Larval Toxicity Assay
From each vial containing 50 eggs, the number of flies emerging was counted twice a day as a measure of nanomaterial effects on egg-to-adult viability and development time. In no case did the carbon nanomaterials alter total survivorship (). Males and females emerged at statistically indistinguishable rates (all terms in an ANOVA for particle mass concentration, sex and interactions were not significant; e.g., P > 0.2). The suspension of C60 in different solvents prior to dispersal in the food matrix had no detectable effect on toxicity in this egg to adult assay (; P > 0.2). Development time was delayed slightly at high nanomaterial doses, but the effect was not significant even if all data for different materials (CB, SWNTs, MWNTs) were pooled and the control, 100 μg/g and 1000 μg/g doses were pooled (data not shown). It is significant that the nanomaterials were not toxic at 1000 μg/g in food, which is a much higher environmental concentration than would be expected in most scenarios associated with the release of nanoparticles by consumers or manufacturers.
FIGURE 2 Drosophila larvae exposed to carbon nanoparticles (NP) in food have little or no toxic effects. (A) No significant effect of material type or concentration on egg-to-adult viability for Drosophila raised in food containing 0, 100, or 1000 ug-nanomaterial/g-food. (more ...)
There are few studies to which the present results can be directly compared. The study of Leeuw et al. (1
) showed no loss of viability or adult weight upon exposure of Drosophila
larvae to 9 ppm SWNTs dispersed in food paste - consistent with our results which extend to 1000 ppm. Recent studies using other organisms include Velzeboer et al. (15
), who report low toxicities of various nanomaterials including C60 and SWNT at concentrations up to 100 μ
g/L in aquatic systems, and Blickley and McClellan-Green (16
), who report low toxicity of fullerene to embryo, larvae and adult Fundulus heteroclitus
. Johansen et al. (17
) report modest but persistent effects of C60 on rapidly growing soil bacteria, while Brunet et al. (18
) report bacterial toxicity of some fullerene formulations - THF/nC60, PVP/C60 but not aqueous C60 or fullerol. The recent comparative study by Kang et al. (19
) report toxicity to gram-negative bacteria for the carbon nanomaterial family in rank order SWNT > C60 > MWNT. Petersen et al. (20
) studied uptake of 14
C labeled SWNTs and MWNTs by aquatic worms and found that the nanomaterials entered the organisms but were purged within a few days and did not persist in tissues. Petersen et al. (21
) further report bioaccumulation and limited depuration of MWNTs after uptake by Daphnia magna
Low toxicity may reflect a low bioavailability of nanomaterials to internal tissue and organs (1
). We did not attempt to quantify uptake, but the optical microscopy showed that at least some of our test nanomaterials do become sequestered in adult Drosophila
tissue after larval exposure (), which indicates transport across the gut lining. It is notable that nanomaterials (carbon black) appear localized in tissues where sensory bristles are dense, and are not evenly distributed across adult tissues. These flies were tested for differences in fecundity after emerging from the food by allowing them to lay eggs for replicate broods of 24 h. No significant effect of nanomaterial exposure was detected (data not shown).
Adult Fly Exposure to Nanomaterials
The different nanomaterials types varied considerably in their effect on adult behavior and survivorship following physical exposure to powder beds in the dry state. CB, toluene washed CB, and SWNTs spontaneously adhered to external surfaces of the flies on contact and engulfed the flies in a fine particle coating (), killing them within several hours. Other materials (C60, MWNT arrays and toluene washed SWNTs), did not adhere to the outer surface of the flies as extensively, and could be removed by the flies through natural grooming behaviors.
FIGURE 3 Representative morphologies observed in adult Drosophila exposed to carbon nanomaterials as dry powders. Top row: whole flies. (A) unexposed; (B) after SWNT exposure with whole body blackened by adherent nanoparticles (particles not visible at this scale). (more ...)
The strongly adhering nanomaterials (CB, toluene washed CB and SWNT) significantly reduced survivorship () relative to unexposed controls. Survivorship for the less-adhering nanomaterials (C60, MWNT arrays, toluene washed SWNTs), was statistically indistinguishable from unexposed control flies (for six treatments excluding CB, W CB and SWNT, χ2 = 7.3, df = 5, P > 0.19). This experiment was repeated in two other assays using slightly different scoring times, with qualitatively similar results. The estimate of time to complete mortality for CB and SWNT () is conservative, as all such flies were dead within ~6 h in repeat experiments where mortality was scored more frequently (not shown).
FIGURE 4 (A) Differential toxicity of nanomaterials to adult Drosophila. Abbreviations as in . “W” denotes toluene-washed samples. For CB and SWNTs, values are staggered around zero on the Y-axis to show different NPs. Toluene washing of (more ...)
Effects of Nanomaterial Exposure on Fly Locomotor Performance
The ability of adult Drosophila to climb test tube walls in the presence of nanomaterial powders was recorded using digital videography. gives the results plotted as average time required for the first fly to reach a height of 6 cm after being knocked into the nanomaterial powder at the bottom of the test tube. In parallel with the adhesion and mortality effects (), CB and SWNTs immobilized the flies at the bottom of the tube completely preventing climbing, while the other nanomaterials that did not adhere to the flies allowed climbing but showed significant material-to-material differences on climbing rates (completion times). In general, control flies (no exposure) climbed the fastest, and exposure to any material slowed climbing time, with toluene washed C60 (W C60) and MWNT arrays having the greatest impact on climbing, and C60, toluene washed SWNT and MWNT (W SWNT; W MWNT) having less effect (). The duration of NP exposure altered the impact of the NPs on climbing speed, as did the use of glass vs plastic vials, or parafilm vs cotton plugs to seal the vials. However, the immobilization effect of CB and SWNT was highly repeatable. The details of these time- and chamber-effects on locomotor performance will be reported in a separate study.
The mechanism of the locomotor impairment warrants further experimentation but may be related to the fine-structure of the coated fly foot. Studies have indicated that secretions from the pads of the foot are important in adhering to smooth surfaces, and that the surface area of contact is related to the magnitude of the attractive force (22
). If nanomaterials interfere with the fine scale pads on the fly foot or hinder the release of fluid, this could limit the adhesive force for climbing up the smooth wall of the glass tubes we used in our experiments.
Material-to-material differences are striking (), with toxicity trends summarized as: CB ~ SWNTs
C60 ~ MWNT-arrays. The effect of toluene washing was material dependent, having no effect for C60 but significantly reducing the toxicity of SWNTs and MWNT arrays. The intention behind toluene washing was to control for the effects of soluble organic matter, if any, present on these high-surface-area hydrophobic materials. We observed, however, that toluene washing collapsed the low-density aggregate structure of the SWNT sample by the action of surface tension during drying to result in a much denser material superstructure. In this form, the availability of tube bundles or small aggregates to detach from the bulk powder and adhere to external fly surfaces appears to be greatly reduced, which is the likely cause of increased survivorship.
Further, we noticed that the main toxicity trend (SWNTs
C60 ~ MWNT-arrays) is consistent with the availability of fine (<20 μ
m) aggregates or free primary nanoparticles. The C60 powder consisted of very large (~ 20–50 μ
m) aggregates with smooth and faceted crystal faces (Supporting Info.
), and was poorly adherent. In contrast, the CB samples (Figure S1B, Supporting Info.
) consist of small aggregates (~2–10 μ
m) with porous, nanostructured surfaces due to irregular packing of the CB 20–50 nm primary nanoparticles. Some insect surfaces have natural micro/nanotextures (23
) including the Drosophila
wing and foot (), and these are capable of interpenetration and interlacing interactions with the textured carbon black aggregates.
The effect of aggregate size is particularly apparent when comparing two different MWNT samples (). Here adult Drosophila interact with: (i) massive vertically aligned MWNT arrays of typical array size >100 μm, and (ii) spherical disordered aggregates of nominal size 5–20 μm. The small spherical aggregates adhere much more extensively, cause whole-body coverage of the flies, and are as toxic as the most toxic materials in (CB, SWNTs). The initial low toxicity of MWNTs in was clearly due to their large-aggregate array structure (MWNT-arrays), and not to an intrinsic property of MWNTs relative to SWNTs or CB.
FIGURE 5 Effect of aggregation state on adhesion and toxicity in adult Drosophila. Columns show two contrasting MWNT aggregation states: left, spherical aggregates of randomly entangled nanotubes; right, large vertically aligned arrays. The small random aggregates (more ...)
mechanisms of nanotoxicity have not been widely reported, one exception being the observation of airway blockage by aggregated nanotubes during intratracheal instillation in rats (24
). Lehmann et al. (25
) report that Drosophila
use spiracle opening to regulate respiratory gas flux in response to metabolic requirements; shows that nanomaterials can partially block spiracles (F,G vs unexposed controls in C-E), which may in turn hinder oxygen diffusion and impair metabolism.
FIGURE 6 Possible mechanism of nanoparticle-induced mortality in adult Drosophila. (A) Location of spiracles in Drosophila: sp1, mesothoracic spiracle; sp2, metathoracic spiracle; sp3 to sp9, abdominal spiracles, image from Lehnmann (25). (B) SEM image shows mesothoracic (more ...)
Overall, aggregate size appears to be the most important variable determining adhesion and toxicity to adult Drosophila
. This is not unexpected as the ratio of adhesion forces to gravitational and inertial detachment forces increases with decreasing particle size. The largest class of adhering aggregates is about 20 μ
m, which is on the order of the threshold between free-flowing (nonaggregating) powders that can be size classified by dry methods (~30 μ
m) and finer particles that undergo spontaneous aggregation. Particles of mean size 7–10 μ
m have been used in pesticide applications where adhesion to insects can cause active transport of the active ingredient to microhabitats that are hard to reach directly (27
). Nanoroughness in the contact area is also known to influence adhesion (28
), and our data () suggest intrinsically stronger adhesion of the nanorough carbon black (aggregates up to 20 μ
m adhere) compared to nanosmooth C60 (only finer aggregates adhere).
In the environment, contact with pure nanomaterial deposits is expected only in hot spots near manufacturing point sources or during intentional application of pest control agents (27
) and, thus, the adult Drosophila
results presented here represent a high-dose limiting case. We anticipate much lower doses in other environmental scenarios, where insects contact airborne nanoparticles or contact soils, sediments, and surfaces containing a nanoparticle fraction through prior deposition. In these scenarios, we expect nanoparticle/insect adhesion and transport similar to microbial transport by flies acting as disease vectors (29
). Figure S2 (in the Supporting Information)
shows a simple experiment in which flies exposed to MWNT spherical aggregates in one test tube move to an adjacent connected clean test tube and through grooming behaviors contaminate the second test tube with easily identifiable nanotube deposits. Clean unexposed flies were observed to be contaminated with nanoparticles through fly-to-fly contact and grooming behaviors. In the environment, such transport and redeposition may bring nanoparticles into contact with human or environmental receptors that would not otherwise be exposed. It is noteworthy that there is an overlapping length scale between the adherent nanoparticle aggregates observed in this study (typically 1–20 μ
m) and pollen (6–100 μ
m) or bacteria (~1 μ
In summary, exposure to the major members of the carbon nanomaterial family (C60, carbon black, SWNTs, and MWNTs) elicit responses in Drosophila that depend sensitively on exposure route (larval ingestion, adult dry contact) and material aggregation state. Larval ingestion leads to systemic uptake and tissue sequestration but is without other significant consequences even at high doses (1 mg/g of food). In contrast, dry exposure of adults to primary particles or small aggregates (<20 μm) leads to whole-body coverage, loss of locomotor function, and mortality, while lower doses lead to active transport and fly-to-fly transmission.