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18.2 Fate and Exposure 599

with another smaller species ( Ceriodaphnia dubia ) using natural organic matter -

stabilized multiwalled CNTs (Figure 18.3 ).

The dietary uptake of single - walled CNTs has also been reported in estuarine

copepods ( Amphiascus tenuiremis ), another zooplankton species [18] . Also reported

here was the presence of compact aggregations of nanotubes in the fecal pellets

of the copepods. Fewer studies exist on the uptake of nanotubes by ﬁ sh; in rainbow

trout, nanotubes ingested from the water column were shown to adhere to the

mucous secretions of the gill tissues (Figure 18.4 ) [19] .

Figure 18.3 Micrographs of the zooplankton Ceriodaphnia

dubia , following exposure to (a) natural organic matter and

(b) natural organic matter and multiwalled carbon nanotubes

(MWCNTs). The dark line visible inside the organism in panel

(b) is the intestinal tract (arrow), containing large amounts

of MWCNTs.

Figure 18.4 (a) Single - walled carbon nanotubes adhered to

the mucous secretions around the gills of rainbow trout;

(b) Phase - contrast micrograph of a mucus smear, showing

nanotubes associated with the mucous proteins. Reproduced

from Ref. [19] .

600 18 Ecological Toxicology of Engineered Carbon Nanoparticles

However, it is unknown what fraction (if any) of the nanotubes crossed the epi-

thelium and were taken up into the soft tissues of the organisms in any of these

studies, although this is largely due to a lack of effective methods for measuring

nanotubes at the low concentrations found in biological tissues. The limit of detec-

tion for analytical methods requires samples to be concentrated to such a high

degree that there may be great uncertainty in the accuracy of their determination.

Fullerenes and some fullerene - derivatives are known to be lipophilic [20] . Fuller-

enes are easily extracted from tissues using organic solvents, and their concentra-

tion determined using spectrophotometry. In one study, Oberdorster et al . [21]

documented an increasing uptake of fullerenes by zooplankton from the water

column over a 96 h period [21] . It has been suggested that adding functional groups

to both fullerenes and nanotubes decreases their toxicity in vitro [22 – 24] , though

this may be due to alterations in the hydrophobicity and thus uptake of the mate-

rial by cells [24] .

18.2.4

Tissue Distribution

Once internalized by the organism, chemical contaminants are distributed to the

tissues via the circulatory system. Again, there is a paucity of data regarding the

tissue distribution of carbon nanoparticles in aquatic organisms. Oberdorster et

al . [21] found that exposure to uncoated fullerenes resulted in an increased oxida-

tive stress in the brains of largemouth bass, leading to the possibility that the

materials were able to cross the blood – brain barrier [11] . These results were sup-

ported by another study, in which the distribution of ﬂ uorescent, nanosized poly-

styrene spheres in the tissues of the medaka, an aquarium ﬁ sh commonly used

in aquatic toxicological studies, was investigated [25] . Kashiwada [25] found that

ﬂ uorescing polystyrene nanoparticles accumulated within the gut and gills of the

ﬁ sh, and were also detected in the brain, testis, liver, and blood. However, the

most intense ﬂ uorescence was observed in the gill tissues, indicating that uptake

across the gill epithelium may be a major route of exposure. A previous study had

shown that rats dosed orally with fullerenes excreted most of the material via the

feces, whereas rats injected intravenously with fullerenes retained the materials

for over one week, with the majority being distributed to the liver [26] . These ﬁ nd-

ings support the hypothesis that, in ﬁ sh, uptake across the gills may be a more

important route of exposure than dietary ingestion.

18.2.5

Food Web

Several chemical compounds found in the environment – and especially those

which are not easily metabolized, such as mercury and polychlorinated biphe-

nyls – have been shown to move through aquatic food chains [27 – 29] . These materi-

als are taken up by organisms at low trophic levels such as bacteria, algae, and

zooplankton and, because they are not easily metabolized and excreted, are passed

18.2 Fate and Exposure 601

on to higher trophic levels when those organisms are subsequently ingested by

predators. This results in increasing concentrations of chemical within organisms

at higher levels of the food chain, a process known as biomagniﬁ cation (Figure 18.5 ).

The biomagniﬁ cation of these materials can pose signiﬁ cant risks to the health

of organisms located at higher trophic levels which might otherwise not be exposed,

including humans [30 – 34] . For example, mercury consumption warnings in

seafood are a result of biomagniﬁ cation of methyl mercury through the food web.

Studies conducted by Templeton et al . [12] and Roberts et al . [18] have indicated

that zooplankton in aquatic systems can ingest nanoparticles from their environ-

ment through normal feeding behavior. Bacteria, which form the bulk of aquatic

bioﬁ lms, have also been shown to take up nanosized particles. Both, bioﬁ lms and

zooplankton occupy lower trophic levels in aquatic ecosystems and provide an

important food base for a variety of organisms, including ﬁ sh. Considering the

lipophilicity of some nanoparticles, and the lack of evidence of true metabolism,

the potential for biomagniﬁ cation clearly exists.

Figure 18.5 Biomagniﬁ cation through an aquatic food chain.

Contaminants may be found at relatively low concentrations

at lower trophic levels (zooplankton). Concentrations will

increase as predatory organisms consume large numbers of

prey items, but do not have the ability to metabolize or

excrete the chemical contaminant.

602 18 Ecological Toxicology of Engineered Carbon Nanoparticles

18.2.6

Effects on the Uptake of Other Contaminants

Due to the relative ease with which some engineered carbon nanoparticles are

taken up by cells, there is also concern regarding the potential for these materials

to facilitate the transport and uptake of other, adsorbed chemical contaminants

into cells. Polycyclic aromatic hydrocarbon s ( PAH s) are a ubiquitous contaminant,

and are discharged into the environment as a result of the incomplete combustion

of fossil fuels. They are generally considered to be carcinogenic and to have a range

of toxic effects [35 – 37] . Yang et al . [38] showed that PAHs readily adsorb to fuller-

enes and nanotubes [38] , while Moore et al . [9] also found that coexposure to a

model PAH (anthracene) and a nanosized polyester sucrose particle increased

both the uptake and cellular toxicity of anthracene in mussels. Contaminants

which are readily adsorbed onto carbon nanoparticles, or are closely associated

with them as a result of manufacturing processes (such as transition metals), may

be delivered more rapidly to cells in the presence of nanosized particles.

18.3

Effects

18.3.1

General

The limited data relating to the toxicological effects of engineered carbon nano-

particles has derived from research groups that have focused largely on mamma-

lian models (both in vitro and in vivo ). Such studies have been carried out primarily

to determine whether these compounds cause, or have the potential to cause,

human health effects following occupational exposure. Although there are excep-

tions, the bulk of the data suggests that engineered carbon nanoparticles are toxic

to some degree, and points to oxidative stress, inﬂ ammatory reactions, and immu-

nological effects as the key features of carbon nanoparticle toxicity [39, 40] .

Relatively few studies, however, have been carried out regarding nanotoxicologi-

cal effects on ecologically important aquatic species, such as ﬁ sh and zooplankton

(Table 18.1 ). The aim of this section is to summarize and review the ecotoxicologi-

cal effects of engineered carbon nanoparticles, focusing primarily on aquatic

eukaryotic systems (e.g., ﬁ sh and zooplankton), as has the bulk of reports made

to date.

18.3.2

Oxidative Stress and Nanoparticles

Aerobic organisms use oxygen to oxidize (burn) carbon - and hydrogen - rich sub-

strates (foods) to obtain the chemical and heat energy that is essential for life.

However, when molecules are oxidized with oxygen, the oxygen molecule itself

18.3 Effects 603

Table 18.1 Summary of the ecotoxicological literature on nanoparticles.

Reference Species Nonmaterial

tested

Preparation

method

Concentrations

tested

Endpoints examined

[11] Micropterus salmoides nC

THF 0.5 – 1.0 ppm Lipid peroxidation, protein oxidation, glutathione

[21] Daphnia magna

Pimephales promelas

Hyalella azteca

Oryzias latipes

Water - stirred 0.5 – 30 ppm Mortality, CYP P450 isozymes, PMP70

[41] Daphnia magna nC

Sonication

THF

0.2 – 880 ppm Mortality, behavior

[42] Daphnia magna

Pimephales promelas

Water - stirred

THF

0.5 ppm Lipid peroxidation, CYP2K1, CYP2M1

[42] Stylonychia mytilus MWCNT AEDP

0.1 – 200 μ g ml

− 1

Cytotoxicity cell morphology, structural alterations

[43] Danio rerio nC

(OH)

DMSO

100 – 500 ppb

500 – 5000 ppb

Development effects, apoptosis

604 18 Ecological Toxicology of Engineered Carbon Nanoparticles

Reference Species Nonmaterial

tested

Preparation

method

Concentrations

tested

Endpoints examined

[44] Bacillus subtilis

Escherichia coli

(OH)

22 – 24

THF

0.04 – 4 mg l

− 1

5 mg l

− 1

Growth

[45] Danio rerio nC

Stirring and

sonication

THF

Dilution of stock

solution

Mortality, behavior, global gene expression

[12] Daphnia magna SWCNT - lipid

coated

LPC

2.5 – 20 mg l

− 1

Mortality

[42] Danio rerio nC

(OH)

16 – 18

Acetone/benzene/

THF

water

1.5 mg l

− 1

50 mg l

− 1

Mortality, developmental effects, hatching rate, heartbeat,

pericardial edema

[19] Oncorhynchius mykiss SWCNT SDS, sonication

0.1 – 0.5 mg l

− 1

Behavior, gill ventilation rates, plasma ions, histopathology,

- ATPase, oxidative stress

[46] Daphnia magna nC

HxC

THF 260 ppb Behavior

THF, tetrahydrofuran; AEDP, 2 - Amino - ethyene - l,1 - bis - phosphonic acid; LPC, lysophophatidylcholine; SDS, sodium dodecylsulfate.

Table 18.1

Continued.

18.3 Effects 605

becomes reduced and forms intermediates called reactive oxygen species ( ROS );

these include free radicals and hydrogen peroxide. Oxidative stress is the term used

to describe the level of oxidative damage in a cell or tissue caused by ROS, the

latter being a class of molecules that are derived from the metabolism of oxygen

and which exist inherently in all aerobic organisms. In fact, ROS are produced

constantly during normal aerobic metabolism, but are normally safely removed by

a variety of biological antioxidants, such as superoxide dismutase ( SOD ), catalase,

or glutathione (GSH) peroxidase. The amount of oxidative stress in a cell or tissue

can be thought of as a balance between the overall generation and the overall

removal/repair of ROS by antioxidants (Figure 18.6 ). Therefore, oxidative stress

within a cell or tissue can result from two factors: (i) an increase in oxidant genera-

tion; and/or (ii) a decrease in antioxidant protection.

Research has shown that xenobiotics such as metals and hydrocarbons can shift

the oxidative stress balance, thus causing an overproduction of ROS or the inhibi-

tion of antioxidant systems [47 – 49] . This shift in balance leads to excessive molecu-

lar damage and tissue injury, an example being lipid peroxidation. The latter

process is deﬁ ned as the free - radical oxidation of polyunsaturated fatty acids in

biological systems, and can be used as an end - point to measure oxidative stress.

The most common biomarkers used to measure oxidative stress include thiobar-

bituric acid reactive substance s ( TBARS ), which measures the presence of lipid

Figure 18.6 Oxidative stress occurs naturally, but biological

systems maintain an equilibrium by counteracting the

deleterious effects of reactive oxygen species ( ROS ) with

antioxidants. Increased oxidative stress, a common mode of

action for a number of chemical toxicants, can occur through

either a depletion of a biological system ’ s antioxidants or an

increase in the number of ROS.

606 18 Ecological Toxicology of Engineered Carbon Nanoparticles

peroxides and the reduction of GSH, an antioxidant which occurs naturally within

cells and is known to scavenge ROS [49 – 52] .

The majority of mammalian - based reports point to oxidative stress as the main

mode of action of engineered carbon nanoparticles (e.g., Ref. [40] ), and so the

assumption is typically made that this is the main mode of action in all species.

With the exception of one report [11] , the present authors are unaware of any other

ecotoxicological literature providing evidence that exposure to engineered carbon

nanoparticles results in oxidative stress. These examinations provide insight into

the possibility that carbon - engineered nanoparticles may affect aquatic organisms

in a different way from mammalian systems. For example, Roberts et al . [12] have

suggested an energetics mechanism and interference with normal feeding behav-

ior in zooplankton.

One major issue which concerns all toxicological studies is to ensure that any

observed effects can be attributed to the compound of interest, and not other con-

founding factors. This is of particular concern when using organic solvents to either

solubilize or stabilize a material in aqueous media. Some carbon nanoparticles,

including C

, have a very low solubility in water; consequently, organic solvents

have been used (notably tetrahydrofuran ; THF ) to increase the solubility of engi-

neered carbon nanomaterials. In the ﬁ rst report addressing the question of whether

exposure to engineered carbon nanoparticles would result in ecotoxicological

effects, Oberdorster [11] used THF to create nC

solutions for exposure to large-

mouth bass. These preliminary study results indicated that nC

caused signiﬁ cant

lipid peroxidation in the brains of the exposed ﬁ sh, since which time the preparation

of nC

using THF has become common practice. However, data obtained by Henry

et al . [45] indicated that, at least in zebraﬁ sh, observable toxic effects could be attrib-

uted to THF and its breakdown products, as opposed to fullerenes [45] . Although

the exact mode of action of THF - prepared nanoparticles is unknown, it has been

suggested that THF might cross the cross the blood – brain barrier, unlike carbon

nanoparticles [53] , thus explaining the ﬁ ndings of Oberdorster [11] .

It is also important to note that not all mammalian - based data have pointed to

oxidative stress as the toxic mode of action of carbon nanoparticles. In fact, it has

been suggested that nC

may function in an opposite manner, and act as a free -

radical scavenger [53] . An in vitro experiment in rats investigated the antioxidant

effect of nC

by pretreating rats with nC

, followed by an injection of carbon

tetrachloride (CCl

), a chemical which is known to cause oxidative stress in the

liver [53] . The study results showed that nC

protected the liver against oxidative

stress, which led the research team to conclude that the mechanism behind the

protective role of nC

could be attributed to the ability of nC

to scavenge large

numbers of free radicals.

18.3.3

Effects on Speciﬁ c Tissues

18.3.3.1 Brain

The brain is arguably the most complex organ of all organisms, and may be incred-

ibly sensitive to insult, with any disruption or interference with correct brain

18.3 Effects 607

functions possibly leading to a loss of ﬁ tness and/or death. Although the effects

of engineered carbon nanoparticles on the brain are poorly understood, behavioral

changes in multiple aquatic species exposed to engineered carbon nanoparticles

have been observed and have provided evidence that these particles might impair

normal brain function [19, 46] . For example, when Smith et al . [19] exposed

rainbow trout ( Oncorhynchus mykiss ) to single - walled CNTs, they observed aggres-

sive behavior in the exposed ﬁ sh that led to severe ﬁ n nipping. Although no bio-

chemical changes were observed in the brains of these ﬁ sh, a histological analysis

identiﬁ ed what appeared to be aneurysms (swelling of the blood vessels), which

indicated that the blood supply – and hence the oxygen supply – of the brain may

have been compromised. Behavioral effects were also observed in Daphnia magna

after exposure to nC

[41, 46] . In these studies, both juvenile and adult D. magna ,

when exposed to nC

, showed an abnormal behavioral response to the exposure

that resulted in sporadic swimming and disorientation.

Following initial studies conducted with mammalian cell lines, the investigations

into the toxic mode of action focused on oxidative stress [5] . As noted above, Ober-

dorster [11] had suggested that nC

could be selectively transported to the brain,

where it would cause toxic effects; this idea was based on the signiﬁ cant increase

in lipid peroxidation that had been shown in the brains of largemouth bass exposed

to nC

. Unfortunately, these results, and their subsequent interpretation, may have

been compromised by the use of an organic solvent in the experiment.

18.3.3.2 Gills

In ﬁ sh, the gills function in similar fashion to the lungs in mammals, and are vital

not only as the main site for gas exchange but also as an important component of

ionoregulation. Carbon - engineered nanoparticles, based on their size and chemi-

cal properties, have the potential to cause harm in both a physical (e.g., abrasions)

and chemical (e.g., disruption of gas exchange) manner. A study conducted by

Smith et al . [19] documented increases in both ventilation rate and mucus secre-

tion, as well as an enlargement of mucocytes on the gills of trout exposed to single -

walled CNTs. This led the authors to conclude that the CNTs had clearly caused

respiratory distress. Interestingly, the trout had responded to the exposure by

increasing a natural defense mechanisms, namely an increased mucus secretion.

This reaction is common in ﬁ sh exposed to aqueous pollutants [54] , and is a short -

term defense mechanism designed to prevent the toxicant from reaching the sensi-

tive gill epithelium [19] .

18.3.3.3 Liver

In higher - level organisms, such as ﬁ sh (and humans), the liver performs the

enormous duty of maintaining the metabolic homeostasis of the body. Its respon-

sibilities include protein synthesis, nutrient homeostasis, and the ﬁ ltration of

particulates. The liver is also the main location for the detoxiﬁ cation of toxicants

and is, therefore, susceptible to chemical exposure and toxicity. Results obtained

from studies in mammals have shown that intravenously administered fullerenes

can be retained in the body for up to one week, with the majority ( > 70%) lodging

in the liver [26] .

608 18 Ecological Toxicology of Engineered Carbon Nanoparticles

Whilst the potential effects of these compounds on correct liver function are

relatively unknown, Smith et al . in 2007, observed apoptosis (programmed cell

death) in the liver of trout following their exposure to single - walled CNTs. Although

the exact toxic mode of action was unknown, the location of the injured liver cells

(close to the blood vessels) raised concern that the nanoparticles were being deliv-

ered systemically via the blood supply [19] .

18.3.3.4 Gut

In theory, the gut would be expected to serve as the main site of uptake for nano-

particles associated with dietary exposure and drinking water. As noted earlier in

the chapter, carbon - engineered nanoparticles are potentially bioavailable and can

enter an organism via a number of pathways, including the ingestion of food and

water. Although, observations have been made of carbon nanoparticle aggregates

in the gut lumen of ﬁ sh [19] and zooplankton [12] , the bioavailability of these

aggregates and their potential to cross the epithelial lining is largely unknown, as

discussed previously.

18.3.4

Developmental Effects

Organisms are typically the most sensitive to insult (chemical or physical) during

their early development and in the early life stages. Chemical compounds

may disrupt cellular signaling, alter apoptosis patterns, or damage DNA, thus

causing organisms to develop abnormally ( teratogenicity ). Teratogens can result

in obvious effects such as physical deformities, or in more subtle changes such

as behavioral abnormalities. These changes early in life can have profound effects

on the ecological ﬁ tness of individuals, and result in deleterious population - level

outcomes.

A host of investigations have utilized zebraﬁ sh embryos when investigating the

developmental effects of engineered carbon nanoparticles [42, 43] . For example,

Usenko et al . [43] observed delayed development, morphological malformations

(e.g., of the body axis, eye, snout, jaw, otic vesicle, notochord, heart, brain, somite,

and ﬁ ns), pericardial edema (Figure 18.7 ), yolk sac edema (Figure 18.7 ), and

behavioral abnormalities (e.g., hyperactivity, hypoactivity, paralysis) in ﬁ sh exposed

to nC

. Of particular interest in this study was the constant abnormal development

of the ﬁ n regions in all exposed organisms, which was indicative of cell signaling

perturbations during early development (Figure 18.7 ). In another study, Zhu et al .

[42] found that hatching rates in nC

- exposed zebraﬁ sh embryos showed notable

developmental delay and toxicity, slower heart rates, and increased pericardial

edema.

The developmental effects such as those observed by Usenko et al . [43] and Zhu

et al . [42] may have far - reaching implications since, in nature, individuals that

experience abnormal development rarely survive to reproduce. Therefore, com-

pounds found to cause developmental effects have the potential to affect entire

populations and communities, and thus deserve special attention.