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Environmental Pollution 159 (2011) 3003e3008

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Environmental Pollution

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Synergistic toxic effect of nano-Al2O3 and As(V) on Ceriodaphnia dubia

Demin Wang a, Ji Hu a,b, Brett E. Forthaus a, Jianmin Wang a,c,*

aDepartment of Civil, Architectural and Environmental Engineering, Missouri University of Science and Technology, Rolla, MO 65409, USAbKey Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, Chinac State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China

a r t i c l e i n f o

Article history:Received 15 December 2010Received in revised form23 March 2011Accepted 14 April 2011

Keywords:NanotoxicologyNano-Al2O3

ArsenicSynergistic toxic effectC. dubia

* Corresponding author.E-mail address: wangjia@mst.edu (J. Wang).

0269-7491/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.envpol.2011.04.019

a b s t r a c t

Engineered nanomaterials (ENMs) alone could negatively impact the environment and human health.However, their role in the presence of other toxic substances is not well understood. The toxicity of nano-Al2O3, inorganic As(V), and a combination of both was examined with C. dubia as the model organisms.Bare nano-Al2O3 particles exhibited partial mortality at concentrations of greater than 200 mg/L. WhenAs(V) was also present, a significant amount of As(V) was accumulated on the nano-Al2O3 surface, andthe calculated LC50 of As(V) in the presence of nano-Al2O3 was lower than that it was without the nano-Al2O3. The adsorption of As(V) on the nano-Al2O3 surface and the uptake of nano-Al2O3 by C. dubia wereboth verified. Therefore, the uptake of As(V)-loaded nano-Al2O3 was a major reason for the enhancedtoxic effect.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The rapid growth of nanotechnology could result in the release oflarge quantities of engineered nanomaterials (ENMs) into the waterenvironment, if appropriate regulations and controls are notimplemented (Oberdörster et al., 2005). The current ecotoxicitystudies of ENMs do not provide sufficient information on thepotential effect of ENMs in a realistic environment (Handy et al.,2008; Klaine et al., 2008; Wiesner et al., 2009). For example, somewater bodies have different toxic substances such as arsenic (Berget al., 2001; Navarro et al., 1993; Smith et al., 2000), Pb (Zietz et al.,2001), Cu (Cao and Hu, 2000), pesticide (Ritter, 1990), etc. Whilethe toxicity of bare ENMs was reported (Sadiq et al., 2009; Strigulet al., 2009; Zhu et al., 2009), the toxic effects from the interactionsof ENMs with the background toxic substances are seldom availablein the literature. These toxic substances can easily accumulate ontothe surface of ENMs (Jegadeesan et al., 2010) andmay be detrimentalto aquatic species. As reported, most ENMs seem safe in syntheticmedia especially at low concentrations (<100 mg/L) (Velzeboeret al., 2008; Zhu et al., 2009). However, these investigations onlyconsidered the interactions of bare ENMs with model organisms.These findings may not be valid in a natural water environmentwhere many other toxic substances are also present. To evaluate the

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environmental safety of ENMs, it is critical to include the possiblecombined effect of ENMs and background toxic substances.

Nano-Al2O3 is one of the most common ENMs with applicationssuch as microelectronics, catalyst support and abrasive (Stanleyet al., 2010). Its elemental form (nano-Al) is also an importantmaterial used for military purposes (Stanley et al., 2010), serving asthe basis for a superior fuel for space launch vehicles. Theseapplications could cause the release of nano-Al2O3 into the envi-ronment. The toxicity of nano-Al2O3 has been examined to someextent in different media and on different model organisms(Stanley et al., 2010; Strigul et al., 2009; Wang et al., 2009). Most ofthe researchers reported that nano-Al2O3 was not toxic, especiallyat low concentrations (Velzeboer et al., 2008; Wang et al., 2009).However, the toxic response of nano-Al2O3, when other toxins suchas As(V) are present, is not available in literature.

Arsenic is one of the major water quality concerns around theworld (Berg et al., 2001; Mohan and Pittman, 2007; Smith et al.,2000; Yadanaparthi et al., 2009). It has been regulated in theprimary drinking water standards issued by U.S. EPA witha maximum contamination level (MCL) of 0.01 mg/L. The toxicity ofarsenic on Ceriodaphnia dubia, a model organism commonly usedfor toxicity assessment, was well documented in literature (Hansenet al., 2002; Huddleston et al., 2009; Naddy et al., 1995; Spehar andFiandt, 1986). We have reported a significant accumulation ofarsenic on aluminum oxide (Guan et al., 2009; Su et al., 2008, 2010),one of the promising adsorbents for removing the arsenic fromsource water for potable purposes (Genç-Fuhrman et al., 2007; Kim

Table 1Characterization of nano-Al2O3.

Parameters Values

Purity* 99.9%Color* WhiteCrystal structure* GammaMorphology* FibrousAdvertised size* 5 nmPrimary size 5e10 nmHydrodynamic size in culture medium 600e1500 nmpHzpc 9.5e10.5Specific surface area* w600 m2/gPreparation method* Solegel

Source: items labeled with * were from the manufacturer, others were measured inthe lab.

D. Wang et al. / Environmental Pollution 159 (2011) 3003e30083004

et al., 2004; Lin and Wu, 2001). Sorption of arsenic onto ENMs hasalso been reported (Jegadeesan et al., 2010; Yadanaparthi et al.,2009). However, the interaction of arsenic with nano-Al2O3 hasnot been examined in toxicology studies.

The interaction of heavymetalswith ligands, such as chelators, iswell understood. The addition of chelators can significantly reducethe toxicity of heavy metals due to the decrease of bioavailability(lower freemetal ion concentrations) (Hockett andMount,1996;Maet al.,1999; Sauvant et al., 2000). Larger particles can also reduce thetoxicity of heavy metals (Ma et al., 2002). In both cases, neitherchelators nor large particles can be accumulated by model organ-isms. However, nano-Al2O3 could be accumulated by C. dubia.Therefore, the toxic response of C. dubiawill be different from thesesituations. For example, the accumulation of different ENMs in thegastrointestinal tract of daphnids was reported (Zhu et al., 2009).The objectives of this research were to determine the synergisticeffect of nano-Al2O3 and As(V) on C. dubia, and to provide insightson the toxicity mechanism of ENMs in a realistic environment.

2. Materials and methods

2.1. Chemicals and nanomaterials

Chemicals used to make synthetic culture media including CaSO4$2H2O (98%),KCl (99%), Na2SeO4 (99%), CuCl2$2H2O, NaHCO3 (100.2%, As< 5.0 mg/kg), MgSO4

(As< 0.001%), and HNO3 (67% in purity), were purchased from Fisher Scientific (Pitts-burgh, PA).NaH2AsO4$7H2Owaspurchased fromAlfaAesar (WardHill,MA).Nano-Al2O3

(gamma, 99.9%) was purchased from Skyspring Nanomaterials Inc. (Houston, TX, USA).Millipore water was produced using a Synergy� ultrapure water system (Billerica, MA).

2.2. Characterization and preparation of the nano-Al2O3 suspensions

The commercial nano-Al2O3 was used as received. However, several character-istic parameters were further determined. The primary size of the nano-Al2O3 ina solid phase was examined using scanning electromicroscopy (SEM) by FEI HeliosNano Lab 600 system (Hillsboro, Oregon, USA). Surface concentrations of differentelements were also analyzed using the same system with energy dispersive spec-troscopy (EDS) technology. The hydrodynamic size in the aqueous phase and pHzpc

were examined using a Zetasizer nano ZS-90 system with dynamic light scatteringtechnology (Malvern Instruments Ltd, Worcestershire, UK). Suspensions of nano-Al2O3 at different concentrations were prepared using a synthetic culture medium.More information can be found in our previous publication (Wang et al., 2011).

Settling property is one of the important characteristics of nanoparticles inaqueous solutions. It was evaluated by measuring the concentration of thesuspension at different time within a period of 48 h. The nano-Al2O3 suspensions, atinitial concentrations of 50 mg/L and 100 mg/L, were prepared by adding nano-Al2O3 into 100 mL culture medium and sonicated before sampling. The samplingposition was at 1 cm below the suspension surface. One milliliter of the suspensionwas drawn at the time interval of 5 min, 15 min, 30 min, 1 h, 2 h, 6 h, 12 h, 24 h and48 h from the beginning, respectively, and the sample was acidified immediatelywith 35% trace metal grade nitric acid. After all the samples were acidified andmixed for at least 24 h, the aluminum ion concentration was analyzed with a flameatomic absorption (FAA) spectroscopy (PerkinElmer 3100) to estimate the nano-Al2O3 concentration in the sample. The method diction limit was 0.1 mg/L as Al. Astandard solution from PerkinElmer was diluted and used for instrument calibra-tion. The experiment was repeated twice and the values were averaged.

2.3. The uptake of nano-Al2O3 by C. dubia

It is speculated that nanoparticles can be accumulated either in the gastroin-testinal tract or on the body surface. The accumulation in the gastrointestinal tractwas visualized using an Olympus CKX41 inverted tissue culture microscope systemfrom Hitschfel Instruments, Inc. (St. Louis, MO, USA). The accumulation on the bodysurface was observed using the FEI Helios Nano Lab 600 system (Hillsboro, Oregon,USA). To quantify the uptake amount, 200 adult C. dubia were collected each timeand exposed to nano-Al2O3 suspensions at concentrations of 10 mg/L, 50 mg/L,100 mg/L, and 200 mg/L, respectively. After 6 h and 24 h of exposure, the mixturecontaining adult C. dubiawas filtered through a membrane filter with a mesh size of0.1 mm to retain C. dubia, which have the size ranging from 0.4 mm to 1.4 mm. Thecollected C. dubia were then acidified with 35% trace metal grade nitric acid. Thevolume of the acid solution was diluted to 10 mL with DI water and mixed for morethan 24 h. The soluble aluminum ion concentration was analyzed using graphitefurnace atomic absorption (GFAA, PerkinElmer AAnalyst� 600). The methoddetection limit was 10 mg/L. The uptake amount of nano-Al2O3 by each adult C. dubiawas estimated based on the measured aluminum concentration.

2.4. Toxicity test

Starter C. dubiawas purchased fromMBL Aquaculture (Sarasota, FL, USA) and thefood (YTC and algae) was purchased from ABS Inc. (Fort Collins, Co, USA). Massculture, individual culture, and toxicity test were performed in a SVC-6AX laminarflow hood (Streamline� Laboratory Products, Fort Myers, FL, USA) to prevent particlecontamination from any other sources. The culture medium was prepared by dis-solving appropriate amounts of NaHCO3, CaSO4$2H2O, MgSO4, KCl, and Na2SeO4 intoMillipore water according to the EPA standard method (USEPA, 2002). It had a pH of7.8 and hardness of 85 mg/L as CaCO3. Healthy neonates, with an age of less than24 h after 7-day individual culturing (three-brood), were used as model organisms.The EPA standard method (USEPA, 2002) was followed to examine the toxic effect.The first set of toxicity test was designed to assess the toxicity of bare nano-Al2O3

alone. Nano-Al2O3 suspensions were prepared by adding appropriate amounts ofnano-Al2O3 particles into a 100 mL culture medium in plastic bottles. The concen-tration range was from 0 (served as a control) to 1000 mg/L. The solutions weremixed with food before performing the toxicity test. Five replicates at eachconcentrationwere performedwith an exposure time of 24 h or 48 h. The second setof the toxicity test was designed to examine the toxicity of nano-Al2O3 in thepresence of the same concentration of inorganic arsenic species. The culturemedium was added with the same amount of As(V) before adding differentconcentrations of nano-Al2O3. Healthy neonates of the model organisms were thenexposed to these mixed culturing media to examine the toxic effect. The last set ofthe toxicity test was designed to examine the toxic effect of inorganic As(V) in thepresence of the same concentration of nano-Al2O3 particles. The same amount ofnano-Al2O3 was added before adding different concentrations of inorganic arsenic.After mixing, healthy neonates were exposed to these synthetic media to examinethe toxic effect.

2.5. Arsenic analysis

After the toxicity test, residual solutions were collected and transferred to 50 mLcentrifugal tubes. The weight of each sample was 30.00� 0.05 g (including the tubeitself). The actual volume of the aliquots was approximately 12 mL each. The aliquotswere centrifuged at 15,000 rpm (12,600 g) for 20 min at 20 �C. After centrifuging,supernatants were collected and acidified using a 0.2% HNO3 solution. Depending onthe initial concentration of the solution, the supernatants may be diluted. Theconcentration of total arsenic was determined using GFAA. A standard solution fromPerkinElmer, with appropriate dilution, was used for instrument calibration. Themethod diction limit was 2 mg/L.

2.6. Data analysis

There are two types of quality control measures for the toxicity test. Thenegative control was considered satisfied if the survival rate was higher than 90% inthe reactor without toxins. The positive control was considered satisfied if themortality was comparable with the result of the same toxin alone. The toxicity datawas analyzed using a TSK program suggested by EPA (USEPA) and TOXCALC Software(v 5.0.32) from Tidepool Scientific Software Company (McKinley Ville, CA, USA). Theconfidence level of the LC50 estimations was 90%.

3. Results and discussion

3.1. Characterization of the nano-Al2O3 suspension

Table 1 summarizes the characteristics of the commercial nano-Al2O3 used in the present research. The primary size in the solidphase, examined with SEM, ranged from 5 nm to 10 nm, which was

Time (hour)

0 10 20 30 40

Co

ncen

tratio

n (m

g/L

)

0

20

40

60

80

100

50 mg/L100 mg/L

Fig. 1. Concentration of suspended nano-Al2O3 at different times. Experiment condi-tions: suspension¼ culture medium and Al2O3, volume¼ 100 mL, pH¼ 7.8, hard-ness¼ 85 mg/L as CaCO3, T¼ 20 �C.

D. Wang et al. / Environmental Pollution 159 (2011) 3003e3008 3005

slightly larger than the advertised size (5 nm). The morphology wasthe same as that advertised as fibrous. However, both the size andshape of the nano-Al2O3 changed in aqueous phase. A hydrodynamicsize of 600e1500 nmwas determined, which was much larger thanthe primary size. The increased size was from the aggregation of

Fig. 2. Accumulation of nano-Al2O3 by C. dubia. A (1e6): Accumulation in the gastrointestinaof Olympus CKX41 with magnification of 10�. B: Accumulation on the carapace. Examined

primary nanoparticles. Furthermore, the morphology also changedfrom fibrous to spherical. The pHzpc of nano-Al2O3 was also deter-mined to be in a range of 9.5e10.5. The measured zeta potential inthe culture mediumwas 3.0 mV.

Size change from 5e10 nm to 600e1500 nm suggests the poorstability of the nano-Al2O3 suspension. Fig. 1 shows the results ofthe settling experiment. The nano-Al2O3 concentration in suspen-sion decreased with time until it reached equilibrium. After 48 h,the residual concentration in the suspension was about one fifth ofthe initial concentration, which was still in the mg/L range. Theequilibrium concentration was also dependent on the initialconcentration of nano-Al2O3.

Therefore, the characteristics of nano-Al2O3 in the aqueousphase were significantly different from that in the solid phase. Thehydrodynamic size of the aggregates was close to that of the algae,food for C. dubia.

3.2. Uptake of nano-Al2O3 by C. dubia

Size distribution from nanometer to micrometer could bringtwo ways of accumulation of nano-Al2O3. The smaller particles innanometer can adhere to the negatively-charged body surface dueto the positive charge and low weight of the particle. The largeaggregates of nano-Al2O3 can be taken by C. dubia along with thefood, e.g. as “fake food”, and accumulated in the gastrointestinaltract. It is believed that C. dubia feed by filtering water with theirthoracic appendages and eat the food that drift by their carapace

l tract after 0.1 h, 0.25 h, 0.5 h, 1.0 h, 6.0 h and 24.0 h. Examined using light microscopeusing electronic microscope of FEI Helios Nano Lab 600 system.

Al2O

3 Concentration (mg/L)

0 200 400 600 800 1000

Mo

rta

lity

(%

)

0

20

40

60

80

100

24h48h

Fig. 3. Toxicity of nano-Al2O3 on C. dubia. Experiment conditions: suspension¼ culturemedium and nano-Al2O3, volume¼ 15 mL, pH¼ 7.8, hardness¼ 85 mg/L as CaCO3,T¼ 25�1 �C.

80

100

24h48h

D. Wang et al. / Environmental Pollution 159 (2011) 3003e30083006

opening (Bitton et al., 1995; Geller and Müller, 1981; Lee et al.,1997). It has also been reported that only appropriate sized foodcan be taken efficiently by daphnia species (Geller and Müller,1981). The size range of food was from hundreds of nanometersto micrometers and the hydrodynamic size of nano-Al2O3 fell inthat range. The accumulations of nano-Al2O3 on the body surfaceand in the gastrointestinal tract were examined using microscopy.Fig. 2 shows pictures of the accumulations. Pictures 2A.1 to 2A.6were taken after exposure to 100 mg/L of nano-Al2O3 at 0.1 h,0.25 h, 0.5 h, 1.0 h, 6.0 h, and 24.0 h, respectively. The color of thegastrointestinal tract changed from gray to solid dark, suggestingan accumulation of nano-Al2O3. Fig. 2B shows the accumulation ofnano-Al2O3 on the body surface. A nano-Al2O3 layer was observed.The sizes of these particles were in the nanometer range, whichwas smaller than the size range taken efficiently by C dubia as “fakefood”.

The accumulation of nanoparticles bymodel organismswas alsoquantified. Two hundred of the adult C. dubia were chosen asa group to be exposed with nano-Al2O3 suspensions. The uptakeamount of each adult organism was estimated indirectly bymeasuring the amount of Al carried by the C. dubia. The result wasshown in Table 2. It should be noted that the mesh size (0.1 mm) ofthe membrane filter used for filtration to collect the adult organ-isms was much larger than the hydrodynamic size of nano-Al2O3(0.6e1.5 mm) but smaller than the size of the C. dubia (0.4e1.4 mm).Therefore, only C. dubia was retained. The maximum uptakeamount was estimated as 15� 5 ng/adult. That is 20% of the dryweight (0.1 mg) of C. dubia. It is worth noting that this is the totalamount including both inside of the gastrointestinal tract and onthe body surface.

3.3. Toxicity of nano-Al2O3

The toxicity of nano-Al2O3 to aquatic species was reported(Stanley et al., 2010; Zhu et al., 2009). However, due to the differentsources and characteristics of the nanoparticles, these results maynot be comparable. The toxicity of the abovementioned nano-Al2O3was examined in this study to obtain consistent data for compar-ison. Fig. 3 shows the mortality at different concentrations. Themortality only occurred at high concentrations (>200 mg/L) after48 h. The first hypothesis was that the mortality was caused by thedissolution of nano-Al2O3, which releases free Al3þ ions. Thetoxicity of aluminum ions on some species had been reported inliterature (Herrmann, 2001; Illé�s et al., 2006; Lione, 1985). Asshown in Fig. 4, free Al3þ ions were toxic to C. dubia. The estimatedLC50 of free Al3þ ions at 24 h and 48 h were 12.04� 0.38 mg/L and11.32� 0.63 mg/L, respectively. Nano-Al2O3 could release free Al3þ

ions and these ions could negatively impact the model organisms.

Table 2Estimate of the uptake amount of nano-Al2O3 by C. dubia.

Measurednano-Al2O3

concentrationa

(mg/L)

Calculateduptakemass by 200adults (mg)

Estimated uptakemass per adult(ng/adult)

Background ND N/A 010 mg/L 6 h 226 2.26 1110 mg/L 24 h 85 0.85 4.250 mg/L 6 h 325 3.25 1650 mg/L 24 h 300 3.0 15100 mg/L 6 h 143 1.43 7.2100 mg/L 24 h 325 3.25 16200 mg/L 6 h 471 4.71 24200 mg/L 24 h 177 1.77 8.9

a Concentration in 10 mL diluted nitric solution.

The residual solution, after the toxicity test, was examined todetermine total soluble aluminum concentration. The maximumconcentration was less than 1.0 mg/L. Therefore, the mortality wasnot from the released free Al3þ ions. In addition, it can be seen inFig. 3 that the mortality did not increase with the concentration,which is not common for soluble toxins. Furthermore, we observedusing a microscope that many living C. dubia were trapped innanoparticle flocs, as well as most dead ones. As a result, wespeculated that the death of C. dubia could have been caused byover-weight rather than by the toxicity of the free Al3þ ions. Theover-weighted model organisms could not find enough food due tothe limited mobility. At low concentrations (<200 mg/L), nano-Al2O3 can be considered non-toxic to C. dubia.

3.4. Toxicity of nano-Al2O3 in the presence of As(V)

While the nano-Al2O3 particles alone were not toxic at lowconcentrations, uptake of these nanoparticles when other toxinsare present could be detrimental. Alumina are promising sorbentsfor As(V) removal for drinking water treatment due to their strong

Al3+

Concentration (mg/L)

0 5 10 15 20 25 30

Mo

rtality (%

)

0

20

40

60

Fig. 4. Toxicity of free Al3þ ions on C. dubia. Experiment conditions: suspen-sion¼ culture medium and nano-Al2(SO4)3, volume¼ 15 mL, pH¼ 7.6e7.8, hard-ness¼ 85 mg/L as CaCO3, T¼ 25�1 �C.

D. Wang et al. / Environmental Pollution 159 (2011) 3003e3008 3007

affinity to arsenic species (Chang et al., 2009; Kim et al., 2004;Yadanaparthi et al., 2009). The accumulated arsenic species couldimpose harm to the aquatic organisms if alumina particles aredigested or adsorbed. The toxicity of nano-Al2O3 in the presence ofconstant concentrations of As(V) were examined. Fig. 5a shows thenano-Al2O3 toxicity result after 24 h of exposure at three differentinitial concentrations of As(V). The toxicity of As(V) without nano-Al2O3 was first examined and the estimated LC50 was found tobe 3.6 mg/L. The concentration of 1.5 mg/L was below the no-observed-adverse-effect-level (NOAEL) in terms of mortality.From Fig. 5a, it can be seen that the toxicity of nano-Al2O3 in thepresence of As(V) were significantly different from that of nano-particles alone. The interaction of As(V) with nano-Al2O3 played animportant role in the toxic effect. For example, in the experimentwith the initial As(V) concentration of 3.6 mg/L, the addition ofnano-Al2O3 initially increased the overall toxicity. However, whenthe added amount was greater than 50 mg/L, the overall toxicitydecreased. This could have been caused by the adsorption of As(V)onto the nano-Al2O3, which significantly reduced the soluble Asconcentrationwhen the nano-Al2O3 additionwas high. Fig. 5b plotsthe mortality as a function of soluble As(V) concentrations for thesame experiment. In this figure, the change in soluble As(V)concentration was caused by the different amount of nano-Al2O3

Nano-Al2O

3 Concentration (mg/L)

0 50 100 150 200

Mo

rtality (%

)

0

20

40

60

80

100

4.5mg/L As + Al2O3

3.6 mg/L As+ Al2O3

1.5 mg/L As + Al2O3

Measured Soluble As Concentration (mg/L)

0 1 2 3 4 5 6

Mo

rtality (%

)

0

20

40

60

80

100

As only4.5 mg/L As +Al2O3

3.6 mg/L As+Al2O3

1.5 mg/L As + Al2O3

a

b

Fig. 5. (a) Toxicity of nano-Al2O3 in the presence of As(V); (b) relationship betweenmortality and soluble As(V) in presence of nano-Al2O3. Experiment conditions: sus-pension¼ culture medium and nano-Al2O3, volume¼ 15 mL, pH¼ 7.8, hardness¼85 mg/L as CaCO3, T¼ 25�1 �C.

added to the reactor. While nano-Al2O3 was non-toxic at concen-trations of less than 200 mg/L, both soluble and adsorbed As(V)contributed to the overall toxic effect. At an initial concentration of1.5 mg/L, both the soluble and sorbed As(V) were very low and nomortality was observed. At an initial concentration of 3.6 mg/L,however, mortality was found even though no nanoparticles wereadded. This is consistent with our published results (Wang et al.,2011) and close to other reported values in literature (Hansenet al., 2002; Naddy et al., 1995). The addition of nano-Al2O3increased the total uptake of As(V) due to the uptake of As(V)-loaded nanoparticles, and the mortality increased accordingly.Also, the addition of nanoparticles decreased the free As(V)concentrations as well. The surface density of As(V) on the nano-particle and the soluble As(V) decreased with the further increasein nanoparticle concentrations. There was a maximum combineduptake of As(V) from both routes where the maximum mortalitywas observed. The decrease of the mortality with the increase ofsoluble As(V) shown in Fig. 5b implies that, under these conditions,very low amounts of nano-Al2O3 were used, and the uptake ofnano-Al2O3 was low. As a result, the toxicity contributed from thesorbed As(V) through nano-Al2O3 uptake was low. At an initialconcentration of 4.5 mg/L, mortality was at a maximum. The effectof nano-Al2O3 was only observed when the added concentrationreached a certain value. Before this value was attained, themortality remained at 100% due to the high As(V) concentration(both soluble and sorbed). When the nanoparticle concentrationwas high enough, both the soluble As(V) concentration and sorbedAs(V) per unit mass of the particle became lower, resulting ina lower mortality.

3.5. Toxicity of As(V) in the presence of nano-Al2O3

Another approach was taken to examine the combined toxiceffect of nano-Al2O3 and As(V) using to fixed concentrations ofnanoparticles. Fig. 6 shows the toxicity results after 48 h of expo-sure to As(V) in the presence of different concentrations of nano-Al2O3. The nanoparticles significantly impacted the toxicity ofAs(V). At low concentrations such as 1.0 mg/L, there was almost noeffect and the results were close to that with As(V) alone. However,when concentrations of nanoparticles increased, the responsecurve shifted to the left. At concentrations of 20 mg/L, 50 mg/L, and100 mg/L, the toxicity of As(V) was significantly enhanced, and theLC50 was reduced from 3.6 mg/L to about 1.0 mg/L. Interestingly, at

Total As(V) Concentration (mg/L)

0 1 2 3 4 5 6

Mo

rtality

0

20

40

60

80

100

As(V) only1 mg/L Al 2O320 mg/L Al 2O350 mg/L Al 2O3100 mg/L Al 2O3200 mg/L Al 2O3

Fig. 6. Toxicity of As(V) in the presence of nano-Al2O3. Experiment conditions: sus-pension¼ culture medium and nano-Al2O3, volume¼ 15 mL, pH¼ 7.8, hardness¼85 mg/L as CaCO3, T¼ 25�1 �C.

D. Wang et al. / Environmental Pollution 159 (2011) 3003e30083008

a concentration of 200 mg/L, the LC50 was higher with a valueof 1.8 mg/L. This observation is also reflected in Fig. 5, whichdemonstrates that a range of nano-Al2O3 concentration can causethe maximum mortality. A high nano-Al2O3 concentration caneffectively reduce the soluble As(V) concentration and the surfacedensity of As(V), thereby reduce the toxicity.

4. Conclusions

Nano-Al2O3 alone does not impose significant health issues,especially at low concentrations. However, it significantly enhancedthe toxicity of As(V), with the enhancement being dependent onthe nano-Al2O3 concentrations. A significant accumulation of As(V)on the nano-Al2O3 surface and the uptake of As(V)-loaded nano-Al2O3 played a very important role in the toxicity response ofC. dubia.

Acknowledgements

This research was partially supported through grants fromLeonard Wood Institute, Fort Leonard Wood, MO, USA (Contract# 281173) and State Key Laboratory of Pollution Control andResource Reuse, Tongji University, China. Ji Hu was supported bythe China Scholarship Council through a state-sponsored scholar-ship program.

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