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Toxicity of nanoparticulate and bulk ZnO, Al2O3 and TiO2 to the nematode Caenorhabditis elegans
Environmental Pollution 157 (2009) 1171–1177
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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Toxicity of nanoparticulate and bulk ZnO, Al2O3 and TiO2 to the nematode Caenorhabditis elegans Huanhua Wang, Robert L. Wick, Baoshan Xing* Department of Plant, Soil and Insect Sciences, University of Massachusetts, Stockbridge Hall, Amherst, MA 01003, USA
ZnO, Al2O3 and TiO2 nanoparticles are more toxic than their bulk counterparts to the nematode, Caenorhabditis elegans.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 August 2008 Received in revised form 30 October 2008 Accepted 3 November 2008
Limited information is available on the environmental behavior and associated potential risk of manufactured oxide nanoparticles (NPs). In this research, toxicity of nanoparticulate and bulk ZnO, Al2O3 and TiO2 were examined to the nematode Caenorhabditis elegans with Escherichia coli as a food source. Parallel experiments with dissolved metal ions from NPs were also conducted. The 24-h median lethal concentration (LC50) and sublethal endpoints were assessed. Both NPs and their bulk counterparts were toxic, inhibiting growth and especially the reproductive capability of the nematode. The 24-h LC50 for ZnO NPs (2.3 mg L1) and bulk ZnO was not significantly different, but significantly different between Al2O3 NPs (82 mg L1) and bulk Al2O3 (153 mg L1), and between TiO2 NPs (80 mg L1) and bulk TiO2 (136 mg L1). Oxide solubility influenced the toxicity of ZnO and Al2O3 NPs, but nanoparticle-dependent toxicity was indeed observed for the investigated NPs. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Toxicity Metal oxide Nanoparticles Caenorhabditis elegans
1. Introduction Nanoparticles (NPs) have unique physicochemical properties such as tiny size, large surface area, surface reactivity, charge, shape, and media interactions. As a result, the properties of NPs differ substantially from their respective bulk materials of the same composition. However, certain novel properties of NPs could lead to adverse biological effects, with the potential to create toxicity (Oberdo¨rster et al., 2005; Nel et al., 2006). The forecasted huge increase in the manufacture and use of NPs makes it likely that increasing human and environmental exposure to NPs will occur (Nowack and Bucheli, 2007); thus, the studies on safety and (eco)toxicity of NPs are of extreme importance (Kahru et al., 2008). Metal oxide nanoparticles are receiving increasing attention for a large variety of applications. Titanium dioxide and zinc oxide NPs are included in toothpaste, beauty products, sunscreens and textiles. Aluminum oxide having good dielectric and abrasive properties is widely used as an abrasive agent or insulator. The concerns of metal oxide NPs are that because of their chemistry, size, and being not biodegradable, they will rapidly distribute throughout the environment with unknown consequences. Until now little is known about the potential toxicity of metal oxide NPs in soil and water. Given the well-known toxicity of the ionic forms of many metals, the solubility of metal oxide NPs may require particular attention, and it is important to distinguish effects of NPs * Corresponding author. Tel.: þ1 413 545 5212; fax: þ1 413 545 3958. E-mail address: [email protected] (B. Xing). 0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.11.004
from dissolved metals when assessing the toxicity of metal oxide NPs. Nematodes are the most abundant metazoa in soil and their function is critical in the soil food web. Nematodes interact closely with other soil organisms and their activity affects primary production, decomposition, energy flow, and nutrient cycling. In natural ecosystems, nematode abundance and community structure analyses were proved to be sensitive indicators of stress caused by soil pollutants and ecological disturbance (Sohlenius, 1980; Neher, 2001). A free-living nematode, Caenorhabditis elegans (C. elegans), is abundant in soil ecosystems and plays a key role in nutrient cycling. C. elegans is a simple multicellular eukaryote whose developmental process and behavior can be readily monitored under a microscope because of its translucent body. After hatching, C. elegans reaches the adult stage by passing through four larval stages (L1, L2, L3 and L4). It is easily grown on Petri plates or in liquid culture with the well-characterized bacterium Escherichia coli (E. coli) as a food source. Its approximate life span is two weeks, with reproduction by either self- or cross-fertilization. C. elegans, being the first multicellular organism to have its genome completely sequenced (The C. elegans Sequencing Consortium, 1998), has served as a superior model in neurobiology, developmental biology and genetics. Aquatic invertebrates have been highlighted as sensitive and relevant test organisms in nanoecotoxicology studies, but most of the available toxicity data are mainly on Daphnia magna (Baun et al., 2008). Several toxicity tests using C. elegans have been developed for ecological risk assessment in soil (Donkin and Dusenbery, 1993) and water (Van Kessel et al.,
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1989). Aquatic test data are applicable to the study of soil contaminants and availability to indigenous invertebrates as the main route of exposure to soil toxicants is probably through the interstitial liquid. To our knowledge, however, there are no reports using C. elegans as a test organism in nanoecotoxicology research. Previous work have investigated the toxicity of ZnO and Al2O3 NPs to plants (Lin and Xing, 2007, 2008), ZnO, CuO and TiO2 NPs to bacteria (Reddy et al., 2007; Heinlaan et al., 2008; Huang et al., 2008), ZnO NPs to freshwater microalga (Franklin et al., 2007), ZnO, TiO2, SiO2 and C60 NPs to D. magna (Adams et al., 2006; Lovern et al., 2007), and CuO NPs to Zebrafish (Griffitt et al., 2007). As indicated above, the effect of metal oxide NPs on nematodes has never been investigated. Toxicity tests on the nematode C. elegans primarily examined free metal ion (Williams and Dusenbery, 1990; Wah Chu and Chow, 2002). Therefore, this study was undertaken to determine whether metal oxide NPs are toxic to C. elegans, and if so, to determine if the toxicity is solely due to dissolution of metal oxide NPs by comparing to their reference toxicants: soluble metal ions and bulk metal oxides of non-nanoparticulate form. The objective of this study is to address the current knowledge gap of C. elegans responses to metal oxide NP exposure. Since the nematodes feed on bacteria and are regarded as particle-ingesting organisms, the current study presents new perspectives in NP-related risk assessment and food web accumulation modeling. 2. Materials and methods 2.1. Preparation of particle suspensions The ZnO, Al2O3 and TiO2 (anatase-type) NPs were purchased from Hongchen Material Sci & Tech. Co., China. Bulk TiO2 (anatase-type), ZnO and Al2O3 were purchased from ACROS, Fisher Scientific and Baker Chemical Co., respectively. Reagent-grade ZnCl2 and AlCl3 were purchased from Fisher Scientific and Aldrich Chemical Co., respectively. The surface areas of the NPs provided by the producers were 50, 180 and 35 m2 g1 for ZnO, Al2O3 and TiO2, respectively. Their surface areas were measured again by N2 sorption at 77 K using a NOVA 1000e instrument (Quantachrome). The multi-point Brunauer–Emmett–Teller (BET) method was employed to calculate the surface areas and the data are listed in Table 1. The stock suspensions of nanoparticulate and bulk ZnO, Al2O3 and TiO2 (16.3, 407.8 and 239.6 mg L1, respectively), were dispersed in ultrapure water by probe sonication (Sonic Dismembrator, Model 100) at 100 W and 40 kHz for 30 min to aid mixing and for forming homogeneous suspensions. The NPs were then serially diluted in sterile ultrapure water and additionally sonicated for 30 min. Small magnetic bars were placed in the suspensions for stirring during dilution to avoid aggregation and deposition of particles. Zinc and aluminum ion (Zn2þ, Al3þ) solutions were prepared by dissolving ZnCl2 and AlCl3 in ultrapure water. Because the solubility of TiO2 is extremely low, the effect of Ti ion was not investigated. The pH for nanoparticulate and bulk ZnO and TiO2 was adjusted with HNO3 and NaOH to 7.0. To avoid precipitation of Al(OH)3, the pH of nanoparticulate and bulk Al2O3 and AlCl3 was adjusted to 5.6, which had little effect on C. elegans because they have a wide pH tolerance range from 3 to 12 (Khanna et al., 1997). 2.2. Characterization of metal oxide NPs The particle shape of nanoparticulate and bulk ZnO, Al2O3 and TiO2 was visualized using transmission electron microscopy (TEM, JEOL 100CX, USA) operated at
80 kV. Also, about 150 individuals for each type of bulk particles were selected to determine their sizes according to the magnification of TEM. The particle size distribution and zeta potential were analyzed with a Nano Zetasizer (Malvern Instrument Ltd., UK) using dynamic light scattering (DLS) technique. 2.3. Preparation of synchronized nematodes C. elegans strain Bristol N2 (wild type) used in this work was provided by the Caenorhabditis Genetics Center (University of Minnesota) and maintained on nematode growth medium (NGM) plates (95 mm 15 mm sterilized-disposable Petri dish, Canada) seeded with E. coli strain OP50 at 20 1 C (Brenner, 1974). The components of NGM were agar, peptone, cholesterol, KH2PO4 and K2HPO4 buffer, NaCl and MgSO4 (Stiernagle, 2006). E. coli was cultured overnight in sterilized LuriaBertani (LB) medium (5 g L1 of yeast extract, 10 g L1 of tryptone, and 10 g L1 NaCl). After 3 days many gravid hermaphrodites, as well as L1 and L2 stage juveniles, were found on the NGM. Our study started with synchronized worms in the L1 stage to avoid the influence of developmental stage on the toxicity test. To obtain worms synchronized to this life stage, the NGM plates were washed with 10.5 mL sterilized water into 15-mL centrifuge tubes and 1.5 mL of 0.5 N NaOH and 3 mL of 1% NaOCl were subsequently added. The tubes were immediately centrifuged at 3000 g for 2 min. The supertanant was pipetted out and the dead nematodes and eggs were pelleted at the bottom. Sterilized water was then added to the pellet and centrifuged again to remove the remaining NaOH and NaOCl mixture. This procedure killed the nematodes but the eggs both inside and outside of the body survived (Fig. 1). After 8 h, the eggs hatched and were termed as L1 larvae. 2.4. Toxicity test Four types of endpoints were assessed, i.e., lethality to the vermiform nematode, length of the worm, number of eggs inside the worm body and offspring per worm. The tested concentrations for each endpoint were 0.4, 0.8, 1.6, 4.1, 6.1 and 8.1 mg L1 for nanoparticulate and bulk ZnO, and 0.7, 1.4, 2.7, 6.8, 10.2 and 13.6 mg L1 for ZnCl2; 10.2, 30.6, 51.0, 102.0, 203.9 and 407.8 mg L1 for nanoparticulate and bulk Al2O3, 0.7, 1.3, 2.7, 6.7, 10.0, 13.3 mg L1 for AlCl3; and 24.0, 47.9, 95.9, 167.8, 239.6 mg L1 for nanoparticulate and bulk TiO2. All the tests were carried out in ultrapure water and no salts were added. At the beginning of each test, approximately 30 L1 juveniles were transferred by micropipette to the Petri plates containing different concentrations of toxicity test particle (i.e., nanoparticulate and bulk ZnO, Al2O3 and TiO2). 250 mL of E. coli in LB broth were centrifuged at 3000 g for 10 min, and the bacterial pellet was used as the concentrated bacteria to feed the nematodes. A loopful of the pelleted bacteria was transferred to the above test plates. We chose an aqueous medium because it was difficult to evenly distribute NPs in an agar medium and the nematodes were able to freely contact with NPs in the aqueous medium; also, C. elegans lives in the interstitial water of soil. The exact number of transferred L1 nematodes was counted with a dissecting microscope. The seeded plates were incubated at 20 1 C under dark condition and shaken at 100 rpm. After 24 h the surviving worms were counted with a dissecting microscope. Death was determined as a lack of response to gentle probing with a needle. For growth and reproduction tests, L1 nematodes were cultured for 5 days under the same conditions as described for the LC50 determination. At the end of the exposure period, about 10 living worms per time were pipetted into a drop of water on a glass slide, covered with a coverglass and heated to approximately 50 C. This treatment resulted in straight and easily measurable worms. The length of the nematodes was measured using a compound microscope equipped with a camera (Olympus DP70) interfaced directly to a computer (Olympus BX51, Japan). The eggs inside the worm body were counted using the same microscope. Reproductive capacity was evaluated by determining the mean number of offspring at all stages beyond the eggs over the entire brood period. 2.5. Metal analysis and comparison of the toxicity of supernatant and suspension
Table 1 Characteristics of particles used in this study. Particle
Purity (%)
Surface areaa (m2 g1)
Diameter (nm)
Hydrodynamic diameterb (nm)
Nano-ZnO Bulk ZnO NanoAl2O3 Bulk Al2O3 Nano-TiO2 Bulk TiO2
>99.5 >99.5 >99.99
54 3.3 188
20 532 60
478–980 (759) 595–976 (737) 470–1126 (763)
>98.5 >99 >99
11 325 7.3
429 50 285
565–974 (889) 338–917 (550) 158–687 (494)
a
Zetac (mv) 0.017 3.6 33.1 5.6 18.9 33.8
Data generated in our lab. b The data are expressed as range (median). c The pH was 7.0 for nanoparticulate and bulk ZnO and TiO2, and 5.6 for nanoparticulate and bulk Al2O3.
A total of 40 mL of each concentration of NP suspensions was centrifuged at 13,000 g for 20 min and filtered (0.2 mm polytetrafluoroethylene filter, Ireland). To determine dissolved metal (defined as metal present in the supernatant), 6 mL of the supernatant was acidified with 250 mL of 1% HNO3 (V:V) and analyzed by an inductively coupled plasma mass spectrometer (Elan 6100, PerkinElmer, USA). The remaining supernatant was also tested for the same four endpoints to elucidate if the toxicity was caused by the particles or the dissolved metal ions. In addition, the time- and concentration-dependent dissolution of nanoparticulate and bulk particles was also investigated using the above procedure. 2.6. Statistical analysis Four experiments with three replicates per concentration were performed for each type of test. The results were presented as mean standard deviation (SD). The software package SPSS 13.0 was used for statistical analyses (SPSS Inc., Chicago, IL). First, c2 test of normality for each replicate and Barlett’s test of homogeneity of variance between replicates of each treatment were made. Then, one-way ANOVA
H. Wang et al. / Environmental Pollution 157 (2009) 1171–1177
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Fig. 1. Eggs of the nematode before (A) and after treating with NaOCl and NaOH solution (B). After being treated the ovary was lysed and the eggs were released.
analyses were performed to assess any significant difference among treatments and between all treatment levels and the control for both endpoints, at a p-value <0.05 or <0.01 significance level. LC50 is defined as the concentration at which survival is reduced by 50% compared with the control. The LC50 values were calculated on logtransformed mortality data using the SPSS probit model.
3. Results and discussion 3.1. Characterization of NPs The characteristics of both nanoparticulate and bulk ZnO, Al2O3 and TiO2 are listed in Table 1. The measured diameter of ZnO, Al2O3 and TiO2 bulk particles were 532, 429 and 285 nm, respectively, much larger than their nano-sized counterparts. However, considerable particle aggregation was observed by TEM for each of the investigated particles (Fig. 2). Using DLS, wide distributions of particle size were observed, from 478 to 980, 470 to 1126 and 338 to 917 nm for ZnO, Al2O3 and TiO2 NPs, respectively, considerably larger than their nominal size 20, 60 and 50 nm, respectively (Table 1). It should be noted that particle sizes measured by the Nano-Sizer are hydrodynamic diameter based on the Stokes–Einstein equation, which are expected to be larger than the actual particle size. Also, the specific surface area of NPs was significantly higher than that of bulk particles (Table 1). Concerns about the bioavailability and toxicity of NPs arise from their assumed occurrence as small particles in the environment. When evaluating nanoparticle-related toxicity, particle size, degree of agglomeration, and specific surface area were factors we considered. Although NP aggregation was extensive in this study, there were still some individual nano-sized particles in solution. Size has important control over other physical and chemical properties such as zeta potential and metal binding (Madden et al., 2006). The zeta potential of NPs was more positive than bulk particles (Table 1). The surface of C. elegans is reported to have an evenly distributed net negative charge at neutral pH, which was diminished but not abrogated at low pH (Himmelhoch et al., 1977; Zuckerman et al., 1979; Himmelhoch and Zuckerman, 1983; Blaxter, 1993). The negative charge is most likely due to sulfated sugar moieties rather than charged amino acid residues in proteins, phosphate groups, or carboxyl groups on sugars (Himmelhoch and Zuckerman, 1983). Therefore, the nematode cuticle may attract more NPs than the bulk particles via electrostatic interactions.
2.5 mg L1 for AlCl3; 79.9 and 135.8 mg L1 for nanoparticulate and bulk TiO2, respectively. The differences in LC50 values among different zinc toxicants were not significant (p < 0.05); but they were significant between Al2O3 NPs and bulk Al2O3, and between TiO2 NPs and bulk TiO2 (p < 0.05). The LC50 value of AlCl3 was significantly lower than that of Al2O3 NPs and bulk Al2O3, probably due to the toxicity by Al ions. The 24-h LC50 values for Zn2þ and Al3þ to C. elegans in aqueous medium were reported as 202 and 79 mg L1 (Williams and Dusenbery, 1990), 257 and 49 mg L1 (Dhawan et al., 2000), and 111 and 18 mg L1 (Wah Chu and Chow, 2002), respectively. The estimated free-ion based LC50 value for Zn to C. elegans was 131 mg L1 (Tatara et al., 1997). Acute toxicity of zinc to various invertebrates and fish has been reported from 40 mg L1 to 58 mg L1 (USEPA, 1995). Our results showed that the LC50 values were much lower than the reported values. TiO2 NPs had no measurable effect at lower doses (10–50 mg L1), whereas there was a significant effect at higher levels (100–250 mg L1) to the in vitro rat liver derived cell line (Hussain et al., 2005). TiO2 NPs were not found to be toxic to MSTO cells until up to 30 mg L1 (Brunner et al., 2006). The toxicity effect was influenced by different assay parameters such as ionic concentration, pH and larval stage. Pure water with no background ions caused more osmotic stress on the nematode which may have reduced their tolerance to toxicants as observed in our study. Developmental stage, salts and food presence have also been found to influence toxicity assays (Donkin and Williams, 1995; Khanna et al., 1997). The presence of food is not necessary for shortterm tests, and thus it may be desirable to omit food to avoid potential complication caused by bacterial adsorption of toxicants. But for longer studies such as a 24-h test, it is necessary to add bacteria to avoid natural death caused by lack of food. We also compared the effect of ZnO NPs in the presence and the absence of K-medium (3.1 g of NaCl and 2.4 g of KCl in 1000 mL of ultrapure water, Williams and Dusenbery, 1990), and the results showed that the nematodes grew better and the toxicity was lower in the presence of K-medium. The reduced toxicity of several heavy metals (Cd, Pb, Cu, and Hg) in the presence of potassium and sodium salts was also reported by Donkin and Williams (1995). The calculated 24-h LC50 value for ZnO NPs in the presence of Kmedium was 4.9 mg L1, higher than that in pure water of 2.2 mg L1. However, salts were not used for other types of particles in this study to decrease the aggregation of NPs.
3.2. Lethality 3.3. Inhibitory effects on growth and reproduction The 24-h concentration–survival curves are shown in Fig. 3. The calculated LC50 values were 2.2, 2.3 and 2.9 mg L1 for nanoparticulate and bulk ZnO and ZnCl2, respectively; 81.6 and 152.9 mg L1 for nanoparticulate and bulk Al2O3, respectively, and
The inhibitory effects of each toxicant to the nematode are shown in Tables 2–4. Compared to the control, exposure to 1.6 mg L1 and 4.1 mg L1 ZnO NPs and bulk ZnO significantly
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Fig. 2. TEM images of nanoparticulate and bulk particles in ultrapure water. The characterization was done at 10 mg L1 for ZnO and TiO2, and 20 mg L1 for Al2O3. ZnO NP (36.3 k magnification, A); bulk ZnO (9.1 k magnification, B); Al2O3 NP (36.3k magnification, C); bulk Al2O3 (21.7 k magnification, D); TiO2 NP (105.8 k magnification, E); and bulk TiO2 (17.9 k magnification, F).
0.0
A
% survival
100
3.5
7.0
10.5
14.0
B
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0 0
2
4
6
8
C
100
0 0
100
200
300
400
0
50
100
150
200
250
Concentration (mg L-1) Fig. 3. Concentration–survival curves of L1 stage C. elegans for 24-h exposure to various toxicants with E. coli added as a food. ZnO NP (:), bulk ZnO (C) and ZnCl2 (-) (A); Al2O3 NP (:) and bulk Al2O3 (C) (using the bottom x-axis) and AlCl3 (-) (using the top x-axis) (B); and TiO2 NP (:) and bulk TiO2 (C) (C). Each test was repeated for four times. The data points and error bars represented the means and standard deviation, respectively, of the four replicates.
H. Wang et al. / Environmental Pollution 157 (2009) 1171–1177 Table 2 Body length, number of eggs inside the worm body, and number of offspring per individual of C. elegans after exposure to different concentrations of ZnO NPs, bulk ZnO and ZnCl2. E. coli was added as a food to C. elegans. Each test was repeated for four times and n ¼ 385. All values are expressed as mean standard deviation (SD) of four replicates.
Table 4 Body length, number of eggs inside the worm body, and number of offspring per individual of C. elegans after exposure to different concentrations of TiO2 NPs and bulk TiO2. E. coli was added as a food to C. elegans. Each test was repeated for four times and n ¼ 425. All values were expressed as mean standard deviation (SD) of four replicates.
Treatment
Body length (mm)
Eggs inside body
Offspring per worm
Control
1220 36
13.1 2.0
29.3 4.6
0.4 mg L1 nano-ZnO 0.8 mg L1 nano-ZnO 1.6 mg L1 nano-ZnO 4.1 mg L1 nano-ZnO 1.6 mg L1 nano-ZnO (supernatant) 4.1 mg L1 nano-ZnO (supernatant)
1137 76 1070 156 821 88* 720 19** 845 27*
19.3 7.6 8.5 3.6* 3.1 1.2** 0.0** 4.8 1.1**
0.4 mg L1 bulk ZnO 0.8 mg L1 bulk ZnO 1.6 mg L1 bulk ZnO 4.1 mg L1 bulk ZnO
1210 104 1028 143 892 42* 715 25**
9.1 3.0 7.5 0.6 3.6 0.9** 0.0**
28.2 3.5 19.0 1.4 10.2 1.8* 0.0**
24.0 mg L1 bulk TiO2 47.9 mg L1 bulk TiO2 95.9 mg L1 bulk TiO2 167.8 mg L1 bulk TiO2
1080 106 901 63* 875 35* 718 23**
8.1 2.5 5.2 1.6* 3.6 1.8** 0.0**
14.5 5.9 7.6 0.5* 3.7 1.2** 0.0**
1
0.7 mg L ZnCl2 1.4 mg L1 ZnCl2 2.7 mg L1 ZnCl2 6.8 mg L1 ZnCl2
760 16**
9.6 2.1 6.8 2.1 3.0 1.2** 0.0** 3.1 1.5** 0.0**
0.0**
*Significantly different from the control within each column at p < 0.05 level. **Significantly different from the control within each column at p < 0.01 level.
reduced the growth of C. elegans, the number of eggs inside the worm and offspring per worm (Table 2). However, the effect on growth and number of eggs inside the worm was not significantly different among ZnO NPs, bulk ZnO and ZnCl2 (p < 0.05). In contrast, it should be noted that the number of offspring per worm exposed to ZnO NPs was significantly less than that of the bulk ZnO at 0.8 and 1.6 mg L1 (p < 0.05). In addition, for the two endpoints of body length and number of eggs inside body, there were no significant differences between the supernatant of 1.6 mg L1 and 4.1 mg L1 ZnO NP and the corresponding unfiltered ZnO NP suspension (p < 0.05). However, the number of offspring per worm of supernatant of 1.6 mg L1 ZnO NP was significantly higher than that of unfiltered ZnO NP suspension (p < 0.05), suggesting that the Table 3 Body length, number of eggs inside the worm body, and number of offspring per individual of C. elegans after exposure to different concentrations of Al2O3 NPs, bulk Al2O3 and AlCl3. E. coli was added as a food to C. elegans. Each test was repeated for four times and n ¼ 479. All values were expressed as mean standard deviation (SD) of four replicates. Treatment
Body length (mm)
Eggs inside body
Offspring per worm
Control
1125 67
10.3 2.1
27.5 3.8
1
30.6 mg L nano-Al2O3 51.0 mg L1 nano-Al2O3 102.0 mg L1 nano-Al2O3 203.9 mg L1 nano-Al2O3 51.0 mg L1 nano-Al2O3 (supernatant) 203.9 mg L1 nano-Al2O3 (supernatant)
1025 85 953 25 881 31* 820 23* 1074 38
9.3 0.6 7.1 0.9 5.9 1.2* 0.0** 8.0 2.1
950 119
3.5 2.2**
30.6 mg L1 bulk Al2O3 51.0 mg L1 bulk Al2O3 102.0 mg L1 bulk Al2O3 203.9 mg L1 bulk Al2O3
1086 31 1225 96 1233 97 1016 125
9.2 1.5 9.5 1.1 7.3 2.3 5.7 1.5*
25.2 5.1 20.7 1.8 7.3 2.0* 3.2 1.5**
0.7 mg L1 AlCl3 1.3 mg L1 AlCl3 2.7 mg L1 AlCl3 6.7 mg L1 AlCl3
1006 25 858 92* 733 41** 612 19**
7.2 2.5 6.1 2.6* 2.9 0.3** 0.0**
13.1 4.6 5.1 1.8* 2.8 0.5** 0.0**
13.5 4.9 6.2 2.4* 3.5 2.1** 0.0** 16.0 1.5 2.1 2.3**
*Significantly different from the control within each column at p < 0.05 level. **Significantly different from the control within each column at p < 0.01 level.
1175
Body length (mm)
Eggs inside body
Offspring per worm
Control
1126 3
11.1 1.2
28.4 3.5
24.0 mg L1 nano-TiO2 47.9 mg L1 nano-TiO2 95.9 mg L1 nano-TiO2 167.8 mg L1 nano-TiO2 167.8 mg L1 nano-TiO2 (supernatant)
1080 24 801 82* 760 29** 740 15** 980 72
9.8 2.3 4.8 1.9* 2.1 0.8** 0** 8.9 1.6
21.7 5.8 3.4 3.1** 0.9 1.2** 0** 18.5 2.4
975 66 923 10 812 59* 770 48**
9.1 3.0 7.9 2.0 5.8 1.5* 2.2 1.3**
22.5 4.2 16.3 5.5 5.6 3.2** 2.3 2.6**
Treatment
*Significantly different from the control within each column at p < 0.05 level. **Significantly different from the control within each column at p < 0.01 level.
ZnO NP itself did cause some toxicity on the nematode. When ZnO concentration increased to 4.1 mg L1, the toxicity of dissolved Zn2þ sharply increased and overshadowed the effect of the nanoparticle itself. Compared to the control, exposure to Al2O3 NPs higher than 102 mg L1 significantly inhibited the growth and number of eggs inside body and offspring per worm of C. elegans (Table 3, p < 0.05). The nematode could not reproduce when exposed to 203.9 mg L1 Al2O3 NPs, while when exposed to 203.9 mg L1 bulk Al2O3, the number of eggs inside the body and offspring per worm were 5.7 and 3.2, respectively. The number of eggs inside body and offspring per worm of Al2O3 NPs at 102.0 and 203.9 mg L1 were significantly lower than that of bulk Al2O3 (p < 0.05), suggesting that the Al2O3 NP itself did pose additional toxicity on the nematode. Except for the significant differences for the three investigated endpoints between Al2O3 NPs and bulk particles discussed above, there were also significant differences between Al2O3 NP suspension and supernatant. For example, 51 mg L1 Al2O3 NP suspension significantly inhibited the number of offspring per worm (p < 0.05), while the supernatant showed no toxicity on this endpoint. Moreover, when the concentration increased to 203.9 mg L1, there were significant differences for the number of eggs inside body and offspring per worm between the Al2O3 NP suspension and supernatant. One of the reasons may stem from the fact that 203.9 mg L1 of Al2O3 NPs dissolved more Al3þ compared to 51 mg L1 Al2O3 NPs; the other reason was that the supernatant still contained a little amount of Al2O3 NPs. Moreover, the toxicity of AlCl3 was much higher than both Al2O3 NPs and bulk Al2O3 for the three investigated endpoints (Table 3 and Fig. 3B). Bulk TiO2 did not show any toxicity to the nematode until the concentration was higher than 95.9 mg L1, while TiO2 NPs even at 47.9 mg L1 showed significant toxicity compared to the control (Table 4, p < 0.01). TiO2 NP suspension at 167.7 mg L1 posed significant toxicity to the nematode, while the toxicity of TiO2 NP supernatant at 167.7 mg L1 was not significantly different from the respective controls for the three tested endpoints (p < 0.01). The number of offspring per worm exposed to 47.9, 95.9 and 167.8 mg L1 TiO2 NPs was significantly lower than those exposed to bulk TiO2 (p < 0.05). The ZnO, Al2O3 and TiO2 NPs were more easily attached onto the surface of E. coli because of their more positive zeta potential than the bulk particles. Due to the reported antibacterial properties of ZnO and TiO2 (Fu et al., 2005), there may be some possibility of indirect effects through food depletion, i.e., E. coli, rather than direct effects on their target organism nematodes. Reddy et al. (2007)
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10
10
B
Dissolved metal (mg L-1)
A 1
1
0.1 0.1 10
100
10
1000
Concentration (mg L-1)
100
Time (h)
Fig. 4. Concentration- and time-dependent dissolution of nanoparticulate and bulk ZnO and Al2O3. The x-axis and y-axis are in log scale. For the concentration-dependent dissolution (A), the dissolution time was 4 days; and for the time-dependent dissolution (B), the concentration was 40.7 and 407.8 mg L1 for ZnO and Al2O3, respectively. ZnO NP (-), bulk ZnO (C), Al2O3 NP (:) and bulk Al2O3 (;).
reported that ZnO NP (w13 nm) showed complete inhibition of E. coli growth at concentrations larger than 276.6 mg L1. Because our experiments were carried out in dark, production of reactive oxygen species may be minimal to cause any toxicity. However, the exact mechanism of NP toxicity to C. elegans needs further study. 3.4. Influence of particle dissolution The concentration- and time-dependent solubility of nanoparticulate and bulk ZnO and Al2O3 are shown in Fig. 4. The dissolution curves between nanoparticulate and bulk ZnO were similar, with 5.8 and 5.6 mg Zn L1 at 96 h for 40.7 mg L1 nanoparticulate and bulk ZnO, respectively. However, Al2O3 had lower dissolution, with a determined solubility of 0.39 and 0.38 mg Al L1 at 96 h for 407.8 mg L1 nanoparticulate and bulk Al2O3, respectively. The nanoparticulate and bulk TiO2 only dissolved approximately 0.020 mg Ti L1 at 96 h. The dissolution rate for the NPs was slightly faster than the bulk particles. It has been suggested that toxic effects of NPs may be attributed to two different actions: (i) a chemical toxicity based on the chemical composition, e.g., release of (toxic) ions and particle surface catalyzed reactions, e.g., formation of reactive oxygen species; or (ii) due to stress or stimuli caused by the surface, size and/or shape of the particles, and it is not straightforward to differentiate between these two types of toxicity, especially in the case when particles (partially) dissolve during culture treatment (Brunner et al., 2006). Franklin et al. (2007) showed that the toxicity of ZnO NPs to a freshwater microalga was attributable to dissolved zinc; while Griffitt et al. (2007) demonstrated that the effects of nanocopper on Zebrafish gill were not mediated solely by dissolution; both ions and nanoparticles were the source of nanosilver’s toxicity to the algae, with nanoparticles furthering the ions’ impacts (Lubick, in press). Our results suggested that the effect of ZnO NPs could be partially related to the release of dissolved Zn2þ (Table 2 and Fig. 3A). Bulk Al2O3 is generally considered to have low solubility, and this is also true for nanoparticulate Al2O3. However, the small particle size and large surface area-to-volume ratio of the NPs make dissolution of Al2O3 from the surface of the NPs possible, and faster than that of the bulk particles (Fig. 4). The toxicity of supernatant from centrifugation was relatively lower than the suspension of Al2O3 NPs, indicating that toxicity of NPs could not be explained by only dissolved ions, and a nanoparticle-specific toxic mechanism may exist. The cell walls of C. elegans are semipermeable. However, permeability of the cell wall might change during reproduction, with the newly synthesized cell wall being more permeable to NPs (Navarro et al., 2008). Electron microscopic analysis of cadmium-treated nematodes (Popham and Webster,
1979) suggested that abnormal ultrastructural changes in the esophagus and intestine may decrease fecundity and growth. We also observed a decrease in fecundity and growth. C. elegans or other nematodes could be attacked by predacious nematodes, insects and parasitic fungi, transferring NPs through the food chain where they could enter organisms at higher trophic levels; thus, the potential effect at the ecosystem level should not be ignored. 4. Conclusions In this research we characterized the toxicity and behavior of nanoparticulate and bulk metal oxide ZnO, Al2O3 and TiO2 to C. elegans in an aqueous exposure medium. We have shown (1) that metal oxide NPs are toxic to C. elegans, especially to its reproductive capability; (2) and that this toxicity could not be adequately explained by dissolution of the particles alone. The present study has taken a first step in the direction of investigating the ecotoxicity of metal oxide NPs to C. elegans and highlights the need for integrated toxicological assessment of metal oxide NPs in soil and aquatic systems. Acknowledgements This research was supported by the Massachusetts Agricultural Experiment Station (MA 0090) and Massachusetts Water Resources Research Center (2007MA73B). References Adams, L.K., Lyon, D.Y., McIntosh, A., Alvarez, P.J., 2006. Comparative toxicity of nano-scale TiO2, SiO2 and ZnO water suspensions. Water Science and Technology 54 (11–12), 327–334. Baun, A., Hartmann, N.B., Grieger, K., 2008. Ecotoxicity of engineered nanoparticles to aquatic invertebrates: a brief review and recommendations for future toxicity testing. Ecotoxicology 17, 387–395. Blaxter, M.L., 1993. Cuticle surface proteins of wild type and mutant Caenorhabditis elegans. Journal of Biological Chemistry 268, 6600–6609. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Brunner, T.J., Wick, P., Manser, P., Spohn, P., Grass, R.N., Limbach, L.K., Bruinink, A., Stark, W.J., 2006. In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility. Environmental Science and Technology 40, 4374–4381. Donkin, S.G., Dusenbery, D.B., 1993. A soil toxicity test using the nematode Caenorhabditis elegans and an effective method of recovery. Archives of Environmental Contamination and Toxicology 25, 145–151. Donkin, S.G., Williams, P.L., 1995. Influence of developmental stage, salts and food presence on various end points using Caenorhabditis elegans for aquatic toxicity testing. Environmental Toxicology and Chemistry 14, 2139–2147. Dhawan, R., Dusenbery, D.B., Williams, P.L., 2000. A comparison of metal-induced lethality and behavioral responses in the nematode Caenorhabditis elegans. Environmental Toxicology and Chemistry 19, 3061–3067.
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Toxicity of nanoparticulate and bulk ZnO, Al2O3 and TiO2 to the nematode Caenorhabditis elegans Huanhua Wang, Robert L. Wick, Baoshan Xing* Department of Plant, Soil and Insect Sciences, University of Massachusetts, Stockbridge Hall, Amherst, MA 01003, USA
ZnO, Al2O3 and TiO2 nanoparticles are more toxic than their bulk counterparts to the nematode, Caenorhabditis elegans.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 August 2008 Received in revised form 30 October 2008 Accepted 3 November 2008
Limited information is available on the environmental behavior and associated potential risk of manufactured oxide nanoparticles (NPs). In this research, toxicity of nanoparticulate and bulk ZnO, Al2O3 and TiO2 were examined to the nematode Caenorhabditis elegans with Escherichia coli as a food source. Parallel experiments with dissolved metal ions from NPs were also conducted. The 24-h median lethal concentration (LC50) and sublethal endpoints were assessed. Both NPs and their bulk counterparts were toxic, inhibiting growth and especially the reproductive capability of the nematode. The 24-h LC50 for ZnO NPs (2.3 mg L1) and bulk ZnO was not significantly different, but significantly different between Al2O3 NPs (82 mg L1) and bulk Al2O3 (153 mg L1), and between TiO2 NPs (80 mg L1) and bulk TiO2 (136 mg L1). Oxide solubility influenced the toxicity of ZnO and Al2O3 NPs, but nanoparticle-dependent toxicity was indeed observed for the investigated NPs. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Toxicity Metal oxide Nanoparticles Caenorhabditis elegans
1. Introduction Nanoparticles (NPs) have unique physicochemical properties such as tiny size, large surface area, surface reactivity, charge, shape, and media interactions. As a result, the properties of NPs differ substantially from their respective bulk materials of the same composition. However, certain novel properties of NPs could lead to adverse biological effects, with the potential to create toxicity (Oberdo¨rster et al., 2005; Nel et al., 2006). The forecasted huge increase in the manufacture and use of NPs makes it likely that increasing human and environmental exposure to NPs will occur (Nowack and Bucheli, 2007); thus, the studies on safety and (eco)toxicity of NPs are of extreme importance (Kahru et al., 2008). Metal oxide nanoparticles are receiving increasing attention for a large variety of applications. Titanium dioxide and zinc oxide NPs are included in toothpaste, beauty products, sunscreens and textiles. Aluminum oxide having good dielectric and abrasive properties is widely used as an abrasive agent or insulator. The concerns of metal oxide NPs are that because of their chemistry, size, and being not biodegradable, they will rapidly distribute throughout the environment with unknown consequences. Until now little is known about the potential toxicity of metal oxide NPs in soil and water. Given the well-known toxicity of the ionic forms of many metals, the solubility of metal oxide NPs may require particular attention, and it is important to distinguish effects of NPs * Corresponding author. Tel.: þ1 413 545 5212; fax: þ1 413 545 3958. E-mail address: [email protected] (B. Xing). 0269-7491/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.11.004
from dissolved metals when assessing the toxicity of metal oxide NPs. Nematodes are the most abundant metazoa in soil and their function is critical in the soil food web. Nematodes interact closely with other soil organisms and their activity affects primary production, decomposition, energy flow, and nutrient cycling. In natural ecosystems, nematode abundance and community structure analyses were proved to be sensitive indicators of stress caused by soil pollutants and ecological disturbance (Sohlenius, 1980; Neher, 2001). A free-living nematode, Caenorhabditis elegans (C. elegans), is abundant in soil ecosystems and plays a key role in nutrient cycling. C. elegans is a simple multicellular eukaryote whose developmental process and behavior can be readily monitored under a microscope because of its translucent body. After hatching, C. elegans reaches the adult stage by passing through four larval stages (L1, L2, L3 and L4). It is easily grown on Petri plates or in liquid culture with the well-characterized bacterium Escherichia coli (E. coli) as a food source. Its approximate life span is two weeks, with reproduction by either self- or cross-fertilization. C. elegans, being the first multicellular organism to have its genome completely sequenced (The C. elegans Sequencing Consortium, 1998), has served as a superior model in neurobiology, developmental biology and genetics. Aquatic invertebrates have been highlighted as sensitive and relevant test organisms in nanoecotoxicology studies, but most of the available toxicity data are mainly on Daphnia magna (Baun et al., 2008). Several toxicity tests using C. elegans have been developed for ecological risk assessment in soil (Donkin and Dusenbery, 1993) and water (Van Kessel et al.,
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1989). Aquatic test data are applicable to the study of soil contaminants and availability to indigenous invertebrates as the main route of exposure to soil toxicants is probably through the interstitial liquid. To our knowledge, however, there are no reports using C. elegans as a test organism in nanoecotoxicology research. Previous work have investigated the toxicity of ZnO and Al2O3 NPs to plants (Lin and Xing, 2007, 2008), ZnO, CuO and TiO2 NPs to bacteria (Reddy et al., 2007; Heinlaan et al., 2008; Huang et al., 2008), ZnO NPs to freshwater microalga (Franklin et al., 2007), ZnO, TiO2, SiO2 and C60 NPs to D. magna (Adams et al., 2006; Lovern et al., 2007), and CuO NPs to Zebrafish (Griffitt et al., 2007). As indicated above, the effect of metal oxide NPs on nematodes has never been investigated. Toxicity tests on the nematode C. elegans primarily examined free metal ion (Williams and Dusenbery, 1990; Wah Chu and Chow, 2002). Therefore, this study was undertaken to determine whether metal oxide NPs are toxic to C. elegans, and if so, to determine if the toxicity is solely due to dissolution of metal oxide NPs by comparing to their reference toxicants: soluble metal ions and bulk metal oxides of non-nanoparticulate form. The objective of this study is to address the current knowledge gap of C. elegans responses to metal oxide NP exposure. Since the nematodes feed on bacteria and are regarded as particle-ingesting organisms, the current study presents new perspectives in NP-related risk assessment and food web accumulation modeling. 2. Materials and methods 2.1. Preparation of particle suspensions The ZnO, Al2O3 and TiO2 (anatase-type) NPs were purchased from Hongchen Material Sci & Tech. Co., China. Bulk TiO2 (anatase-type), ZnO and Al2O3 were purchased from ACROS, Fisher Scientific and Baker Chemical Co., respectively. Reagent-grade ZnCl2 and AlCl3 were purchased from Fisher Scientific and Aldrich Chemical Co., respectively. The surface areas of the NPs provided by the producers were 50, 180 and 35 m2 g1 for ZnO, Al2O3 and TiO2, respectively. Their surface areas were measured again by N2 sorption at 77 K using a NOVA 1000e instrument (Quantachrome). The multi-point Brunauer–Emmett–Teller (BET) method was employed to calculate the surface areas and the data are listed in Table 1. The stock suspensions of nanoparticulate and bulk ZnO, Al2O3 and TiO2 (16.3, 407.8 and 239.6 mg L1, respectively), were dispersed in ultrapure water by probe sonication (Sonic Dismembrator, Model 100) at 100 W and 40 kHz for 30 min to aid mixing and for forming homogeneous suspensions. The NPs were then serially diluted in sterile ultrapure water and additionally sonicated for 30 min. Small magnetic bars were placed in the suspensions for stirring during dilution to avoid aggregation and deposition of particles. Zinc and aluminum ion (Zn2þ, Al3þ) solutions were prepared by dissolving ZnCl2 and AlCl3 in ultrapure water. Because the solubility of TiO2 is extremely low, the effect of Ti ion was not investigated. The pH for nanoparticulate and bulk ZnO and TiO2 was adjusted with HNO3 and NaOH to 7.0. To avoid precipitation of Al(OH)3, the pH of nanoparticulate and bulk Al2O3 and AlCl3 was adjusted to 5.6, which had little effect on C. elegans because they have a wide pH tolerance range from 3 to 12 (Khanna et al., 1997). 2.2. Characterization of metal oxide NPs The particle shape of nanoparticulate and bulk ZnO, Al2O3 and TiO2 was visualized using transmission electron microscopy (TEM, JEOL 100CX, USA) operated at
80 kV. Also, about 150 individuals for each type of bulk particles were selected to determine their sizes according to the magnification of TEM. The particle size distribution and zeta potential were analyzed with a Nano Zetasizer (Malvern Instrument Ltd., UK) using dynamic light scattering (DLS) technique. 2.3. Preparation of synchronized nematodes C. elegans strain Bristol N2 (wild type) used in this work was provided by the Caenorhabditis Genetics Center (University of Minnesota) and maintained on nematode growth medium (NGM) plates (95 mm 15 mm sterilized-disposable Petri dish, Canada) seeded with E. coli strain OP50 at 20 1 C (Brenner, 1974). The components of NGM were agar, peptone, cholesterol, KH2PO4 and K2HPO4 buffer, NaCl and MgSO4 (Stiernagle, 2006). E. coli was cultured overnight in sterilized LuriaBertani (LB) medium (5 g L1 of yeast extract, 10 g L1 of tryptone, and 10 g L1 NaCl). After 3 days many gravid hermaphrodites, as well as L1 and L2 stage juveniles, were found on the NGM. Our study started with synchronized worms in the L1 stage to avoid the influence of developmental stage on the toxicity test. To obtain worms synchronized to this life stage, the NGM plates were washed with 10.5 mL sterilized water into 15-mL centrifuge tubes and 1.5 mL of 0.5 N NaOH and 3 mL of 1% NaOCl were subsequently added. The tubes were immediately centrifuged at 3000 g for 2 min. The supertanant was pipetted out and the dead nematodes and eggs were pelleted at the bottom. Sterilized water was then added to the pellet and centrifuged again to remove the remaining NaOH and NaOCl mixture. This procedure killed the nematodes but the eggs both inside and outside of the body survived (Fig. 1). After 8 h, the eggs hatched and were termed as L1 larvae. 2.4. Toxicity test Four types of endpoints were assessed, i.e., lethality to the vermiform nematode, length of the worm, number of eggs inside the worm body and offspring per worm. The tested concentrations for each endpoint were 0.4, 0.8, 1.6, 4.1, 6.1 and 8.1 mg L1 for nanoparticulate and bulk ZnO, and 0.7, 1.4, 2.7, 6.8, 10.2 and 13.6 mg L1 for ZnCl2; 10.2, 30.6, 51.0, 102.0, 203.9 and 407.8 mg L1 for nanoparticulate and bulk Al2O3, 0.7, 1.3, 2.7, 6.7, 10.0, 13.3 mg L1 for AlCl3; and 24.0, 47.9, 95.9, 167.8, 239.6 mg L1 for nanoparticulate and bulk TiO2. All the tests were carried out in ultrapure water and no salts were added. At the beginning of each test, approximately 30 L1 juveniles were transferred by micropipette to the Petri plates containing different concentrations of toxicity test particle (i.e., nanoparticulate and bulk ZnO, Al2O3 and TiO2). 250 mL of E. coli in LB broth were centrifuged at 3000 g for 10 min, and the bacterial pellet was used as the concentrated bacteria to feed the nematodes. A loopful of the pelleted bacteria was transferred to the above test plates. We chose an aqueous medium because it was difficult to evenly distribute NPs in an agar medium and the nematodes were able to freely contact with NPs in the aqueous medium; also, C. elegans lives in the interstitial water of soil. The exact number of transferred L1 nematodes was counted with a dissecting microscope. The seeded plates were incubated at 20 1 C under dark condition and shaken at 100 rpm. After 24 h the surviving worms were counted with a dissecting microscope. Death was determined as a lack of response to gentle probing with a needle. For growth and reproduction tests, L1 nematodes were cultured for 5 days under the same conditions as described for the LC50 determination. At the end of the exposure period, about 10 living worms per time were pipetted into a drop of water on a glass slide, covered with a coverglass and heated to approximately 50 C. This treatment resulted in straight and easily measurable worms. The length of the nematodes was measured using a compound microscope equipped with a camera (Olympus DP70) interfaced directly to a computer (Olympus BX51, Japan). The eggs inside the worm body were counted using the same microscope. Reproductive capacity was evaluated by determining the mean number of offspring at all stages beyond the eggs over the entire brood period. 2.5. Metal analysis and comparison of the toxicity of supernatant and suspension
Table 1 Characteristics of particles used in this study. Particle
Purity (%)
Surface areaa (m2 g1)
Diameter (nm)
Hydrodynamic diameterb (nm)
Nano-ZnO Bulk ZnO NanoAl2O3 Bulk Al2O3 Nano-TiO2 Bulk TiO2
>99.5 >99.5 >99.99
54 3.3 188
20 532 60
478–980 (759) 595–976 (737) 470–1126 (763)
>98.5 >99 >99
11 325 7.3
429 50 285
565–974 (889) 338–917 (550) 158–687 (494)
a
Zetac (mv) 0.017 3.6 33.1 5.6 18.9 33.8
Data generated in our lab. b The data are expressed as range (median). c The pH was 7.0 for nanoparticulate and bulk ZnO and TiO2, and 5.6 for nanoparticulate and bulk Al2O3.
A total of 40 mL of each concentration of NP suspensions was centrifuged at 13,000 g for 20 min and filtered (0.2 mm polytetrafluoroethylene filter, Ireland). To determine dissolved metal (defined as metal present in the supernatant), 6 mL of the supernatant was acidified with 250 mL of 1% HNO3 (V:V) and analyzed by an inductively coupled plasma mass spectrometer (Elan 6100, PerkinElmer, USA). The remaining supernatant was also tested for the same four endpoints to elucidate if the toxicity was caused by the particles or the dissolved metal ions. In addition, the time- and concentration-dependent dissolution of nanoparticulate and bulk particles was also investigated using the above procedure. 2.6. Statistical analysis Four experiments with three replicates per concentration were performed for each type of test. The results were presented as mean standard deviation (SD). The software package SPSS 13.0 was used for statistical analyses (SPSS Inc., Chicago, IL). First, c2 test of normality for each replicate and Barlett’s test of homogeneity of variance between replicates of each treatment were made. Then, one-way ANOVA
H. Wang et al. / Environmental Pollution 157 (2009) 1171–1177
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Fig. 1. Eggs of the nematode before (A) and after treating with NaOCl and NaOH solution (B). After being treated the ovary was lysed and the eggs were released.
analyses were performed to assess any significant difference among treatments and between all treatment levels and the control for both endpoints, at a p-value <0.05 or <0.01 significance level. LC50 is defined as the concentration at which survival is reduced by 50% compared with the control. The LC50 values were calculated on logtransformed mortality data using the SPSS probit model.
3. Results and discussion 3.1. Characterization of NPs The characteristics of both nanoparticulate and bulk ZnO, Al2O3 and TiO2 are listed in Table 1. The measured diameter of ZnO, Al2O3 and TiO2 bulk particles were 532, 429 and 285 nm, respectively, much larger than their nano-sized counterparts. However, considerable particle aggregation was observed by TEM for each of the investigated particles (Fig. 2). Using DLS, wide distributions of particle size were observed, from 478 to 980, 470 to 1126 and 338 to 917 nm for ZnO, Al2O3 and TiO2 NPs, respectively, considerably larger than their nominal size 20, 60 and 50 nm, respectively (Table 1). It should be noted that particle sizes measured by the Nano-Sizer are hydrodynamic diameter based on the Stokes–Einstein equation, which are expected to be larger than the actual particle size. Also, the specific surface area of NPs was significantly higher than that of bulk particles (Table 1). Concerns about the bioavailability and toxicity of NPs arise from their assumed occurrence as small particles in the environment. When evaluating nanoparticle-related toxicity, particle size, degree of agglomeration, and specific surface area were factors we considered. Although NP aggregation was extensive in this study, there were still some individual nano-sized particles in solution. Size has important control over other physical and chemical properties such as zeta potential and metal binding (Madden et al., 2006). The zeta potential of NPs was more positive than bulk particles (Table 1). The surface of C. elegans is reported to have an evenly distributed net negative charge at neutral pH, which was diminished but not abrogated at low pH (Himmelhoch et al., 1977; Zuckerman et al., 1979; Himmelhoch and Zuckerman, 1983; Blaxter, 1993). The negative charge is most likely due to sulfated sugar moieties rather than charged amino acid residues in proteins, phosphate groups, or carboxyl groups on sugars (Himmelhoch and Zuckerman, 1983). Therefore, the nematode cuticle may attract more NPs than the bulk particles via electrostatic interactions.
2.5 mg L1 for AlCl3; 79.9 and 135.8 mg L1 for nanoparticulate and bulk TiO2, respectively. The differences in LC50 values among different zinc toxicants were not significant (p < 0.05); but they were significant between Al2O3 NPs and bulk Al2O3, and between TiO2 NPs and bulk TiO2 (p < 0.05). The LC50 value of AlCl3 was significantly lower than that of Al2O3 NPs and bulk Al2O3, probably due to the toxicity by Al ions. The 24-h LC50 values for Zn2þ and Al3þ to C. elegans in aqueous medium were reported as 202 and 79 mg L1 (Williams and Dusenbery, 1990), 257 and 49 mg L1 (Dhawan et al., 2000), and 111 and 18 mg L1 (Wah Chu and Chow, 2002), respectively. The estimated free-ion based LC50 value for Zn to C. elegans was 131 mg L1 (Tatara et al., 1997). Acute toxicity of zinc to various invertebrates and fish has been reported from 40 mg L1 to 58 mg L1 (USEPA, 1995). Our results showed that the LC50 values were much lower than the reported values. TiO2 NPs had no measurable effect at lower doses (10–50 mg L1), whereas there was a significant effect at higher levels (100–250 mg L1) to the in vitro rat liver derived cell line (Hussain et al., 2005). TiO2 NPs were not found to be toxic to MSTO cells until up to 30 mg L1 (Brunner et al., 2006). The toxicity effect was influenced by different assay parameters such as ionic concentration, pH and larval stage. Pure water with no background ions caused more osmotic stress on the nematode which may have reduced their tolerance to toxicants as observed in our study. Developmental stage, salts and food presence have also been found to influence toxicity assays (Donkin and Williams, 1995; Khanna et al., 1997). The presence of food is not necessary for shortterm tests, and thus it may be desirable to omit food to avoid potential complication caused by bacterial adsorption of toxicants. But for longer studies such as a 24-h test, it is necessary to add bacteria to avoid natural death caused by lack of food. We also compared the effect of ZnO NPs in the presence and the absence of K-medium (3.1 g of NaCl and 2.4 g of KCl in 1000 mL of ultrapure water, Williams and Dusenbery, 1990), and the results showed that the nematodes grew better and the toxicity was lower in the presence of K-medium. The reduced toxicity of several heavy metals (Cd, Pb, Cu, and Hg) in the presence of potassium and sodium salts was also reported by Donkin and Williams (1995). The calculated 24-h LC50 value for ZnO NPs in the presence of Kmedium was 4.9 mg L1, higher than that in pure water of 2.2 mg L1. However, salts were not used for other types of particles in this study to decrease the aggregation of NPs.
3.2. Lethality 3.3. Inhibitory effects on growth and reproduction The 24-h concentration–survival curves are shown in Fig. 3. The calculated LC50 values were 2.2, 2.3 and 2.9 mg L1 for nanoparticulate and bulk ZnO and ZnCl2, respectively; 81.6 and 152.9 mg L1 for nanoparticulate and bulk Al2O3, respectively, and
The inhibitory effects of each toxicant to the nematode are shown in Tables 2–4. Compared to the control, exposure to 1.6 mg L1 and 4.1 mg L1 ZnO NPs and bulk ZnO significantly
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Fig. 2. TEM images of nanoparticulate and bulk particles in ultrapure water. The characterization was done at 10 mg L1 for ZnO and TiO2, and 20 mg L1 for Al2O3. ZnO NP (36.3 k magnification, A); bulk ZnO (9.1 k magnification, B); Al2O3 NP (36.3k magnification, C); bulk Al2O3 (21.7 k magnification, D); TiO2 NP (105.8 k magnification, E); and bulk TiO2 (17.9 k magnification, F).
0.0
A
% survival
100
3.5
7.0
10.5
14.0
B
100
80
80
80
60
60
60
40
40
40
20
20
20
0
0 0
2
4
6
8
C
100
0 0
100
200
300
400
0
50
100
150
200
250
Concentration (mg L-1) Fig. 3. Concentration–survival curves of L1 stage C. elegans for 24-h exposure to various toxicants with E. coli added as a food. ZnO NP (:), bulk ZnO (C) and ZnCl2 (-) (A); Al2O3 NP (:) and bulk Al2O3 (C) (using the bottom x-axis) and AlCl3 (-) (using the top x-axis) (B); and TiO2 NP (:) and bulk TiO2 (C) (C). Each test was repeated for four times. The data points and error bars represented the means and standard deviation, respectively, of the four replicates.
H. Wang et al. / Environmental Pollution 157 (2009) 1171–1177 Table 2 Body length, number of eggs inside the worm body, and number of offspring per individual of C. elegans after exposure to different concentrations of ZnO NPs, bulk ZnO and ZnCl2. E. coli was added as a food to C. elegans. Each test was repeated for four times and n ¼ 385. All values are expressed as mean standard deviation (SD) of four replicates.
Table 4 Body length, number of eggs inside the worm body, and number of offspring per individual of C. elegans after exposure to different concentrations of TiO2 NPs and bulk TiO2. E. coli was added as a food to C. elegans. Each test was repeated for four times and n ¼ 425. All values were expressed as mean standard deviation (SD) of four replicates.
Treatment
Body length (mm)
Eggs inside body
Offspring per worm
Control
1220 36
13.1 2.0
29.3 4.6
0.4 mg L1 nano-ZnO 0.8 mg L1 nano-ZnO 1.6 mg L1 nano-ZnO 4.1 mg L1 nano-ZnO 1.6 mg L1 nano-ZnO (supernatant) 4.1 mg L1 nano-ZnO (supernatant)
1137 76 1070 156 821 88* 720 19** 845 27*
19.3 7.6 8.5 3.6* 3.1 1.2** 0.0** 4.8 1.1**
0.4 mg L1 bulk ZnO 0.8 mg L1 bulk ZnO 1.6 mg L1 bulk ZnO 4.1 mg L1 bulk ZnO
1210 104 1028 143 892 42* 715 25**
9.1 3.0 7.5 0.6 3.6 0.9** 0.0**
28.2 3.5 19.0 1.4 10.2 1.8* 0.0**
24.0 mg L1 bulk TiO2 47.9 mg L1 bulk TiO2 95.9 mg L1 bulk TiO2 167.8 mg L1 bulk TiO2
1080 106 901 63* 875 35* 718 23**
8.1 2.5 5.2 1.6* 3.6 1.8** 0.0**
14.5 5.9 7.6 0.5* 3.7 1.2** 0.0**
1
0.7 mg L ZnCl2 1.4 mg L1 ZnCl2 2.7 mg L1 ZnCl2 6.8 mg L1 ZnCl2
760 16**
9.6 2.1 6.8 2.1 3.0 1.2** 0.0** 3.1 1.5** 0.0**
0.0**
*Significantly different from the control within each column at p < 0.05 level. **Significantly different from the control within each column at p < 0.01 level.
reduced the growth of C. elegans, the number of eggs inside the worm and offspring per worm (Table 2). However, the effect on growth and number of eggs inside the worm was not significantly different among ZnO NPs, bulk ZnO and ZnCl2 (p < 0.05). In contrast, it should be noted that the number of offspring per worm exposed to ZnO NPs was significantly less than that of the bulk ZnO at 0.8 and 1.6 mg L1 (p < 0.05). In addition, for the two endpoints of body length and number of eggs inside body, there were no significant differences between the supernatant of 1.6 mg L1 and 4.1 mg L1 ZnO NP and the corresponding unfiltered ZnO NP suspension (p < 0.05). However, the number of offspring per worm of supernatant of 1.6 mg L1 ZnO NP was significantly higher than that of unfiltered ZnO NP suspension (p < 0.05), suggesting that the Table 3 Body length, number of eggs inside the worm body, and number of offspring per individual of C. elegans after exposure to different concentrations of Al2O3 NPs, bulk Al2O3 and AlCl3. E. coli was added as a food to C. elegans. Each test was repeated for four times and n ¼ 479. All values were expressed as mean standard deviation (SD) of four replicates. Treatment
Body length (mm)
Eggs inside body
Offspring per worm
Control
1125 67
10.3 2.1
27.5 3.8
1
30.6 mg L nano-Al2O3 51.0 mg L1 nano-Al2O3 102.0 mg L1 nano-Al2O3 203.9 mg L1 nano-Al2O3 51.0 mg L1 nano-Al2O3 (supernatant) 203.9 mg L1 nano-Al2O3 (supernatant)
1025 85 953 25 881 31* 820 23* 1074 38
9.3 0.6 7.1 0.9 5.9 1.2* 0.0** 8.0 2.1
950 119
3.5 2.2**
30.6 mg L1 bulk Al2O3 51.0 mg L1 bulk Al2O3 102.0 mg L1 bulk Al2O3 203.9 mg L1 bulk Al2O3
1086 31 1225 96 1233 97 1016 125
9.2 1.5 9.5 1.1 7.3 2.3 5.7 1.5*
25.2 5.1 20.7 1.8 7.3 2.0* 3.2 1.5**
0.7 mg L1 AlCl3 1.3 mg L1 AlCl3 2.7 mg L1 AlCl3 6.7 mg L1 AlCl3
1006 25 858 92* 733 41** 612 19**
7.2 2.5 6.1 2.6* 2.9 0.3** 0.0**
13.1 4.6 5.1 1.8* 2.8 0.5** 0.0**
13.5 4.9 6.2 2.4* 3.5 2.1** 0.0** 16.0 1.5 2.1 2.3**
*Significantly different from the control within each column at p < 0.05 level. **Significantly different from the control within each column at p < 0.01 level.
1175
Body length (mm)
Eggs inside body
Offspring per worm
Control
1126 3
11.1 1.2
28.4 3.5
24.0 mg L1 nano-TiO2 47.9 mg L1 nano-TiO2 95.9 mg L1 nano-TiO2 167.8 mg L1 nano-TiO2 167.8 mg L1 nano-TiO2 (supernatant)
1080 24 801 82* 760 29** 740 15** 980 72
9.8 2.3 4.8 1.9* 2.1 0.8** 0** 8.9 1.6
21.7 5.8 3.4 3.1** 0.9 1.2** 0** 18.5 2.4
975 66 923 10 812 59* 770 48**
9.1 3.0 7.9 2.0 5.8 1.5* 2.2 1.3**
22.5 4.2 16.3 5.5 5.6 3.2** 2.3 2.6**
Treatment
*Significantly different from the control within each column at p < 0.05 level. **Significantly different from the control within each column at p < 0.01 level.
ZnO NP itself did cause some toxicity on the nematode. When ZnO concentration increased to 4.1 mg L1, the toxicity of dissolved Zn2þ sharply increased and overshadowed the effect of the nanoparticle itself. Compared to the control, exposure to Al2O3 NPs higher than 102 mg L1 significantly inhibited the growth and number of eggs inside body and offspring per worm of C. elegans (Table 3, p < 0.05). The nematode could not reproduce when exposed to 203.9 mg L1 Al2O3 NPs, while when exposed to 203.9 mg L1 bulk Al2O3, the number of eggs inside the body and offspring per worm were 5.7 and 3.2, respectively. The number of eggs inside body and offspring per worm of Al2O3 NPs at 102.0 and 203.9 mg L1 were significantly lower than that of bulk Al2O3 (p < 0.05), suggesting that the Al2O3 NP itself did pose additional toxicity on the nematode. Except for the significant differences for the three investigated endpoints between Al2O3 NPs and bulk particles discussed above, there were also significant differences between Al2O3 NP suspension and supernatant. For example, 51 mg L1 Al2O3 NP suspension significantly inhibited the number of offspring per worm (p < 0.05), while the supernatant showed no toxicity on this endpoint. Moreover, when the concentration increased to 203.9 mg L1, there were significant differences for the number of eggs inside body and offspring per worm between the Al2O3 NP suspension and supernatant. One of the reasons may stem from the fact that 203.9 mg L1 of Al2O3 NPs dissolved more Al3þ compared to 51 mg L1 Al2O3 NPs; the other reason was that the supernatant still contained a little amount of Al2O3 NPs. Moreover, the toxicity of AlCl3 was much higher than both Al2O3 NPs and bulk Al2O3 for the three investigated endpoints (Table 3 and Fig. 3B). Bulk TiO2 did not show any toxicity to the nematode until the concentration was higher than 95.9 mg L1, while TiO2 NPs even at 47.9 mg L1 showed significant toxicity compared to the control (Table 4, p < 0.01). TiO2 NP suspension at 167.7 mg L1 posed significant toxicity to the nematode, while the toxicity of TiO2 NP supernatant at 167.7 mg L1 was not significantly different from the respective controls for the three tested endpoints (p < 0.01). The number of offspring per worm exposed to 47.9, 95.9 and 167.8 mg L1 TiO2 NPs was significantly lower than those exposed to bulk TiO2 (p < 0.05). The ZnO, Al2O3 and TiO2 NPs were more easily attached onto the surface of E. coli because of their more positive zeta potential than the bulk particles. Due to the reported antibacterial properties of ZnO and TiO2 (Fu et al., 2005), there may be some possibility of indirect effects through food depletion, i.e., E. coli, rather than direct effects on their target organism nematodes. Reddy et al. (2007)
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10
10
B
Dissolved metal (mg L-1)
A 1
1
0.1 0.1 10
100
10
1000
Concentration (mg L-1)
100
Time (h)
Fig. 4. Concentration- and time-dependent dissolution of nanoparticulate and bulk ZnO and Al2O3. The x-axis and y-axis are in log scale. For the concentration-dependent dissolution (A), the dissolution time was 4 days; and for the time-dependent dissolution (B), the concentration was 40.7 and 407.8 mg L1 for ZnO and Al2O3, respectively. ZnO NP (-), bulk ZnO (C), Al2O3 NP (:) and bulk Al2O3 (;).
reported that ZnO NP (w13 nm) showed complete inhibition of E. coli growth at concentrations larger than 276.6 mg L1. Because our experiments were carried out in dark, production of reactive oxygen species may be minimal to cause any toxicity. However, the exact mechanism of NP toxicity to C. elegans needs further study. 3.4. Influence of particle dissolution The concentration- and time-dependent solubility of nanoparticulate and bulk ZnO and Al2O3 are shown in Fig. 4. The dissolution curves between nanoparticulate and bulk ZnO were similar, with 5.8 and 5.6 mg Zn L1 at 96 h for 40.7 mg L1 nanoparticulate and bulk ZnO, respectively. However, Al2O3 had lower dissolution, with a determined solubility of 0.39 and 0.38 mg Al L1 at 96 h for 407.8 mg L1 nanoparticulate and bulk Al2O3, respectively. The nanoparticulate and bulk TiO2 only dissolved approximately 0.020 mg Ti L1 at 96 h. The dissolution rate for the NPs was slightly faster than the bulk particles. It has been suggested that toxic effects of NPs may be attributed to two different actions: (i) a chemical toxicity based on the chemical composition, e.g., release of (toxic) ions and particle surface catalyzed reactions, e.g., formation of reactive oxygen species; or (ii) due to stress or stimuli caused by the surface, size and/or shape of the particles, and it is not straightforward to differentiate between these two types of toxicity, especially in the case when particles (partially) dissolve during culture treatment (Brunner et al., 2006). Franklin et al. (2007) showed that the toxicity of ZnO NPs to a freshwater microalga was attributable to dissolved zinc; while Griffitt et al. (2007) demonstrated that the effects of nanocopper on Zebrafish gill were not mediated solely by dissolution; both ions and nanoparticles were the source of nanosilver’s toxicity to the algae, with nanoparticles furthering the ions’ impacts (Lubick, in press). Our results suggested that the effect of ZnO NPs could be partially related to the release of dissolved Zn2þ (Table 2 and Fig. 3A). Bulk Al2O3 is generally considered to have low solubility, and this is also true for nanoparticulate Al2O3. However, the small particle size and large surface area-to-volume ratio of the NPs make dissolution of Al2O3 from the surface of the NPs possible, and faster than that of the bulk particles (Fig. 4). The toxicity of supernatant from centrifugation was relatively lower than the suspension of Al2O3 NPs, indicating that toxicity of NPs could not be explained by only dissolved ions, and a nanoparticle-specific toxic mechanism may exist. The cell walls of C. elegans are semipermeable. However, permeability of the cell wall might change during reproduction, with the newly synthesized cell wall being more permeable to NPs (Navarro et al., 2008). Electron microscopic analysis of cadmium-treated nematodes (Popham and Webster,
1979) suggested that abnormal ultrastructural changes in the esophagus and intestine may decrease fecundity and growth. We also observed a decrease in fecundity and growth. C. elegans or other nematodes could be attacked by predacious nematodes, insects and parasitic fungi, transferring NPs through the food chain where they could enter organisms at higher trophic levels; thus, the potential effect at the ecosystem level should not be ignored. 4. Conclusions In this research we characterized the toxicity and behavior of nanoparticulate and bulk metal oxide ZnO, Al2O3 and TiO2 to C. elegans in an aqueous exposure medium. We have shown (1) that metal oxide NPs are toxic to C. elegans, especially to its reproductive capability; (2) and that this toxicity could not be adequately explained by dissolution of the particles alone. The present study has taken a first step in the direction of investigating the ecotoxicity of metal oxide NPs to C. elegans and highlights the need for integrated toxicological assessment of metal oxide NPs in soil and aquatic systems. Acknowledgements This research was supported by the Massachusetts Agricultural Experiment Station (MA 0090) and Massachusetts Water Resources Research Center (2007MA73B). References Adams, L.K., Lyon, D.Y., McIntosh, A., Alvarez, P.J., 2006. Comparative toxicity of nano-scale TiO2, SiO2 and ZnO water suspensions. Water Science and Technology 54 (11–12), 327–334. Baun, A., Hartmann, N.B., Grieger, K., 2008. Ecotoxicity of engineered nanoparticles to aquatic invertebrates: a brief review and recommendations for future toxicity testing. Ecotoxicology 17, 387–395. Blaxter, M.L., 1993. Cuticle surface proteins of wild type and mutant Caenorhabditis elegans. Journal of Biological Chemistry 268, 6600–6609. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Brunner, T.J., Wick, P., Manser, P., Spohn, P., Grass, R.N., Limbach, L.K., Bruinink, A., Stark, W.J., 2006. In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica, and the effect of particle solubility. Environmental Science and Technology 40, 4374–4381. Donkin, S.G., Dusenbery, D.B., 1993. A soil toxicity test using the nematode Caenorhabditis elegans and an effective method of recovery. Archives of Environmental Contamination and Toxicology 25, 145–151. Donkin, S.G., Williams, P.L., 1995. Influence of developmental stage, salts and food presence on various end points using Caenorhabditis elegans for aquatic toxicity testing. Environmental Toxicology and Chemistry 14, 2139–2147. Dhawan, R., Dusenbery, D.B., Williams, P.L., 2000. A comparison of metal-induced lethality and behavioral responses in the nematode Caenorhabditis elegans. Environmental Toxicology and Chemistry 19, 3061–3067.
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