analysis, behavior and ecotoxicity of carbon-based nanomaterials in the aquatic environment

Trends Trends in Analytical Chemistry, Vol. 28, No. 6, 2009

Analysis, behavior and ecotoxicityof carbon-based nanomaterialsin the aquatic environmentSandra Perez, Marinel.la Farre, Damia Barcelo

Due to their unique properties, carbon-based nanomaterials (CNMs) have attracted considerable interest in many fields of

research, including materials sciences, microelectronics and biomedicine. The potential, the growing use and the mass produ-

ction of fullerenes and carbon nanotubes have stimulated research on their potential impact on the environment and human

health. To gather proper information about hazards of CNMs, it is important to have reliable analytical data on them, to find out

how they behave in the environment and to evaluate ecotoxicological information about them.

This review presents the latest research carried out to assess the risks of engineered CNMs in the aquatic environment,

including analytical methods and ecotoxicity assessment. We pay special attention to the surface properties of CNMs, which are

vitally important for their aggregation behavior, their mobility in aquatic systems, their interactions with aquatic organisms, and

their possible entry into the food chain. We also consider interactions with natural organic matter and other interactions that can

alter aggregation behavior in water.

ª 2009 Elsevier Ltd. All rights reserved.

Keywords: Aggregation; Aquatic environment; Carbon-based nanomaterial; Ecotoxicity; Emerging pollutant; Environmental analysis; Human health;

Mass spectrometry; Mobility; Surface property

Sandra Perez,

Marinel.la Farre,

Damia Barcelo*

Department of Environmental

Chemistry, IDAEA-CSIC c/ Jordi

Girona, 18-26, 08034

Barcelona,

Spain

Damia Barcelo

Catalan Institute of

Water Research,

ICRA C/ Pic de Peguera,

15, 17003 Girona,

Spain

820

*Corresponding author.

Tel.: +34 934 006 100x435;

Fax: +34 932 045 904;

E-mail: [email protected]

1. Introduction

Nanomaterials (NMs) are defined asmaterials that have structural featureswith at least one dimension of 100 nm orless, and include nanofilms and nano-coatings (<100 nm in one dimension),nanotubes and nanowires (<100 nm intwo dimensions) and NMs (<100 nm inthree dimensions) [1]. NMs differ in size,shape, composition and origin, and theycan comprise organic or inorganic, crys-talline or amorphous particles. They canbe found as single particles, aggregates,powders or dispersed in a matrix, overcolloids, suspensions and emulsions,nanolayers and films, and coated or stabi-lized as fullerenes and their derivates [2].

NMs can be classified in three maingroups: (i) natural; (ii) incidental; and, (iii)engineered. Natural NMs are those thatcan occur naturally and may have been inthe environment for millions of years (e.g.,fullerenes have been detected in geologicaldeposits from the Cretaceous-Tertiaryboundary [3]). In addition, in a meltsample from an ice core dated as being

0165-9936/$ - see front matter ª 2009 Elsev

about 10,000 years old, carbon nanotubes(CNTs) and fullerenes were detected inGreenland [4]. Nanodiamonds (NDs) havebeen also found in the Younger DryasBoundary Sediment Layer in NorthAmerica [5]. Incidental NMs are producedunintentionally during many industrialprocesses, or as consequence of enginepollution (e.g., welding fume and diesel-emission particulates are sources of inci-dental NMs). Finally, engineered NMs(ENMs) and nanostructures are producedintentionally and differ because they arebeing fabricated from the ‘‘bottom up’’.

During the past decade, interest in NMshas risen dramatically because of theirexceptional physico-chemical properties.NMs are characterized by large surface-area-to-volume ratios, with about 40–50% of the atoms being on the surface;this results in greater reactivity, comparedwith bulk materials, or quantum effects.They are used in many industrial areas(e.g., materials science, personal-careproducts and electronics) and will providea promising technology in many otherareas (e.g., medicine [6]).

ier Ltd. All rights reserved. doi:10.1016/j.trac.2009.04.001

mailto:[email protected]

Figure 1. The fate of nanomaterials in the environment [11,12].

Trends in Analytical Chemistry, Vol. 28, No. 6, 2009 Trends

Classification of NMs for commercial purposes includesmetal NMs, metal-oxide nanopowders, semiconductorsand alloys, carbon-based NMs (CNMs) (e.g., fullerenes)and nanorods (CNTs and nanowires). In addition,nanolayers are the subject of the most important topicswithin nanotechnology. Through nanoscale engineeringof surfaces and layers, a vast range of functionalities andnew physical effects (e.g., magnetoelectronic or optical)can be achieved. Furthermore, nanoscale design of sur-faces and layers is often necessary to optimize interfacesbetween different material classes (e.g., compoundsemiconductors on silicon wafers) and to obtain thespecial properties desired. Other supramolecular struc-tures (e.g., dendrimers, micelles or liposomes) are alsoNMs [6].

At present, research on NMs is focused on develop-ment of new NMs or their applications in different areas(e.g., biomedicine [7]). However, concern has arisenabout the presence of NMs in the environment. Forexample, the US Environmental Protection Agency(EPA) and the European Community (EC) are payingattention to the study of the fate, transport, and healtheffects of the NMs in the environment. However, theirenvironmental study is still in its infancy because there isa lack of analytical methods able to detect and toquantify the wide range of NMs and their unique prop-erties (e.g., there is only one paper available reportingfullerene concentrations in suspended solids of waste-water [8]). Also, NMs can be modified in the environ-ment by the action of light, oxidants or microorganismsor can be coated with organic matter [9]. Moreover, NMswill inevitably aggregate or agglomerate into largermasses, thereby losing their nanoscale-related properties

and increasing the difficulty of monitoring them in theenvironment. Although NMs are not yet regulated, theyare already included in lists of emerging pollutants [10].Through modeling a range of NMs arising from con-sumer products, estimations of the potential environ-mental concentrations have been published (Fig. 1 andTable 1) [11,12].

Because NMs are involved in a new technology andpresent properties different from common contaminantsof larger dimensions, the risks of NMs have to be eval-uated differently. Due to the great increase in the pro-duction volume and widespread use of NMs, they canpose a potential threat for the environment and humanhealth. Since NMs differ in origin, size and material, NMsare expected to exhibit different biological effects. Inaddition, NMs of the same bulk material but with dif-ferent crystal structure, surface coating or size can showdifferent effects. For example, the toxicity of CNTs is af-fected by the degree and the kind of agglomeration [13].

Because only biological effects of NMs are known andno data on presence of NMs in the aquatic environmentare available, their environmental risks are hard topredict. First simulations of environmental exposure ofthree NMs [i.e. silver, titanium dioxide (TiO2) and CNTs]have been reported [12]. According to these calcula-tions, CNTs presented no significant environmental risk.However, effects for TiO2 and silver could not be ex-cluded. Determination of these NMs in the environmentis required for further risk assessment [14].

CNMs (e.g., fullerenes, CNTs and NDs) may be amongthe most useful ENMs for different applications. Atpresent, fullerenes and CNTs are widely used, mainly inelectro-optical devices, as polymers and films. CNTs can

http://www.elsevier.com/locate/trac 821

Table 1. Predicted concentrations of nanomaterials in the environment. Extracted from soil, water and air [11,12]

Type of nanomaterial Water [lg/L] Soil [lg/kg] Air [lg/m3]

Ag 0.010–0.03 0.02–0.43 0.0017–0.0014AlO3 0.0002 0.01Au 0.14 5.99Carbon nanotubes 0.0005–0.0008 0.001–0.02 0.0015–0.0023CeO2 <0.0001 <0.01Fullerenes 0.31 13.1Hydroxyapatite 10.1 422Latex 103 4307Organo-silica 0.0005 0.02SiO2 0.0007 0.03TiO2 0.7–24.5 0.4–1030 0.0015–0.042ZnO 76 3194


occur in a variety of forms [e.g., single-walled CNTs(SWCNTs) or multi-walled CNTs (MWCNTs), filled orsurface-modified]. In addition, their macroscopic prop-erties make them suitable for production of plastics,catalysts, components in electronics and composites.

Another emerging group of CNMs comprises NDs,which are strongly expected to become versatile mate-rials for biological applications (e.g., drug carriers[15,16]), coatings on medical implants [17] and imagingprobes [18–20]). However, to date, very few studies havedescribed their fate and environmental presence inrealistic scenarios. For example, understanding the bio-logical consequences of ND is crucial for realizing itsbiorelevant applications and ascertaining its possiblehazards to organisms. Just one study has been carriedout reporting the evaluation of biodistribution and fate ofNDs in mouse in vitro studies. In this study, Yuan et al.[21] stated that NDs were predominantly accumulatedin liver and lung.

Ecotoxicological evaluation of fullerenes and CNTs isdifficult using existing tools. In addition, the hydropho-bicity and van der Waals interactions of CNTs maysuggest aggregation and sedimentation in aquaticsystems, whereas surface modifications (e.g., functionalgroups and coating) used to improve their propertiesmay increase their stability in aqueous systems, there-fore influencing their environmental fate and toxicology.

Although fullerenes and CNTs are considered ENMs,they are also natural particles (fullerenes) or have closerelatives in the environment (CNTs) [22]. Regardless ofbeing classified together in terms of composition, it isclear that fullerenes and CNTs may behave very differ-ently in the environment. In aqueous systems, both tendto aggregate and therefore more efforts are made tomodify the surfaces of CNTs to improve stability inaqueous suspensions [23].

In this paper, we review research on analysis, envi-ronmental health and safety carried out in recent yearsto assess the impact of CNMs, including analyticalmethods and approaches to assessing their ecotoxico-logical impact.

822 http://www.elsevier.com/locate/trac

2. Analytical methods

The determination of NMs in complex environmentalmatrices is challenging due to their unique propertiesthat affect sampling, extraction and quantification [24].Sampling of NMs is a demanding task for several rea-sons:� adsorption in common vessels should be avoided due

to the surface charge of the NMs and possible chargesof the bottle walls at specific pHs [25];

� NMs are present in the environment in dynamic non-equilibrium systems that are often sensitive to chemi-cal, or even physical, disturbances, so it is preferableto perform in situ analysis or apply methodologiesthat cause minimum perturbation from the sampling[26]; and,

� discrimination between existing ambient NMs andENMs is an important factor in the sampling strategy.

2.1. Preparation of aqueous dispersionsBefore developing an analytical methodology, someproperties of the NMs have to be taken into account (e.g.,aggregation state, elemental composition, mass concen-tration, shape, size, solubility, structure, surface chemis-try and charge). For example, fullerenes have lowsolubility (�1.3 · 10�11 lg/L in water [27]), but they areable to form water-stable C60 aggregates with diameters inthe range 25–500 nm. Consequently, fullerenes cannotdissolve in pure water, and a stable aqueous dispersion offullerenes can be prepared from only organic stable col-loidal dispersions. Several approaches have been reportedfor producing aqueous suspensions of fullerenes withoutstabilizing agents or apparent functionalization. Forexample, an aqueous dispersion was prepared usingbenzene and the solution diluted first with tetrahydrofu-ran (THF), then with acetone, and finally with water [28].The aqueous suspension was obtained after distilling outthe organic solvent. Other authors prepared a solution offullerenes in a mixture of toluene and water, sonicated forseveral hours until the toluene was evaporated [29,30].


Other methods proposed the use of THF for preparingaqueous fullerene dispersions for toxicological tests[31,32]. Deguchi et al. [31] proposed solubilization ofC60 and C70 in THF and stirring overnight under anargon atmosphere. The excess of solid was filteredthrough PTFE 0.45 lm to give a saturated solution, andthen diluted with water; and, finally, THF was removed.In other efforts [33], a fullerene powder was added towater and stirred for 4–6 weeks until the solution turneddark brown. This effort then stopped and the solutionwas allowed to settle for 4 h, after which it was decantedto obtain the suspended solids. Finally, the concentrationobtained in the suspensions was determined by UV-Vis,and the size distribution by transmission electronmicroscopy (TEM) and dynamic light scattering. Each ofthese methodologies generates n-fullerenes with verydifferent physico-chemical characteristics. Aggregatesproduced by each of these methods differed with respectto size, structure, charge and hydrophobicity [34]. Someof these differences can probably be attributed toincomplete removal of organic solvent. Other factors thatcan influence aggregation are the pH of the sample,dissolved oxygen, effects on ionic strength, and the effectof organic macromolecules [35].

For preparation in organic solvent, a tedious methodwas developed by Deguchi et al. [31] and a modified onewas used by other authors [36,37]. To increase theaffinity of fullerenes in water, a derivatization could beperformed. Derivatized fullerenes are often expected toform rather stable, molecularly-dispersed suspensions, asa result of hydrophilization or encapsulation of the ful-lerenes to prevent contact between the fullerene cores[38]. For example, the stability in water of hydroxylatedfullerene (fullerol) depends on their aggregation, andthat depends on the pH of the sample and the effects oforganic macromolecules, particle size and charge [39].

Preparation of aqueous solutions of CNTs can beclassified into three groups:� dispersion upon oxidative treatment;� non-covalent stabilization; and,� covalent stabilization [40].

Oxidative treatments are the most common treatmentsused for solubilization of CNTs. For example, for prepa-ration of stable, aqueous, suspended SWCNTs from soot,a method using arc discharge [41] and purifying theSWCNTs with 3.3 N nitric acid was proposed [42]. Thisprocess oxidizes SWCNTs at their termini, leading toincorporation of hydroxyl and carboxylic-acid functionalgroups and rendering the tubes dispersible in water. Theconcentration of the dispersion was further determinedby UV-Vis at 365 nm [43]. In non-covalent solubiliza-tion, the CNTs are dispersed through derivatization orcomplexation with micelles, polymers or other aggregatesystems. After arc-discharge evaporation of the CNTsfrom graphite rods [13], the addition of the surfactant(Tween 80) in ultrapure water was used in order to

obtain the suspension of CNTs. In another work to pre-pare dispersions of SWCNTs and medium-walled CNTs(MWCNTs), an aqueous solution of hydroxypropyl me-thyl cellulose was added to 1 mg of each CNT and thensonicated [44].

2.2. Sample preparationWhile a range of methods is accessible for detection andcharacterization of NMs, a number of challenges willarise when analyzing these materials in environmentalmatrices due to the analytical artifacts caused by samplepreparation, lack of reference materials and the matrix ofthe sample. Few methods have been reported. Forexample, pristine fullerenes are comparatively soluble inorganic solvents, so they can be extracted by exploitingtheir solubility in toluene [37]. However, for theirextraction from environmental matrices, properties,such as adsorption to biological molecules or aggrega-tion, have to be taken into account.

For extraction of fullerenes, C60 and C70, in materials[45], toluene was used a solvent. Three different proce-dures were applied to the samples:� sonication with toluene for four hours;� Soxhlet with toluene; and,� centrifugation [45].

To calculate recoveries, the materials were spiked withfree C60 and C70 and treated like real samples, showingrecoveries of 92% and 91%, respectively. Fullerenes fromC60 to C98 were extracted first with toluene and thenwith trichlorobenzene [46,47]. The trichlorobenzenesolution was evaporated under nitrogen and solventexchanged to toluene:methanol (55:45) and filteredthrough PTFE and 1 lm [46].

In another publication from the same group, they useda fractionated extraction of soot with toluene and thentrichlorobenzene in order to obtain only fullerenes largerthan C70 in the second extraction [47]. The trichloro-benzene solution was filtered through a PTFE membraneand the solution was evaporated to dryness, reconsti-tuted in toluene:methanol (55:45) and filtered again.After that, the sample solution was fractionated into fivefractions by preparative-scale liquid chromatography(LC) with the monomeric octadecyl-silica phase andfullerenes up to C98 were determined and identified inthe soot samples [47].

Supercritical fluid extraction and supercritical fluidchromatography were used for extraction of fullerenes[48]. Using mixtures of CO2 and toluene as extractionmedium and mobile phase, isolation, purification andcollection of fullerenes from carbon soot can besimplified, and made convenient and environmentallysafe.

Fullerenes, C60, have been selectively extracted from atoluene solution with cyclic dimers of zinc porphyrins1C6 and the extraction of C P C76 from a toluene solu-tion with zinc porphyrins 2C5–2C7 [49].



Liquid-liquid extraction (LLE) is a classical method forextracting organic molecules from environmental sam-ples. Traditional and modified LLEs were used for deter-mination of C60 in the biological samples [50]. Tocompare these two methodologies, water solutions con-taining C60 were prepared for a modified methodology ofDeguchi et al. [31] using different types of filter with0.22 lm pore size (see above). To a 10 mL glass vial,2 mL of the C60 in water and 4.5 mL of THF were added;they were then shaken at 500 rpm for 10 min and cen-trifuged, and the residue evaporated. They tried two typesof evaporation (i.e. until dryness or partial), and it wasfound that evaporation to dryness caused a dropped inefficiency in the extraction methodology, including whensilanized glassware was used [50]. When the solution ofC60 in toluene was evaporated partially, the recoverieswere 97–100%. However, the extraction efficiencydropped when evaporation to dryness was used [50].Other authors also used evaporation for extracting C60

from water, and compared it to two other methodologies{i.e. salt addition with LLE and solid-phase extraction(SPE) [51]}. Evaporation of the aqueous sample, followedby partition of dry fullerenes into toluene, showed thataqueous C60 were non-volatile and heat stable in water[29]. Although the recoveries obtained from the threetechniques were similar for ultrapure water, low recoverywas obtained for the evaporation technique for theextraction of C60 from tap water. In addition, the limits ofdetection (LODs) for the evaporation technique werehigher than those for SPE (i.e. 2.8 lg/L and 0.3 lg/L,respectively [51]). Consequently, this evaporation tech-nique was rejected for the determination of C60 in waters.

Regarding the use of LLE, it showed LODs for C60 inultrapure water of the same magnitude as those for theevaporation technique. Although the recovery efficiencyof C60 in ultrapure water and tap water were similar tothose obtained with SPE, when LLE was used to extractC60 from treated effluents from wastewater, it led to theformation of emulsions that were difficult to separate,thus yielding lower recoveries [51].

Other authors proposed the use of LLE for extractingfullerenes, C60 to C98, from artificial freshwater, yieldingrecoveries of 93% [52].

For determination of fullerenes, namely C60, C70 andN-methylfulleropyrrolidine C60, in real suspendedmaterial from wastewater samples, a ultrasonicationmethod relying on extraction with toluene was reported[8]. They compared extraction efficiencies in three typesof water, showing that the recoveries obtained weregenerally above 60% for both surface water and waste-waters. The overall variability of the method was below15% for the three fullerenes and all tested particulatematrices (i.e. ultra-pure water, surface water andwastewater). Method quantification limits were in therange 0.2–1 ng/L for particulate from effluents ofwastewater-treatment plants (WWTPs).


It has been suggested that CNTs will have limitedpotential for aqueous association and transport becauseof aggregation and adsorption to sediment particles.However, research is progressing to functionalize CNTsto enhance their dispersion potential [53] (e.g., incor-porating hydroxyl and carboxylic-acid functional groupsto make the tubes dispersible in water [42]). They canalso be stabilized in the aqueous phase by well-charac-terized surfactants and polymers, as well as havingsimilar interactions with organic molecules present inthe environment [9].

Generally, to separate CNMs, microfiltration, field-flowfractionation, and size-exclusion chromatography (SEC)have been used. One recent work evaluated extraction ofcarboxylic CNTs from surface water using a filter modi-fied with MWCNTs as a preconcentrator [54]. To preparethe filters, a dispersion of MWCNTs prepared in TritonX100 was filtered through a nylon filter with a pore sizeof 0.45 lm. They were then washed with methanol anddried under air stream. MWCNTs can interact withSWCNTs through p-p interactions, showing a highcapacity to adsorb SWCNTs [54]. SEC was also used asan extraction/purification method to obtained free andsized separated multi-walled nanotubes from aqueousdispersions [55]. This chromatographic technique is aneffective, non-destructive method for purification andsize separation of CNTs, with further applications beingits use for HPLC separation.

2.3. Separation and detectionSeparation of fullerenes to obtain the individual mem-bers is a critical step, for which electrophoresis andchromatography have been used. Capillary electropho-resis (CE) and electrochromatography have been em-ployed to separate fullerenes [56] in both non-aqueoussolutions and aqueous buffer in order to solubilize ful-lerenes with surfactants [57]. High-performance liquidchromatography (HPLC) is the method of choice foranalyzing fullerenes using octadecyl or other modifiedsilica-based stationary phases and novel stationaryphases (e.g., bonded-phase ligand) [58,59].

There are several techniques available to characterizeand to detect fullerenes and their derivatives {namely,13C nuclear magnetic resonance (NMR), X-ray diffrac-tion (XRD), UV-Vis, and Fourier transform infraredspectroscopy (FTIR) and mass spectrometry (MS)[60,61]}. A number of reviews have described the rangeof techniques for characterizing NMs [1,26]. However,this article reviews the techniques used for quantifyingfullerenes and CNTs in the aquatic environment. Forfullerene quantification, UV-Vis has been for a long timebased upon using an absorbance peak in the UV spec-trum in the range 300–400 nm [61,62]; recently, MStechniques coupled to a system using liquid or solidphases was used because of its selectivity and degree ofinformation.


Regarding surface-sampling/ionization techniques,matrix-assisted laser desorption ionization (MALDI) isconsidered the most successful MS technique for fuller-ene analysis [63,64]. However, to analyze fullerenes, it isnecessary to identify appropriate matrices to obtainhigher yields of molecular ions of analytes. For example,the new matrix 9-nitroanthracene was recently used asa suitable matrix for detecting fluorofullerenes withMALDI in negative mode [64]. Other new matrices haveprovided good results in detecting substituted fullerenes[63].

Laser-desorption MS greatly fragments molecular/anion or cation radicals (e.g., in fullerene derivatives),resulting in problems in interpreting the spectra. Softtechniques using atmospheric pressure ionization (API)have a potential advantage in this case because of itssoftness. API, including electrospray ionization (ESI) andatmospheric pressure chemical ionization (APCI), iscurrently used in LC-MS systems for detection of fuller-enes. However, due to the low polarity of C60 fullerene,APCI was considered more suitable for their analysis[65].

Introducing benzene to the APCI source ensured thatthe dominant ionization mechanism for fullerenes inpositive-ion mode was charge transfer from the C6H6

+

ions that dominated the plasma [66]. However, somedegree of ionization via proton transfer from waterclusters or benzene was also apparent. Also, using a non-aqueous reversed-phase HPLC-APCI-MS separationmethod in negative mode, fullerenes and bromo-substi-tuted fullerenes were separated and detected efficiently[67].

Although it is assumed that neutral or non-polaranalytes (e.g., fullerenes) are generally not detectable byESI-MS, some groups have demonstrated successfuldetection of fullerenes using this approach [47,68]. Be-cause fullerene molecules show high electron affinityand low ionization potential, they can be pre-ionized insolution prior to ESI by adding an electron donor or anelectron acceptor [68,69].

Using ESI without any chemical pre-ionization ofsamples in solution, some authors have detected fuller-enes. The ESI-generation mechanism shows that anelectrospray source works as controlled current electro-chemical cell, and molecular radical anions and cationscan be formed by electrochemical oxidation at the metal/solution interface of the ESI needle [70]. With an elec-trospray capillary needle of thin metal and a time-of-flight (ToF) detector, fullerenes were ionized in bothnegative and positive mode [70].

In another work, without any modification, Jinnoet al. were able to detect fullerenes. A method using anunmodified LC-ESI-MS with an octadecyl-silica station-ary phase and a mixture of toluene:acetonitrile as mobilephase, was able to separate and to detect higher fuller-enes successfully [47]. Temperature effects in the sample

preparation were examined and the results indicatedthat separation of higher fullerenes was achieved at15�C.

Using API, only two methods have been reported fordetermination of fullerenes in waters [8,51]. Ultrapurewater samples, spiked at the lg/L level, were extractedwith SPE (see above) and were eluted in the HPLC sys-tem isocratically using toluene:acetonitrile (55:45) on areversed-phase Nova-pack C18 column. The LOD of themethodology using LC-APCI-MS in negative mode was0.30 lg/L for fullerene C60 [51].

Another method using mobile phase toluene:methanol(55:45) on a reversed-phase Targa C18 column and ESIwith an LC-MS2 instrument used selected ion monitoring(SIM) mode for determination of fullerenes in water [52].The instrumental limit of quantification (LOQ) was0.002 lg/L, while the whole-method LOQ was 0.04 lg/L,which is one order of magnitude lower than that achievedwith the previous APCI method [51].

Also, using ESI-MS in negative mode, LC coupled to anhybrid triple-quadrupole linear ion-trap MS, three ful-lerenes (i.e. C60, C70 and N-methylfulleropyrrolidine C60)were detected in water [8]. Chromatographic separationwas achieved with a Purospher Star RP-18 end-cappedcolumn and the analytes were eluted isocratically usinga mobile phase comprising a mixture of toluene:metha-nol (1:1) This method employed multiple reactionmonitoring (MRM) mode, but the ion selected in thethird quadrant was identical to the precursor ion, due tothe chemical stability of fullerenes. Due to the sensitivityof the instrument, the method LOQs were in the range1–10 ng/L in ultrapure water. The method was devel-oped for the determination of fullerenes in suspendedsoils from WWTP effluents (see extraction methodabove) achieving method LOQs in the range 0.2–1 ng/L[8].

Atmospheric pressure photoionization (APPI) is an-other ionization mode that has properties and mecha-nisms different from those of API. APPI has been used fordetection of fullerenes in toluene solutions with electron-capture ionization because fullerenes present positiveelectron affinity. APPI achieved sensitive detection usingdirect injection to obtain an LOD of 0.15 pg [71,72]. ThisLOD is lower than those achieved by other techniquesand is similar to those obtained by MALDI.

Microscopic, spectroscopic, instrumental separationmethods and UV-Vis detection are used to characterizeCNTs, [54]. Microscopic techniques {e.g., TEM, scanningelectron microscopy (SEM) and atomic force microscopy(AFM) [6,55,73]}, spectroscopic techniques {e.g., Ra-man and near-infrared spectroscopy [74]} and thermo-gravimetric analysis are the methods of choice.Separation techniques prior to detection techniques (e.g.,UV-Vis and Raman detection) encompass CE and LC. AnLC method for purifying SWCNTs was based on usingTHF as the mobile phase [75].



More frequently, CE is used for CNT separation. Twoworks separated CNTs in water using CE with UV-Visdetection at 210 nm and 240 nm, respectively. CNTseparation was accomplished with a background elec-trolyte solution containing a polymer in ammoniumacetate at pH 8.03 [44].

In another work, determination of CNTs in surfacewaters was performed using the electrophoretic separa-tion of carboxylic SWCNTs using an ammonium-acetatesolution at pH 7.5 and was accomplished in 5 min.Recoveries for the whole method using spiked sampleswere in the range 70–85% and the LOD was 0.8 mg/L[54]. The authors proposed the combination of the CEwith NIR-fluorescence or Raman spectroscopy as pow-erful tools for determination of CNTs in the environment.

3. Occurrence and behavior

The behavior of ENMs in environmental matrices in-volves several processes [25,76] that may also influenceecotoxicity [77]. Handy et al. [77] provided a detaileddiscussion of the chemistry of ENMs and the implicationsfor ecotoxicity tests. These processes may be assigned tothe following major groups:

(i) aggregation chemistry and the ability to formstable dispersions in aqueous systems;

(ii) the effects of particle shape, size, surface area andsurface charge on aggregation chemistry;

(iii) adsorption of NMs onto surfaces, including theexterior surfaces of organisms; and,

(iv) the effect of other abiotic factors on all the above,including the influence of changing environmen-tal pH, salinity (or ionic strength), water hard-ness, and natural organic matter.

The surface properties of CNMs are among the mostimportant factors governing their stability and mobilityas colloidal suspensions or their aggregation into largerparticles and deposition in aquatic systems. Stable col-loidal suspensions of CNMs are a prerequisite for efficientinteractions with some aquatic organisms, such as algae,which may lead to uptake or toxic effects.

Particle aggregation and deposition are closely-relatedphenomena [78]. Research on NM aggregation anddeposition suggests that the principles of colloidaltransport in aqueous media (i.e. Smoluchovsky�s equa-tions and Derjaguin and Landau, Verwey and Overbeek(DLVO) theory) may still apply. According to these the-ories, particle deposition and aggregation kinetics can bedefined as a two-step process (i.e. transport followed byattachment). The transport of colloidal particles isdetermined by convection, diffusion (Brownian motion)and external forces, whereas attachment onto otherparticles or surfaces is controlled by the colloidal inter-action forces operating at short distances.


The processes of deposition and aggregation are alsoinfluenced by NM surface properties, which mainly de-pend on, e.g., temperature, ionic strength, pH, particleconcentration and size, steric repulsion or attraction,hydration effects, hydrophobic interactions, magneticattraction, thus increasing their complexity.

Hydrophobic properties and van der Waals interac-tions of CNMs suggest aggregation and sedimentation inaquatic systems; however, engineered surface modifica-tions may increase their aqueous stability. A recentstudy demonstrated the influence of functional groups inMWCNTs. The presence of functional groups attached toMWCNTs increased their stability in aqueous systems(increasing order of stability: hydroxyl>carboxyl>raw),especially in combination with natural organic matter(NOM). Stabilized MWCNTs in high concentrations ofNOM were relevant for water transport and toxicitystudies. However, results from this study indicate thatfunctionalized CNTs were less detrimental to filter-feed-ing organisms than non-functionalized CNTs. In addi-tion, particle size and concentration were important fordetermining toxicity to benthic organisms, but, in thiscase, raw MWCNTs were less ecotoxic in sediments thanblack carbon, with lethality occurring at extremely highconcentrations.

In general, for the different classes of NMs, NOM mayinfluence their surface speciation and charge, changingtheir aggregation/deposition properties. NOM can besorbed to the NM surface by different interactions (e.g.,hydrogen bonding, electrostatic attraction and hydro-phobic interaction [79,80]). NOM comprises fulviccompounds, flexible biopolymers and rigid biopolymers.Fulvic compounds and flexible biopolymers have a highsurface-charge density and they increase the stability ofNMs through electrostatic or steric repulsion [9,76].However, rigid biopolymers can induce aggregation ordeposition by forming gels.

Some studies reported that CNTs, such as SWCNTs,can be taken up from test solutions in tests under con-trolled conditions [81,82]. Other studies also reportedaccumulation of CNMs through adhesion to exoskeletonand deposition on the organs of aquatic organisms (e.g.,tract and brains [83–85]). These results suggest thataquatic exposures of aquatic organisms to such CNMscould pose a risk of bioaccumulation, especially for filter-feeding copepods (e.g., D. magna). These CNTs can thenenter the food chain. Lovern et al. [86], studied the ef-fects on D. magna exposed to four solutions using US EPA48-h acute-toxicity tests. Images of the particle solutionswere recorded using TEM, and the median lethal con-centration, lowest-observable-effect concentration, andno-observable-effect concentration were determined.Exposure of D. magna to C60 and TiO2 saw mortalityincrease with concentration, whereas fullerenes showhigher levels of toxicity at lower concentrations [86].


There have been reports of in vivo biomodification andaccumulation in aquatic organisms [81,87]. Robertset al. [81] reported in vivo bio modification produced bylipid-coated CNTs. Bioaccumulation in Lumbriculus var-iegatus was recently reported by Petersen et al. [120],who showed that, after 6 h of exposure, CNTs werefound in the gut but not absorbed in the tissues. Thesame study reported uptake and depuration behaviorswith Eisena foetida.

Tissue interaction and toxicity are of great impor-tance, since CNMs (e.g., C60 derivatives) have beenconsidered carriers able to transport other molecules tospecific organs and to cross the blood-brain barrier. Thisproperty has been studied in order to obtain efficientdrug carriers, but it should be also considered with re-spect to the capability of efficiently carrying toxic com-pounds. Their possible toxicity, linked to their highadsorption properties due to their size, and their capa-bility of transporting other substances can be causes ofconcern in relation to possible ecotoxicity and damage tohuman health. However, so far, few studies have con-sidered the potential carrier effect of NMs in inverte-brates. Baun et al. [88] studied changes in D. magnamobility after exposure to organic pollutants (i.e. methylparathion, phenanthrene and pentachlorophenol) in thepresence and the absence of aqueous suspensions of C60

NMs. It was found that the toxicity of methyl parathionwas not affected by the presence of C60 aggregates,whereas a 1.9 times decrease in toxicity was observed forpentachlorophenol. For phenanthrene, an 85% sorptionto C60 aggregates was observed, but the toxicity ofphenanthrene was increased by 60% in the presence ofC60 aggregates. This showed that the sorbed phenan-threne was available for the organisms [88].

Furthermore, CNTs are known to be solubilized bybiopolymers [89,90], so the interactions between ENMsand NOM may finally determine the fate of ENMs inaquatic systems. The formation of larger aggregates byhigh molecular-weight NOM compounds will favor re-moval of ENMs into sediments and is likely to decreasetheir bioavailability. By contrast, solubilization by nat-ural surfactants (e.g., lower-molecular-weight NOMcompounds) will increase their mobility and further thebioavailability of ENMs.

Artificially synthesized organic compounds are nowbeing used to stabilize ENM suspensions in aquatic sys-tems [91–94]. These compounds are generally hydro-phobic surfactants and amphiphilic compounds can thenencapsulate NMs and form stable micelles with theirhydrophilic end outside. The formation of a stable, finely-dispersed, aqueous colloidal solution of fullerenes C60

and C70 was reported in the presence of c-cyclodextrin[95].

Similarly, poly(vinylpyrrolidone) can also stabilize C60

and CNT suspensions by wrapping round the aggregates[96,97]. CNTs can form stable suspensions with the

addition of sodium dodecylbenzene sulfonate (SDBS) andsodium dodecyl sulfate (SDS) [98–101].

Different studies have focused on the dynamicmechanical properties of CNTs [102,103]. In thesestudies, it was shown that, in the presence of appropriateorganic compounds, CNTs will have a longer residencetime in aquatic systems, and this may influence theirbioavailability and ecotoxicology.

In a recent study [101], the cytotoxicity of SWCNTssuspended in various surfactants was investigated byphase-contrast light microscopy in combination withabsorbance spectroscopy. Results of this study indicatedthat individual SWCNTs suspended in the surfactants,SDS and SDBS, were toxic to 1321N1 human astrocy-toma cells due to the toxicity of SDS and SDBS on thenanotube surfaces, whereas SWCNTs alone were nottoxic.

Only one paper (to be published) has dealt withdetermination of fullerenes in environmental samples[8]. This work was developed for determination of ful-lerenes in suspended solids from wastewater effluents of22 WWTPs in Catalonia, north-east Spain. Half theanalyzed samples contained fullerenes, and nine of themhad concentrations in the lg/L range.

4. Aquatic ecotoxicity

As for fullerenes, the potential and the growing use ofCNTs and their mass production have raised questionsabout their safety and environmental impact. Researchon the toxicity of CNTs has just begun and the data arestill fragmentary and subject to criticism. Preliminaryresults highlight the difficulties in evaluating the toxicityof CNMs. Different characteristics (e.g., structure, sizedistribution and surface area, surface chemistry, surfacecharge, agglomeration state and purity) have consider-able impact on the reactivity of CNTs. However, avail-able data show that, under some conditions, CNTs cancross membrane barriers and suggest that, if NMs reachorgans, they can induce harmful effects (e.g., inflam-matory and fibrotic reactions). Further studies on well-characterized materials are therefore necessary todetermine the safety of CNTs as well as their environ-mental impact.

There are three areas of primary concern in terms oftoxicity of fullerenes and CNTs, as follows.

i) Some ENMs (especially fullerenes) are redox-activecompounds [104], and there are controversial opin-ions about the possible toxicity of fullerenes associ-ated with redox properties.

ii) NMs partition into cell membranes, especially mito-chondria both in vivo and in vitro [105,106].

iii) Research on NMs in mammalian systems showsthat there is a selective transport mechanism fromthe olfactory nerve into the olfactory bulb [107].



This pathway also exists in rodents and fish for sol-uble metals [108]. Recently, Oberdorster hypothe-sized that this neuronal translocation pathwaycould also exist in fish for redox-active, lipophilicfullerenes, causing oxidative damage in the brain.This study showed that juvenile largemouth bassexposed to 0.5-ppm aqueous uncoated fullerenes(nC60) for 48 h had a significant increase in lipidperoxidation of the brain and glutathione (GSH)depletion in the gill.

However, interpretation of the aquatic ecotoxicity testsof CNMs presents a major difficulty because of their lowwater solubility, so the results can differ, depending onthe dispersion methods (stirring, sonication, use ofchemical dispersants, or a combination of these meth-ods) used to obtain standard solutions, or the samplepreparations.

The use of solvents (e.g., THF) or dispersants (e.g.,SDS) has the advantage of being able to obtain stabledispersions in a short time, without any other steps.However, these solvents can themselves be toxic, andother toxic impurities can be present in them. The use ofextra solvents may deform structures, thereby changingtoxicity. Methods based on the use of solvents need tokeep a consistent ratio of solvent and test materials.

Sonication has also been used a great deal because itdoes not involve solvents. However, sonication timesshould be adjusted according the concentration. Inaddition, sonication can change shapes and conse-quently toxicity. However, natural samples can containelectron donors and sonication in presence of thesesubstances could result in reactive oxygen species. Stir-ring and shaking do not requires solvents, but their maindisadvantages are long times are needed, dispersions arequite unstable, and they can change shapes and conse-quently toxicity, and, in both cases, additional stepscould be required.

Zhu et al. [83] reported acute toxicity to D. magna andfathead minnow from nC60 solubilized in THF and stirredin water. The 48-h acute toxicity test with D. magnashowed LC50 values for THF-solubilized nC60 at least oneorder of magnitude less (0.8 mg/L) than that for water-stirred nC60 (>35 mg/L). These findings suggest that THFincreased the toxicity of C60 particles. Fortner et al. [37]argued that this could be due to residual THF beingtrapped in the centre of C60 aggregates, or some otherunpredicted effect of THF on particle shape or size.

The use of less toxic compounds, but with low dis-persion has been explored. For example, Smith et al. [83]studied toxicity produced by SWCNTs to rainbow trout.In this case, stock solutions of dispersed SWCNTs wereprepared using a combination of solvent SDS, and soni-cation. SWCNT exposure during 10 days under a semi-static test caused a dose-dependent rise in ventilationrate, gill pathologies (edema, altered mucocytes, and


hyperplasia) and mucus secretion with SWCNT precipi-tation on the gill mucus. No major hematological orblood disturbances were observed in terms of red andwhite blood-cell counts, hematocrits, whole-bloodhemoglobin, and plasma Na+ or K+.

The potential for CNMs to cause oxidative injury infish remains controversial. Some recent works demon-strated that toxic effects were more likely to result fromdegradation products of the THF used to prepare theaggregates [109]. This study evidenced the lack of tox-icity of C60 in an experiment in which larval zebrafish(Danio rerio) were exposed to C60 aggregates (no THF orsolvents present). In addition, after a 72-h exposure,changes in global gene expression in larval zebrafishwere minimal and no detoxication pathways were acti-vated.

Indirect non-specific toxic effects of CNMs, which in-clude physical irritation and occlusion of surface tissues(e.g., gills), have been found in some studies with aquaticorganisms [87]. CNTs accumulated on gill surfaces andled to irritation and lesions. Smith et al. [82] concludedthat CNTs acted as a respiratory toxicant in rainbowtrout. As we argued in previous sections, the size and theshape of CNMs can affect the potential for exposure andtoxicity in organisms. For example, Cheng et al. reportedthat zebrafish embryos appear to be protected fromaggregates of CNTs, because aggregates are larger andunable to pass through nm-size pores in the chorion[110]. Fish-gill surfaces may be more susceptible toirritation by CNMs of a defined size range, or specific sizesor shapes; and, it may be more difficult for fish to dis-lodge some shapes or sizes of CNMs from the cell surface.

Other expressions of ecotoxicity remain unstudied orwith very few works carried out. For example, very fewworks report study of possible genotoxicity associatedwith CNMs. Recently, characterization and in vivo tox-icity evaluation of double-walled CNTs (DWCNTs) inlarvae of the amphibian Xenopus laevis was conducted byMouchet et al. [111]. In this case, acute toxicity (mor-tality), growth inhibition, and genotoxicity as theexpression of the clastogenic and/or aneugenic effectswere observed in erythrocytes in the running blood after12 days of exposure. The results of this study showedthat toxicity was related to physical blockage of the gillsand/or digestive tract.

The main difficulties in analysis of CNMs result fromthe different methods of preparation, the lack of estab-lished methods to test the toxicity of NMs and the lack ofpure standards. A significant concern regarding inves-tigations of NMs is the inadequate assessment of purityto assure that NMs are free from contaminants, partic-ularly metal catalysts used during fabrication, which canbe present in materials obtained from commercial sup-pliers [112]. In addition, to date, it is very difficult toevaluate any environmentally realistic CNT concentra-tions. Table 2 summarizes data from ecotoxicity tests.

Table 2. Summary of ecotoxicological studies of engineered carbon-based nanomaterials

Particle Test Measurements and remarks Ref.

Fullerene C60,SWCNTs, MWCNTs

Profound cytotoxicity of SWCNTswas observed in alveolar macrophage(AM) after a 6-h exposure in vitro.

Dispersion due to sonicationSWCNTs>MWCNTs>C60

[113]

Fullerene C60 Antibacterial activity In this work, toxicities using different dispersionapproaches were compared

[96]

Fullerene C60 Daphnia magna EC50 = 460 lg/L (THF) [86]EC50 = 7.9 mg/L (sonication)

Fullerene C60 Daphnia magna EC50 = 0.8 lg/L (THF) [83]Fathead minnow EC50 > 35 mg/L (stirring)

Fullerene C60 Daphnia magna Fixed exposure concentrations tested. [87]Hyalella azteca For D. magna effects were observed.

Dispersion: Stirring in ultra pure waterFullerene C60 Daphnia magna Exposure to 260 lg/L increased heart rate of D. magna [114]

Solvent THFFullerene C60 Pseudomona putida Bacterial membrane lipid composition and behavior [115]

Bacillus subtilis Growth-inhibiting concentrations at 0.5 mg/L P.putida, 0.75 mg/L B. subtilis

FullerenesC60, C70, C60(OH)24

Danio rerio (Zebrafish) Development of embryos and physiological changes. [116]200 lg/L of C60 and C70 induced malformations,pericardial edema and mortality. C60(OH)24 was lesstoxic that C60.

Fullerene C60 Daphnia magna Dispersion: Stirring in ultra-pure water [88]Addition of 5–8 mg/L of fullerene produced anincrease in phenanthrene toxicity

C60 watersuspensions (nC 60)

Exerts ROS-independentoxidative stress in bacteria

Protein oxidation, changes in cell membranepotential, and interruption of cellular respiration

[117]

C60, SWCNT, MWCNT Daphnia magna [118]C60, SWCNT, Decomposes bacteria, micro-algae,

microinvertebrates and fishAqueous solution filtered [119]

SWCNTs Amphiascus tenuiremis Dispersion, oxidation and dispersion in water. [43]Mortality of 36% at 10 mg/L

SWCNTs Rainbow trout Dispersion with sodium dodecyl sulfate [82]Respiratory toxicity and possible neurotoxic effects

SWCNTs Danio rerio (Zebrafish) Several conditions were tested [110]Development of embryos

MWCNT, Multi-walled carbon nanotube; SWCNT, Single-walled carbon nanotube.


5. Conclusions

CNMs are used for a wide range of applications fromcommerce to medicine. Difficulties in determining CNTsbegin with sampling and preparation of solutions. Thenature of the fullerene-water or CNT-water interactionsdramatically changes the state of fullerenes. For theirdetermination in natural waters, the dispersion step ofthese carbonaceous materials is crucial, and severalmethods have been proposed. For the preparation offullerene dispersions, organic solvent has to be used be-fore water, and, for CNT dispersions, the CNTs have to beoxidized, derivatized or complexed due to their low sol-ubility.

Several methods for characterizing CNMs have beenproposed, but only two for their determination in theaquatic environment, so environmental analysis is in itsinfancy. Research efforts are needed in the analyticaldetermination of CNMs.

There is a need to define scenarios of exposure. Re-search on establishing appropriate ecotoxicity-teststrategies and methods should first define realistic con-ditions and then test ecotoxicity under these conditions.The fate and the behavior of NMs in the aquatic envi-ronment need to be considered, as does the influence onaggregation and deposition processes of NMs, and pos-sible changes in the bioavailability of other toxicantspresent in the same environmental compartment.

In recent years, several studies have focused onassessing the ecotoxicological risk of new ENMs inaquatic environments. However, this research is still atan initial stage of development and several issues need tobe resolved, because of using different dispersal methodsfor preparing test solutions in ecotoxicity tests.

Another critical point is chemical characterization oftest materials.

Preliminary toxicological data and predicted concen-trations suggest that NMs are not acute toxicants, but



call for warnings of possible sub-lethal and long-termeffects, and possible synergistic effects with other toxicpollutants present in the same environmental compart-ments.

Finally, to date, no data are available on marine eco-systems. There is a need to collect data on marine andestuarine species, where the physico-chemical propertiesof NMs in these habitats may differ from those propertiesin fresh water.

AcknowledgementsThe work presented in this article was supported by theSpanish Ministry of Science and Innovation, CEMAGUA(CGL2007-64551/HID) and INNOVA MED (INCO-2006-584517728). This work reflects only the authors� views,and the European Community is not liable for any usethat may be made of the information contained therein.SP acknowledges the contract from the Ramon y CajalProgram of the Spanish Ministry of Science and Inno-vation.

References[1] K. Tiede, A.B.A. Boxall, S.P. Tear, J. Lewis, H. David, M.

Hassellov, Food Addit. Contam., A 25 (2008) 795.

[2] J. Jiang, G. Oberdorster, P. Biswas, J. Nanopart. Res. 11 (2009)

77.

[3] L. Becker, J.L. Bada, R.E. Winans, J.E. Hunt, T.E. Bunch, B.M.

French, Science (Washington, DC) 265 (1994) 642.

[4] L.E. Murr, E.V. Esquivel, J.J. Bang, G. de la Rosa, J.L. Gardea-

Torresdey, Water Res. 38 (2004) 4282.

[5] D.J. Kennett, J.P. Kennett, A. West, C. Mercer, S.S.Q. Hee, L.

Bement, T.E. Bunch, M. Sellers, W.S. Wolbach, Science (Wash-

ington, DC) 323 (2009) 94.

[6] M. Farre, K. Gajda-Schrantz, L. Kantiani, D. Barcelo, Anal.

Bioanal. Chem. 393 (2009) 81.

[7] N.A. Rizvi, G.J. Riely, C.G. Azzoli, V.A. Miller, K.K. Ng, J. Fiore, G.

Chia, M. Brower, R. Heelan, M.J. Hawkins, M.G. Kris, J. Clin.

Oncol. 26 (2008) 639.

[8] M. Farre, S. Perez, K. Gajda-Schrantz, V. Osorio, L. Kantiani, A.

Ginebreda, D. Barcelo, Environ. Sci. Technol. (submitted, 2009).

[9] H. Hyung, J.D. Fortner, J.B. Hughes, J.-H. Kim, Environ. Sci.

Technol. 41 (2007) 179.

[10] S.D. Richardson, Anal. Chem. 80 (2008) 4373.

[11] A.B.A. Boxall, K. Tiede, Q. Chaudhry, Nanomedicine 2 (2007)

919.

[12] N.C. Mueller, B. Nowack, Environ. Sci. Technol. 42 (2008)

4447.

[13] P. Wick, P. Manser, L.K. Limbach, U. Dettlaff-Weglikowska, F.

Krumeich, S. Roth, W.J. Stark, A. Bruinink, Toxicol. Lett. 168

(2007) 121.

[14] B. Nowack, Nano Today 4 (2009) 11.

[15] O.A. Shenderova, V.V. Zhirnov, D.W. Brenner, Crit. Rev. Solid

State Mater. Sci. 27 (2002) 227.

[16] A. Kruger, Angew. Chem., Int. Ed. Engl. 45 (2006)

6426.

[17] R.J. Narayan, W. Wei, C. Jin, M. Andara, A. Agarwal, R.A.

Gerhardt, C.C. Shih, C.M. Shih, S.J. Lin, Y.Y. Su, R. Ramamurti,

R.N. Singh, Diamond Relat. Mater. 15 (2006) 1935.

[18] S.J. Yu, M.W. Kang, H.C. Chang, K.M. Chen, Y.C. Yu, J. Am.

Chem. Soc. 127 (2005) 17604.


[19] C.C. Fu, H.Y. Lee, K. Chen, T.S. Lim, H.Y. Wu, P.K. Lin, P.K. Wei,

P.H. Tsao, H.C. Chang, W. Fann, Proc. Nat. Acad. Sci. U.S.A.

104 (2007) 727.

[20] F. Neugart, A. Zappe, F. Jelezko, C. Tietz, J.P. Boudou, A.

Krueger, J. Wrachtrup, Nano Lett. 7 (2007) 3588.

[21] Y. Yuan, Y. Chen, J.H. Liu, H. Wang, Y. Liu, Diamond Relat.

Mater. 18 (2009) 95.

[22] B. Nowack, T.D. Bucheli, Environ. Pollut. 150 (2007) 5.

[23] S.J. Klaine, P.J.J. Alvarez, G.E. Batley, T.F. Fernandes, R.D.

Handy, D.Y. Lyon, S. Mahendra, M.J. McLaughlin, J.R. Lead,

Environ. Toxicol. Chem. 27 (2008) 1825.

[24] K. Tiede, M. Hassellov, E. Breitbarth, Q. Chaudhry, A.B.A. Boxall,

J. Chromatogr., A 1216 (2009) 503.

[25] J.R. Lead, K.J. Wilkinson, Environ. Chem. 3 (2006) 159.

[26] M. Hassellov, J. Readman, J. Ranville, K. Tiede, Ecotoxicology 17

(2008) 344.

[27] D. Heymann, Fullerene Sci. Technol. 4 (1996) 509.

[28] W.A. Scrivens, J.M. Tour, K.E. Creek, L. Pirisi, J. Am. Chem. Soc.

116 (1994) 4517.

[29] G.V. Andrievsky, M.V. Kosevich, O.M. Vovk, V.S. Shelkovsky,

L.A. Vashchenko, J. Chem. Soc., Chem. Commun. (1995)

1281.

[30] Y. Prilutski, S. Durov, L.C. Bulavin, V. Pogorelov, Y. Astashkin,

V. Yashchuk, T. Ogul�Chansky, E. Buzaneva, G. Andrievsky, Mol.

Cryst. Liq. Cryst. Sci. Technol., Sect. A 324 (1998) 65.

[31] S. Deguchi, R.G. Alargova, K. Tsujii, Langmuir 17 (2001) 6013.

[32] R.G. Alargova, S. Deguchi, K. Tsujii, J. Am. Chem. Soc. 123

(2001) 10460.

[33] J. Brant, H. Lecoanet, M. Hotze, M. Wiesner, Environ. Sci.

Technol. 39 (2005) 6343.

[34] J.A. Brant, J. Labille, J.-Y. Bottero, M.R. Wiesner, Langmuir 22

(2006) 3878.

[35] M.R. Wiesner, E.M. Hotze, J.A. Brant, B. Espinasse, Water Sci.

Technol. 57 (2008) 305.

[36] P. Spohn, C. Hirsch, F. Hasler, A. Bruinink, H.F. Krug, P. Wick,

Environ. Poll. 157 (2009) 1134.

[37] J.D. Fortner, D.Y. Lyon, C.M. Sayes, A.M. Boyd, J.C. Falkner, E.M.

Hotze, L.B. Alemany, Y.J. Tao, W. Guo, K.D. Ausman, V.L.

Colvin, J.B. Hughes, Environ. Sci. Technol. 39 (2005) 4307.

[38] Y. Gao, Z. Tang, E. Watkins, J. Majewski, H.-L. Wang, Langmuir

21 (2005) 1416.

[39] J.A. Brant, J. Labille, C.O. Robichaud, M. Wiesner, J. Colloid

Interface Sci. 314 (2007) 281.

[40] M. Valcarcel, B.M. Simonet, S. Cardenas, B. Suarez, Anal.

Bioanal. Chem. 382 (2005) 1783.

[41] C. Journet, W.K. Maser, P. Bernier, A. Loiseau, M.L. de la

Chapelle, S. Lefrant, P. Deniard, R. Lee, J.E. Fischer, Nature

(London) 388 (1997) 756.

[42] X. Xu, R. Ray, Y. Gu, H.J. Ploehn, L. Gearheart, K. Raker, W.A.

Scrivens, J. Am. Chem. Soc. 126 (2004) 12736.

[43] R.C. Templeton, P.L. Ferguson, K.M. Washburn, W.A. Scrivens,

G.T. Chandler, Environ. Sci. Technol. 40 (2006) 7387.

[44] B. Suarez, B.M. Simonet, S. Cardenas, M. Valcarcel, J. Chroma-

togr., A 1128 (2006) 282.

[45] D. Heymann, L.P.F. Chibante, R.E. Smalley, J. Chromatogr., A

689 (1995) 157.

[46] K. Jinno, H. Matsui, H. Ohta, Y. Saito, K. Nakagawa, H.

Nagashima, K. Itoh, Chromatographia 41 (1995) 353.

[47] K. Jinno, Y. Sato, H. Nagashima, J. Itoh, J. Microcolumn Sep. 10

(1998) 79.

[48] K. Jinno, H. Nagashima, K. Itoh, M. Saito, M. Buonoshita,

Fresenius� J. Anal. Chem. 344 (1992) 435.

[49] Y. Shoji, K. Tashiro, T. Aida, J. Am. Chem. Soc. 126 (2004)

6570.

[50] X.-R. Xia, N.A. Monteiro-Riviere, J.E. Riviere, J. Chromatogr., A

1129 (2006) 216.


[51] Z. Chen, P. Westerhoff, P. Herckes, Environ. Toxicol. Chem. 27

(2008) 1852.

[52] C.W. Isaacson, C.Y. Usenko, R.L. Tanguay, J.A. Field, Anal.

Chem. 79 (2007) 9091.

[53] A.J. Kennedy, M.S. Hull, J.A. Steevens, K.M. Dontsova, M.A.

Chappell, J.C. Gunter, C.A. Weiss, Environ. Toxicol. Chem. 27

(2008) 1932.

[54] B. Suarez, Y. Moliner-Martınez, S. Cardenas, B.M. Simonet, M.

Valcarcel, Environ. Sci. Technol. 42 (2008) 6100.

[55] G.S. Duesberg, M. Burghard, J. Muster, G. Philipp, Chem.

Commun. (1998) 435.

[56] K.W. Whitaker, M.J. Sepaniak, Electrophoresis 15 (1994) 1341.

[57] J.M. Treubig, P.R. Brown, J. Chromatogr., A 873 (2000) 257.

[58] P. Zarzycki, H. Ohta, Y. Saito, K. Jinno, Chromatographia 64

(2006) 79.

[59] Y. Saito, H. Ohta, K. Jinno, Anal. Chem. 76 (2004) 266 A.

[60] A.D. Maynard, R.J. Aitken, T. Butz, V. Colvin, K. Donaldson, G.

Oberdorster, M.A. Philbert, J. Ryan, A. Seaton, V. Stone, S.S.

Tinkle, L. Tran, N.J. Walker, D.B. Warheit, Nature (London) 444

(2006) 267.

[61] J.M. Treubig, P.R. Brown, J. Chromatogr., A 960 (2002) 135.

[62] K. Jinno, T. Uemura, H. Ohta, H. Nagashima, K. Itoh, Anal.

Chem. 65 (1993) 2650.

[63] L. Zhou, H. Deng, Q. Deng, L. Zheng, Y. Cao, Rapid Commun.

Mass Spectrom. 19 (2005) 3523.

[64] A.V. Streletskiy, I.V. Goldt, I.V. Kuvychko, I.N. Ioffe, L.N.

Sidorov, T. Drewello, S.H. Strauss, O.V. Boltalina, Rapid Com-

mun. Mass Spectrom. 18 (2004) 360.

[65] J.F. Anacleto, R.K. Boyd, S. Pleasance, M.A. Quilliam, J.B.

Howard, A.L. Lafleur, Y. Makarovsky, Can. J. Chem. 70 (1992)

2558.

[66] J.F. Anacleto, R.K. Boyd, M.A. Quilliam, J. High Resolut.

Chromatogr. 16 (1993) 85.

[67] J.R. Deye, A.N. Shiveley, S.A. Oehrle, K.A. Walters, J. Chroma-

togr., A 1181 (2008) 159.

[68] S.R. Wilson, Y. Wu, J. Am. Chem. Soc. 115 (1993) 10334.

[69] G.J. Van Berkel, K.G. Asano, Anal. Chem. 66 (1994) 2096.

[70] V. Kozlovski, V. Brusov, I. Sulimenkov, A. Pikhtelev, A.

Dodonov, Rapid Commun. Mass Spectrom. 18 (2004) 780.

[71] S. Kawano, H. Murata, H. Mikami, K.H.W. Mukaibatake, Rapid

Commun. Mass Spectrom. 20 (2006) 2783.

[72] L. Song, A.D. Wellman, H. Yao, J. Adcock, Rapid Commun. Mass

Spectrom. 21 (2007) 1343.

[73] J.E. Canas, M. Long, S. Nations, R. Vadan, L. Dai, M. Luo, R.

Ambikapathi, E.H. Lee, D. Olszyk, Environ. Toxicol. Chem. 27

(2008) 1922.

[74] R.Y. Sato-Berru, E.V. Basiuk (Golovataya-Dzhymbeeva), J.M.

Saniger, J. Raman Spectrosc. 37 (2006) 1302.

[75] S. Niyogi, H. Hu, M.A. Hamon, P. Bhowmik, B. Zhao, S.M.

Rozenzhak, J. Chen, M.E. Itkis, M.S. Meier, R.C. Haddon, J. Am.

Chem. Soc. 123 (2001) 733.

[76] K.L. Chen, M. Elimelech, J. Colloid Interface Sci. 309 (2007)

126.

[77] R.D. Handy, T.B. Henry, T.M. Scown, B.D. Johnston, C.R. Tyler,

Ecotoxicology 17 (2008) 396.

[78] M.R. Wiesner, G.V. Lowry, P. Alvarez, D. Dionysiou, P. Biswas,

Environ. Sci. Technol. 40 (2006) 4336.

[79] S.J. Hug, B. Sulzberger, Langmuir 10 (1994) 3587.

[80] L. Ojamae, C. Aulin, H. Pedersen, P.O. Kall, J. Colloid Interface

Sci. 296 (2006) 71.

[81] A.P. Roberts, A.S. Mount, B. Seda, J. Souther, R. Qiao, S. Lin,

C.K. Pu, A.M. Rao, S.J. Klaine, Environ. Sci. Technol. 41 (2007)

3028.

[82] C.J. Smith, B.J. Shaw, R.D. Handy, Aquatic Toxicol. 82 (2007)

94.

[83] S. Zhu, E. Oberdorster, M.L. Haasch, Mar. Environ. Res. 62

(2006).

[84] A. Baun, N.B. Hartmann, K. Grieger, K.O. Kusk, Ecotoxicology

17 (2008) 387.

[85] G. Oberdorster, Amino Acids 33 (2007).

[86] S.B. Lovern, R. Klaper, Environ. Toxicol. Chem. 25 (2006) 1132.

[87] E. Oberdorster, S. Zhu, T.M. Blickley, P. McClellan-Green, M.L.

Haasch, Carbon 44 (2006) 1112.

[88] A. Baun, S.N. SA�rensen, R.F. Rasmussen, N.B. Hartmann, C.B.

Koch, Aquatic Toxicol. 86 (2008) 379.

[89] S.S. Karajanagi, D.Y. Kim, R.S. Kane, J.S. Dordick, Abstracts

Paper Am. Chem. Soc. 227 (2004).

[90] A. Liu, I. Honma, M. Ichihara, H. Zhou, Nanotechnology 17

(2006) 2845.

[91] F. Dubois, B. Mahler, B. Dubertret, E. Doris, C. Mioskowski, J.

Am. Chem. Soc. 129 (2007) 482.

[92] X. Zhang, H. Sun, Z. Zhang, Q. Niu, Y. Chen, J.C. Crittenden,

Chemosphere 67 (2007) 160.

[93] Y. Wang, J.F. Wong, X. Teng, X.Z. Lin, H. Yang, Nano Lett. 3

(2003) 1555.

[94] W.W. Yu, E. Chang, J.C. Falkner, J. Zhang, A.M. Al-Somali, C.M.

Sayes, J. Johns, R. Drezek, V.L. Colvin, J. Am. Chem. Soc. 129

(2007) 2871.

[95] G.V. Andrievsky, V.K. Klochkov, A.B. Bordyuh, G.I. Dovbeshko,

Chem. Phys. Lett. 364 (2002) 8.

[96] D.Y. Lyon, L.K. Adams, J.C. Falkner, P.J.J. Alvarez, Environ. Sci.

Technol. 40 (2006) 4360.

[97] M.J. O�Connell, P. Boul, L.M. Ericson, C. Huffman, Y. Wang, E.

Haroz, C. Kuper, J. Tour, K.D. Ausman, R.E. Smalley, Chem.

Phys. Lett. 342 (2001) 265.

[98] L. Jiang, L. Gao, J. Sun, J. Colloid Interface Sci. 260 (2003) 89.

[99] M.F. Islam, E. Rojas, D.M. Bergey, A.T. Johnson, A.G. Yodh,

Nano Lett. 3 (2003) 269.

[100] V.C. Moore, M.S. Strano, E.H. Haroz, R.H. Hauge, R.E. Smalley, J.

Schmidt, Y. Talmon, Nano Lett. 3 (2003) 1379.

[101] J. Dong, W.H. Mao, G.P. Zhang, F.B. Wu, Y. Cai, Plant Soil

Environ. 53 (2007) 193.

[102] H. Sun, X. Zhang, Q. Niu, Y. Chen, J.C. Crittenden, Water Air

Soil Pollut. 178 (2007) 245.

[103] Y. Song, M.L.A.V. Heien, V. Jimenez, R.M. Wightman, R.W.

Murray, Anal. Chem. 76 (2004) 4911.

[104] V.L. Colvin, Nat. Biotechnol. 21 (2003) 1166.

[105] R.J. DeLorenzo, F.H. Ruddle, Biochem. Genet. 4 (1970)

259.

[106] S. Foley, C. Crowley, M. Smaihi, C. Bonfils, B.F. Erlanger, P. Seta,

C. Larroque, Biochem. Biophys. Res. Commun. 294 (2002)

116.

[107] E. Oberdorster, Environ. Health Perspect. 112 (2004)

1058.

[108] H. Tjalve, J. Henriksson, NeuroToxicology 20 (1999) 181.

[109] T.B. Henry, F.M. Menn, J.T. Fleming, J. Wilgus, R.N. Compton,

G.S. Sayler, Environ. Health Perspect. 115 (2007) 1059.

[110] J. Cheng, E. Flahaut, H.C. Shuk, Environ. Toxicol. Chem. 26

(2007) 708.

[111] F. Mouchet, P. Landois, E. Sarremejean, G. Bernard, P. Puech, E.

Pinelli, E. Flahaut, L. Gauthier, Aquatic Toxicol. 87 (2008)

127.

[112] V.E. Kagan, Y.Y. Tyurina, V.A. Tyurin, N.V. Konduru, A.I.

Potapovich, A.N. Osipov, E.R. Kisin, D. Schwegler-Berry, R.

Mercer, V. Castranova, A.A. Shvedova, Toxicol. Lett. 165 (2006)

88.

[113] G. Jia, H. Wang, L. Yan, X. Wang, R. Pei, T. Yan, Y. Zhao, X.

Guo, Environ. Sci. Technol. 39 (2005) 1378.

[114] S.B. Lovern, J.R. Strickler, R. Klaper, Environ. Sci. Technol. 41

(2007) 4465.

[115] J. Fang, D.Y. Lyon, M.R. Wiesner, J. Dong, P.J.J. Alvarez,

Environ. Sci. Technol. 41 (2007) 2636.

[116] C.Y. Usenko, S.L. Harper, R.L. Tanguay, Carbon 45 (2007)

1891.



[117] D.Y. Lyon, P.J.J. Alvarez, Environ. Sci. Technol. 42 (2008)

8127.

[118] X. Zhu, L. Zhu, Y. Chen, S. Tian, J. Nanopart. Res. 11 (2009)

67.


[119] C. Blaise, F. Gagne, J.F. Ferard, P. Eullaffroy, Environ. Toxicol. 23

(2008) 591.

[120] E.J. Petersen, Q. Huang, W.J. Weber Jr., Environ. Health

Perspect. 116 (2008) 496.

analysis, behavior and ecotoxicity of carbon-based nanomaterials in the aquatic environment

Documents