resulting in higher toxicity than the larger particles.¹¹ Adsorption rates expressed as the number of NPs per unit cell surface area and unit time were much greater for small hematite NPs (26 and 53 nm) than large ones (76 and 98 nm), though the large NPs with a greater mass weight had a higher affinity toward the Caco-2 human intestinal cells according to the adsorption rate of the adsorbed mass of NPs.¹²¹

Regarding the internalization of NPs, it was suggested that nanoparticle size and shape can mediate receptor–ligand binding constants, receptor recycling rates, and exocytosis during the uptake of gold NPs into mammalian cells.³⁴ When pores on the cell surfaces are the entrances, size plays a vital role, because only NPs with a specific size can fit in the pores and pass through them. It was observed that QD aggregates with a certain hydrodynamic diameter range were too large to be internalized, while those with a much smaller diameter could enter the bacterial cells.¹²² NPs with larger surface areas undoubtedly have more opportunities and sites to come into contact with cells. SWCNTs exhibited higher toxicity to the cell membrane of bacteria than MWCNTs due to the much larger surface area.¹²³ Moreover, a research study indicated that smaller Ag NPs with a higher surface area could react with cell membranes more easily and thus become more toxic to the bacteria than any other larger NPs.¹²⁴ Theoretically, NPs with different shapes could exhibit diverse interactions with aquatic organisms. Rigid and sharp CNTs could likely penetrate into cells easily.¹²⁵ Ag nanoplates were considered to be more toxic than nanospheres and nanowires due to their stronger reactivity.¹²⁶

From the above description, it can be concluded that NPs with a smaller size have a higher potential to be adsorbed and internalized by aquatic organisms. However, small-sized NPs are prone to aggregation in water and the aggregation can likely influence the nano–bio interactions. When the NPs aggregate in water, the apparent particle size becomes large; this makes it difficult or even impossible for the NPs to pass through the cell wall via the natural pores.⁷⁶ Aggregation of Ag NPs can change the organism exposure levels by reducing the nominal (added) dose to which the organisms are exposed, while the specific surface area available to interact with biota will be larger in the dispersed form.¹²⁷ In addition, dosimetric selection is another factor to be considered, because different concentrations or particle number density caused by different dosages is supposed to impact the particulate gathering state and the interpretation of the size effect of NPs.³⁴ It was suggested that the increasing number of nanometer particulates in air pollution may enhance the interfacial interactions between reactive particle surfaces and epithelial cells.¹²⁸ However, there is very limited information available to date on the size effect of NPs as influenced by NP dose, which should be an area of focus for future nanotoxicity research, especially on the microcosmic interactions.¹²⁹

The adsorption of NPs on the organisms by electrostatic forces can be regulated by the surface charges of the organisms and the NPs. Therefore, the surface charge or zeta potential of the NPs is one of the key characteristics of the NPs that may dominate the nano–bio interactions as addressed in Section 3.1. The functional groups on the NP surfaces are also very important to the nano–bio interactions because they decide the potential specific interactions (like hydrogen bonding) and the active sites on the nano–bio interfaces; they may also change the surface charges of the NPs and influence the electrostatic interactions.⁸⁴ A study indicated that cationic Au NPs were moderately toxic, while anionic ones were nontoxic.¹³⁰

Surface coating by surfactants or polymers would change the surface properties of the NPs, and thereupon change the environmental behaviors of the NPs and the nano–bio interactions.¹ It was observed that an organic polymer coating on carboxyl QDs provided protection from the metal toxicity during laboratory exposure to freshwater organisms.¹³¹ However, on the contrary, it was also reported that polymer-coated CuO NPs were more toxic than the uncoated NPs toward a green alga, which was mainly attributed to the increased capacity of the polymer-coated CuO NPs to penetrate the algal cell.⁴⁰

7.2 Environmental conditions

Environmental conditions (such as pH, ionic strength, and coexisting substances) can change the surface properties of both NPs and aquatic organisms, and thereupon influence the nano–bio interactions. Solution pH and ionic strength are key parameters that can determine the surface charge properties of the NPs,^1,132 and can therefore regulate the electrostatic interactions between NPs and organisms. However, very few studies have been specifically performed to examine the effects of solution pH and ionic strength on the nano–bio interactions. A study⁵³ revealed that a low solution pH (<8) favored the adsorption of positively charged Ag NPs (point of zero charge = 8) on algal surfaces due to electrostatic attraction, whereas a high pH (>8) inhibited the adsorption because of the electrostatic repulsion between the negatively charged NPs and algae. Moreover, it was indicated that a low pH could facilitate the accumulation of Cd from CdTe/CdS QDs in Chlamydomonas reinhardtii.⁹ Counter-ions can depress the electric double-layer and lower the absolute zeta potential of the colloidal NPs,¹ and thereby affect the electrostatic interaction between NPs and organisms. The depressed electric double-layer will facilitate the aggregation of NPs,¹ which can also influence the nano–bio interactions.

Ubiquitous natural organic matter (NOM) can coat the released NPs, and change their surface properties and environmental behaviors,^1,133,134 and therefore can definitely influence the nano–bio interactions to a great extent. NOM-coating can deliver negative charges to the NPs; therefore, theoretically it can inhibit the adsorption of the NPs to the negatively charged organisms. It has been confirmed that both dissolved and surface bound humic acid could increase the negativity of the surface charges of TiO₂ NPs, and thereby limit their adsorption and toxicity to algal cells.⁶⁵ Actually, mitigation of the nanotoxicity has been widely observed in the presence of NOM, such as the toxicity of nC₆₀ to bacteria,¹³⁵ Ag NPs to bacteria¹³⁶ and early life stage zebrafish (Danio rerio) and Daphnia magna,¹³⁷ Fe⁰ NPs to bacteria,^64,138 CeO₂ NPs to algae,¹³⁹ and CdSe/ZnS QDs to Daphnia magna.⁴¹ However, it has also been reported that dissolved organic matter could increase the toxicity of CuO NPs toward algae because of the higher Cu²⁺ release, decreased degree of NP aggregation and enhanced NP internalization.¹⁴⁰ The effect of NOM on the nano–bio interaction and nanotoxicity warrants more specific investigation.

NPs are excellent sorbents for both organic and inorganic chemicals due to their huge specific surface areas. Therefore, coexisting contaminants in the aquatic environment are likely to be adsorbed by the NPs, which may alter the surface properties of the NPs and the potential interactions with organisms. To date, however, no study has been focused on the effect of the coexisting contaminants on the nano–bio interactions or the nanotoxicity; while, a few researchers have examined the effect of NPs on the bioavailability of the coexisting contaminants. In these stuidies, NPs were observed to act as the “Trojan horse”¹⁴¹ for delivery of the adsorbed contaminants into the aquatic organisms, such as C₆₀ facilitating the internalization and accumulation of arsenic in zebrafish hepatocytes,¹⁴² TiO₂ NPs enhancing the accumulation of cadmium in viscera and gills of carp,¹⁴³ and nano-charcoal favoring the uptake of tributyltin and dibutyltin in Daphnia magna.¹⁴⁴

Temperature and light can influence the interfacial interactions to some degree. Concerning global warming, the temperature of the earth and the sensitive water keeps changing; although it is only slightly, the effect on the whole aquatic environment cannot be ignored. It was concluded that temperature had different effects on the formation and zeta potential of Ag NP agglomerates, and the increase of temperature tended to enhance the toxic effects on two green algae (Chlorella vulgaris and Dunaliella tertiolecta).¹⁴⁵ In addition, temperature fluctuation may induce a change in the suspending conditions of NPs, the habitat and some properties of the aquatic organisms, and the adsorption energy of the nano–bio interface.¹⁴⁶ The nano–bio interactions and nanotoxicity of some photoactive NPs (like TiO₂) could be affected in the presence of ultraviolet radiation.¹⁴⁷

8 Analytical methods

To date, there are many methods for the characterization of the surface properties of NPs and organisms.^148–150 However, it is still a big challenge to correctly and precisely analyze the nano–bio interfaces and interactions. Microscopic observations were generally employed to analyze the nano–bio interfaces and interactions (Table 4). The used microscopes include a light microscope (LM), fluorescence microscope, transmission electron microscope (TEM), scanning electron microscope (SEM), scanning tunnel microscope (STM), atomic force microscope (AFM), laser scanning confocal microscope (LSCM), and coherent anti-Stokes Raman scattering microscope (CARS). The majority of these microscopes can be used to directly observe the apparent surface morphology of the objects, distribution of the NPs, and the association relationship between the NPs and organisms, all of which are substantially helpful for the analysis of the nano–bio interfaces and interactions. Gilbert et al.¹⁵⁹ complemented X-ray 2D imaging methods with AFM to differentiate NPs transported inside the cells with those adhered to the cell surface. Reuel et al.¹⁶⁰ detected the association of SWCNTs with living cells using 3D tracking with an orbital tracking microscope. Some fluorescent QDs can be used in LED systems and flat screen computer displays, thus synchrotron X-ray 2D and 3D elemental imaging was performed to investigate the interfacial interactions between these QDs and daphnia.⁹¹ Flow cytometry was also an effective method used for the rapid detection of the internalization of NPs in live bacteria.^47,161 However, direct observation is not enough to perform precise research. Adsorption experiments can be performed to study the association of NPs with cells and biomolecules.^162–164 There are still many problems concerning the methodology used for the investigation of nano–bio interfacial interactions which remain to be solved. Sometimes, it is really difficult to distinguish whether the metal accumulated in the organisms is the NPs or just the corresponding released ions,⁹⁹ though a study reported that synchrotron X-ray microspectroscopy could be used to analyze Au particles versus Au ions in soil.¹⁶⁵ Therefore, the methods used to define the interfaces and explore the interactions, especially the observation technology and comprehensive methods concerning microcosmic world, still need to be developed.

Table 4 Microscopes used to observe the interactions between NPs and aquatic organisms

Observation technique	NPs	Organisms	Interactions	Ref.
CARS	ZnO, CeO2, TiO2	Zebrafish (Danio rerio), rainbow trout (Oncorhynchus mykiss)	Internalization and localization	115,151
	CeO2, TiO2, Ag	Trout hepatocytes	Internalization	102
TEM	C60	Daphnia magna	Attachment and internalization	152
	TiO2	Nitrogen-fixing cyanobacteria (Anabaena variabilis)	Internalization and distribution	46
	TiO2	Abalone (Haliotis diversicolor supertexta) embryos	Internalization	88
	Au	Escherichia coli, Bacillus subtilis	Attachment	153
SEM	Cu, Ag, TiO2	Zebrafish	The change of gill filament after NPs exposure	111
LM	MWCNTs	Ceriodaphnia dubia	Accumulation and distribution	84
	TiO2	Daphnia magna	Uptake	154
CLSM	QDs	Ciliate (T. pyriformis)	Uptake and depuration	155
Epifluorescence microscopy	QDs	Ceriodaphnia dubia, Pseudokirchneriella subcapitata	Internalization, distribution and adsorption	131
Transmission and confocal fluorescence microscopy	Fluorescent core–shell silica nanoparticles	Zebrafish (Danio rerio)	Adsorption	156
Bright-field and fluorescent microscopy	CuO, ZnO, NiO, and Co3O4	Zebrafish embryos and larvae	Adsorption and distribution	157
Fluorescence stereomicroscope	Nano-sized latex particles	Medaka (Oryzias latipes) embryos and larvae	Internalization and distribution	158

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9 Summary and perspectives

The interactions at the nano–bio interfaces are regarded as a prerequisite and of vital importance to the nanotoxicity, but they still lack thorough research to date. We have briefly described the NP–cell interactions at the nano–bio interfaces, reviewed the research advances on the nano–bio interactions between NPs and aquatic organisms, and introduced the recorded methods for the interfacial interaction research. NPs may be adsorbed on the cell surfaces mainly through hydrophobic, electrostatic, receptor–ligand and hydrogen bonding interactions, and thereafter could be internalized into the cells via penetration, endocytosis, transient holes or membrane disruption. The NPs adsorbed or internalized by the microorganisms could be transferred through the food chain and risk the whole ecosystem. The surface properties and suspending state of the NPs and the conditions of the aquatic environment have profound influences on the interfacial interactions. By controlling the environmental conditions, such as pH, ionic strength, NOM, and temperature as well as the NP surface properties, the nano–bio interactions can be mastered.

However, there are many uncertainties concerning nano–bio interfacial interactions that merit more specific investigations, with a few summarized below as research directions for the future.

(1) The mechanisms of the interfacial interactions between NPs and aquatic organisms have not been well illustrated. The adsorption, internalization, and NP–biomolecule interactions mentioned in the present paper are just the tip of the iceberg, and more research into nano–bio interfaces is needed to fully understand the nano–bio interactions. Conditions (such as solution pH, ionic strength, and especially NOM) can influence the nano–bio interaction and therefore can be used to investigate the interaction mechanism. Research on the NP–biomolecule interaction should be strengthened to probe the molecular mechanisms of the interfacial interactions.

(2) The interfaces and interfacial interactions vary from one species to another. Different algae or bacteria exhibit quite different surface properties and contain diverse chemical substances; invertebrates and fish have many kinds of outer and inner interfaces that can meet NPs. Therefore, the interspecies differences deserve more attention to improve the knowledge of the nano–bio interactions. Future research should also be directed to the contribution of extracellular substances that may govern the nano–bio interfaces and the interfacial interactions.

(3) Though there are a few studies providing evidence of the transfer of NPs through aquatic food chains, the detailed pathway and the interfacial interactions involved are still unclear. It is really crucial to study the nano–bio interactions on a series of interfaces concerning the aquatic food chains. The relationship between the nano–bio interaction and the nanotoxicity should be elucidated. Methods modulating the nano–bio interaction are warranted to control the nanotoxicity. Manufacturers are encouraged to produce environmentally benign NPs that have a lower potential of being accumulated by the organisms and transported through the food chains.

(4) The methodology used for nano–bio interfacial interaction research is still in its infancy. The commonly adopted microscope observations are helpful to visualize the location and distribution of the NPs at the bio interfaces and in the organisms, however, they could not be used to quantitatively or qualitatively analyze the nano–bio interaction forces. Novel techniques and/or methods are demanded to characterize the nanoscale interfaces, quantify and locate the adsorption of NPs, reveal the transmembrane internalization process, and trace NP distribution in the organism and their transport in the food chain.

Acknowledgements

This work was supported by National Natural Science Foundation of China (no. 21077089), Zhejiang Provincial Natural Science Foundation of China (LR12B07001), Zhejiang Provincial “Qianjiang Talent Program” (no. 2010R10041), Program for New Century Excellent Talents in University (NCET-10-0731), Fundamental Research Funds for the Central Universities, and Zhejiang Provincial Innovative Research Team of Water Treatment Functional Materials and their Application.

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