Article pubs.acs.org/est
Trophic Transfer of Au Nanoparticles from Soil along a Simulated Terrestrial Food Chain. Jason M. Unrine,†,‡,* W. Aaron Shoults-Wilson,§ Oksana Zhurbich,†,‡ Paul M. Bertsch,†,‡,∥ and Olga V. Tsyusko†,‡ †
Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546, United States Center for Environmental Implications of NanoTechnology (CEINT), Duke University, Durham, North Carolina 27708, United States § Department of Biological, Chemical and Physical Sciences, Roosevelt University, Chicago, Illinois 60605, United States ∥ Tracy Farmer Institute for Sustainability and the Environment, University of Kentucky, Lexington, Kentucky 40546 United States ‡
S Supporting Information *
ABSTRACT: To determine if nanoparticles (NPs) could be transferred from soil media to invertebrates and then to secondary consumers, we examined the trophic transfer of Au NPs along a simulated terrestrial food chain. Earthworms (Eisenia fetida) were exposed to Au NPs in artificial soil media and fed to juvenile bullfrogs (Rana catesbeina). Earthworm Au concentrations were continuously monitored so that the cumulative dose to bullfrogs could be accurately estimated throughout the experiment. We exposed a second group of bullfrogs to equivalent doses of Au NPs by oral gavage to compare the bioavailability of NPs through direct exposure to trophic exposure. We observed accumulation of Au in liver, kidney, spleen, muscle, stomach, and intestine in both treatment groups. Tissue concentrations decreased on average of approximately 100-fold with each trophic-step. The total assimilated dose averaged only 0.09% of the administered dose for direct exposure (oral gavage), but 0.12% for the trophic exposure. The results suggest that manufactured NPs present in soil may be taken up into food chains and transferred to higher order consumers. They also suggest that Au NPs may be more bioavailable through trophic exposure than direct exposure and that trophic transfer may influence the biodistribution of particles once absorbed.
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from hydroponic suspension,13 as well as the trophic transfer and biomagnification of Au NPs to insect larvae (Manduca sexta).1 Trophic transfer has also been established for CdSe quantum dots from bacteria (Pseudomonas aeruginosa) to protozoans (Tetrahymena thermophile).2 Trophic transfer from Daphnia magna to Danio rerio had been observed for TiO2NPs14 and CdSe quantum dots.15 Our previous food chain study with M. sexta used hydroponic media as the base rather than soil media.1 The purpose of the present study was to investigate the possibility of trophic transfer of NPs along a more realistic, simulated laboratory terrestrial food chain consisting of soil media, a primary consumer (the earthworm E. fetida), and a secondary vertebrate consumer (the bullfrog Rana catesbeiana). Rana catesbeiana is a large carnivorous amphibian species native to eastern North America and introduced in many other locations around the
INTRODUCTION Incorporation of nanomaterials into increasing numbers of consumer products has led to concerns about the potential adverse impacts of their release to the environment and possible entry of nanomaterials into food chains.1,2 Over the past several years there have been a large number of studies that have investigated the toxicological effects of nanomaterials in aquatic organisms,3 but far fewer studies have focused on terrestrial organisms.4 Several recent estimates suggest that a large proportion of metal and metal oxide nanomaterials will enter wastewater streams and ultimately be partitioned to sewage sludge.5,6 In many parts of the world, including the United States, the majority of sewage sludge is applied to agricultural fields as an organic amendment and fertilizer.7 Our previous studies demonstrated that both Au and oxidized Cu nanoparticles (NPs) could be taken up by earthworms (Eisenia fetida) from soil.8,9 We have also observed some evidence for adverse effects of intact Ag NPs10 and dissolved Ag ions released from Ag NPs11,12 on earthworm behavior and reproduction, respectively. Additionally, we have demonstrated uptake of Au NPs by plants (Nicotiana tabacum) © 2012 American Chemical Society
Received: Revised: Accepted: Published: 9753
June 22, 2012 August 14, 2012 August 17, 2012 August 17, 2012 dx.doi.org/10.1021/es3025325 | Environ. Sci. Technol. 2012, 46, 9753−9760
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world.16 They are known to consume virtually any prey item that they can swallow ranging from small invertebrates to small vertebrates including mammals and birds. Although they are primarily aquatic, earthworms are one of the prey items have been found in the stomach contents of wild R. catesbeiana.17 They were chosen primarily because of the ease of maintaining and feeding them in the laboratory. We chose to use Au NPs as a model nanomaterial because they serve as an ideal probe of particle uptake due to their insolubility and the low natural abundance of Au. Our previous studies have indicated that similar Au NP suspensions are not subject to oxidative dissolution under ambient or physiological conditions in our assays and remain intact before, during, and after uptake from soil or aqueous media into plants and invertebrates.1,4,9,13 Previous food chain studies have only demonstrated uptake into a producer from aqueous media.1,2,14 The secondary objective was to determine the relative bioavailability of NPs through trophic versus direct exposure. Gastrointestinal uptake of Au NPs has previously been established in mice,18 and from soil to earthworms;9 therefore, we hypothesized that Au NPs could be transferred from soil through a food chain to a secondary vertebrate consumer. We also hypothesized that trophic transfer would render NPs more bioavailable than through direct exposure, since NP surfaces could reasonably be expected to be modified by biomolecules. This would make them more readily recognized by cell surface receptors, which is consistent with previous studies demonstrating the importance of the “protein corona” in membrane-bound receptor recognition during receptor-mediated endocytosis of Au NPs.19 Differences in residence time between a realistic dietary exposure and a bolus dose might also be expected to contribute to differences in bioavailability. It is also possible for a trophic filtration effect to occur, in which a subpopulation within a heterogeneous particle population would be more efficiently transferred in the initial trophic step, thus making transfer in the second trophic step more efficient. We tested these hypotheses by maintaining earthworms in a soil media containing Au NPs, tracking uptake in earthworms over time, feeding these earthworms to juvenile bullfrogs, and then sacrificing the bullfrogs to determine uptake and biodistribution of Au. Since our previous studies established that oxidative dissolution of Au NPs does not occur in soil or in organisms,1,9,13 we could be certain that detection of Au was equivalent to detection of Au NPs. To test our second hypothesis, we estimated the cumulative dose at each feeding and administered an equivalent dose of pristine NPs in aqueous suspension using oral gavage (i.e., directly into the stomach using a syringe and gavage needle inserted into the esophagus).
dynamic light scattering (DLS; Malvern Instruments Zetasizer Nano ZS, Malvern, United Kingdom). Gold concentrations in stock solutions were determined by inductively coupled plasma mass spectrometry (ICP-MS) after dissolution in concentrated aqua regia using previously described methods.9 The presence of unreacted Au (III) ions in solution was assessed by measuring Au concentration in the supernatant after ultracentrifugation at 239 311g for 2 h. This would have removed Au particles larger than about 1.2 nm. Soil Dosing and Animal Exposures. Earthworms (E. fetida) from our laboratory culture were maintained in either control soil media or soil media containing 200 mg Au NPs kg−1 dry mass. Although this concentration is much higher than what would be expected for most types of NPs in soils currently,5 it may be applicable to NP spills and it was also necessary in order to detect the Au NPs along the trophic chain. The soil medium was formulated to allow for adequate growth of earthworms in a relatively high density culture for an extended period of time and to minimize the mass of Au NPs needed for the experiment. The media consisted of 60% silica sand, 20% peat moss, 20% dried and ground organic cow manure (as a food source), and 0.01% CaCO3 to adjust the pH to 6.0 ± 0.5. The Au NP dosed medium was brought to 50% v/ w using 18 MΩ deionized water to which Au NP suspension was previously added and shaken to result in the desired final concentration. The medium was then mixed by hand and several subsamples were taken for analysis to ensure homogeneity. Control medium was treated identically without the addition of Au NP suspension. We added 250 earthworms to exposure chambers containing 7.5 kg wet mass of either control or Au NP treated medium and maintained them in a temperature control chamber at 20 °C. Uptake of Au into earthworms was monitored by sampling 5 worms per time point at various intervals (0, 1, 2, 5, 7, 9, 11, 13, 15, 25, and 59 days) and allowing them to void their gut contents for 24 h on moistened paper towels before digestion and analysis (see section on Tissue and Soil Analyses). Earthworms for feeding to bullfrogs were taken from the samples on days 5, 7, 9, 11, 13, and 15. Earthworms from control media were sampled on day 7 to ensure that no Au was present. On day 60, we transferred a cohort of 75 earthworms to 7.5 kg dry mass of clean exposure media and sampled at regular intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 16, 18, 20, and 59 days) to determine physiological elimination rates for Au NPs. We randomly assigned juvenile (newly metamorphosed) bullfrogs (Rana catesbeiana, Rana Ranch, Twin Falls, ID) to treatments. There were three treatments; exposure to Au NPs via diet (trophic exposure, TE, n = 8), exposure to Au NPs via oral gavage (direct exposure, DE, n = 7), and control (C, n = 3). Animals were housed in 4 L polypropylene exposure chambers with screen lids in an environmental chamber with 12 h of light per day at 22 °C. The exposure chambers contained 2 L of reconstituted moderately hard water (60 mg L−1 MgSO4, 96 mg L−1NaHCO3, 4 mg L−1 KCl, 60 mg L−1 CaSO4, pH 7.6),21 as well as a sponge to allow the bullfrogs to exit the water. On a daily basis the bullfrogs were checked, weighed, and the aqueous medium was exchanged for fresh medium. On even exposure days, the controls and the direct exposure group received randomly assigned control E. fetida, while the trophic exposure treatments received randomly assigned Au NP exposed worms. On odd exposure days, controls and trophic exposure treatments received a control oral gavage consisting of DI water, while the directly exposed animals received an
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METHODS AND MATERIALS Nanoparticle Characterization. We obtained tannic acid capped Au NPs from Nanocomposix (San Diego, CA) with a nominal primary particle size of 12 nm and a nominal concentration of 1 g L−1. The primary particle size was characterized by the manufacturer using transmission electron microscopy (TEM) and ultraviolet-visible (UV-vis) spectroscopy. The wavelength of absorption due to the surface plasmon resonance of Au NPs is directly related to the primary particle size.20 We also verified the primary particle size reported by the manufacturer using high resolution TEM (JEOL 2010F, Tokyo, Japan) as well as the purity of the particles using energy dispersive spectroscopy (TEM). The hydrodynamic diameter of the particles in the stock suspension was determined using 9754
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zero, based on lack of elimination in the earthworms, as well as lack of elimination of other insoluble NPs, such as CeO2 demonstrated in previous studies.23 This conservative estimate will yield the highest possible modeled trophic transfer factor given the other parameters. Typically, biodynamic models estimate concentrations at steady-state; however, this approach could not be used here since we assume that there is no elimination and that growth is slow, thus steady-state would not be achieved within the lifespan of the animal. Also, since growth is determinant in bullfrogs,24 steady-state would not be achieved due to growth dilution.
equivalent Au NP dose via oral gavage. The volume of oral gavage was 0.5 mL and was delivered using a 1.0 mL 100% polypropylene syringe and a stainless steel gavage needle. This volume is similar to the volume of the earthworms and was not expected to have any adverse effects. The concentration of Au NPs was adjusted by diluting with DI water based on analyses of a subsample of the prior day’s Au NP-exposed earthworms so that an equivalent Au NP dose was delivered. Actual concentration and volume of Au NP suspension delivered via the gavage apparatus was nearly identical to the expected concentration and volume. The earthworms were either fed whole to the bullfrogs or in some instances the worms need to be chopped into 2−3 pieces before feeding. If necessary, forcefeeding techniques were used to ensure that all tissue was consumed. The animals were observed after feeding or gavage to ensure that the dose was not regurgitated. After 14 days of exposure, all animals were sacrificed using intraperitoneal injection of tricane-methanesulfonate. The animals were immediately necropsied and the stomach, intestine, liver, spleen, and a small subsample of muscle from the quadriceps as well as the remaining carcass were lyophilized. The carcasses were then homogenized using a stainless steel grinder, decontaminating between samples with acidic (Citranox) detergent and 18 MΩ deionized water. All animal care and use procedures were conducted according to a protocol approved by the University of Kentucky Institutional Care and Use Committee (animal welfare assurance number A333601). Tissue and Soil Au Analyses. Earthworms and soils were digested in polypropylene centrifuge tubes using a microwave digestion system (MARS Xpress, CEM, Matthews, NC. Briefly, we first digested whole earthworms, lyophilized frog tissue samples, or 250 mg dry mass of soil using 5 mL of concentrated trace-metal grade HNO3 acid ramping to 110 °C and holding under reflux for 10 min. This was followed by adding 5 mL of concentrated trace-metal grade HCl and heating under identical conditions. Reagent blanks were included with each digestion set. The samples were appropriately diluted and analyzed using ICP-MS. Quality control measures included determination of spike recovery for each analytical set, internal standardization, using analyses of laboratory control tissue samples containing known mass concentrations of Au, and use National Institute of Standards and Technology (NIST) traceable Au standards with cross-calibration verification using a matrix blank fortified with an NIST traceable Au standard from an independent source. Data Analyses. We estimated the applied dose for the trophic exposure animals by multiplying the mass of E. fetida fed to the bullfrogs by the mean concentration of Au in the subsamples from the earthworms used to feed them on a particular day. These doses were then summed across the entire exposure period for each animal. The gavage doses were calculated based on the concentration and volume of gavage dose and summed across the exposure period for each animal. Student’s t tests were used to test for significant differences in weight change, applied dose, and Au concentration in each tissue type. The significance level was set at α = 0.05. Because our exposures were conducted over a short time period, we modeled concentrations over time to determine if biomagnification would likely ever be observed over the bullfrog lifetime. This was accomplished employing a modified biodynamic model22 that expresses the trophic transfer factor as a function of time based on accumulation and growth according to eqs 1−4. We assume the elimination rate constant (ke) to be
TTF(t ) = Cfrog(t )/Cworm(t )
(1)
Cfrog(t ) = A frog (t )/M frog(t )
(2)
M frog(t ) = Ma /(1 + be−kt )
(3)
A frog (t ) = (Cworm(t ) × AE × IR × M frog (t )) + A frog(t − 1) (4)
Where TTF(t) is the trophic transfer factor at time t, the Cfrog(t) is the concentration of Au in the frog at time t, and the Cworm(t) is the concentration in the earthworms at time t. For simplification, we assume that the earthworm tissue concentration is constant at 2 μg Au g−1. Similarly in eq 2, Afrog(t) is the amount of Au in the frogs at time t in μg and Mfrog(t) is the mass of the frog at time t. Mfrog at time t is calculated using a logistic growth function (eq 3), where Ma is the asymptotic mass at maturity (about 500 g for a bullfrog at maturity), and b and k are constants determining the y-intercept (about 10 g at metamorphosis25) and inflection point, respectively, which was fitted to yield a reasonable hypothetical growth curve based on what has been observed previously.24 The Afrog(t) is calculated using the modified biodynamic model (eq 4). We calculate the assimilation efficiency (AE) based on the reconstructed body burden and the estimated dose from the total mass of worms fed to the animals multiplied by the mean worm Au concentration. Finally, the ingestion rate (IR) is assumed to be about 5% of body mass based on dietary requirements for bullfrogs.26 The calculations were conducted iteratively to generate growth curves and corresponding TTF each day for 7 years.
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RESULTS AND DISCUSSION Au NP Characterization. The Au NPs had a geometric (TEM) diameter of 14.6 ± 3.1 nm (mean ± standard deviation throughout text; n = 104 from 10 images; Supporting Information (SI) Figure S1−2). The NPs were composed of pure Au based on EDS analysis (SI Figure S3). The ζ potential of the stock suspension (diluted to 50 mg Au L−1) was −31.1 ± 1.76 mV (n = 3) at pH 5.8. The Z-average hydrodynamic diameter was 21.66 ± 0.12 nm (n = 3). The maximum fraction of dissolved Au, as measured using ultracentrifugation, was 0.087 ± 0.077% (wt/wt; n = 3). One replicate had a value of 0.002% Au suggesting that mean may be skewed toward higher values due to resuspension while decanting the supernatant. Our previous study of similar Au NPs demonstrated that little or no Au (III) is taken up from the soil by earthworms from these suspensions.9 The same study also demonstrated that there is some aggregation of the NPs in soil pore water, but that some intact primary particles can remain. Uptake and Elimination of Au NPs in E. fetida. We observed a rapid initial increase in the Au concentrations in 9755
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Figure 1. Uptake and elimination curves for Eisenia fetida exposed to 15 nm diameter Au nanoparticles in soil media (FW = fresh weight). Error bars indicate standard deviation (n = 5 per point).
earthworms for the first 15 days of exposure to a maximum concentration of 9.5 mg Au kg−1 wet tissue mass, followed by a decline in concentrations from days 15 to 60 to a concentration of 2.6 mg Au kg−1 wet tissue mass (Figure 1). This decline occurred despite the fact that the earthworms had not yet been transferred to clean media for determining elimination. We speculate that some of the Au measured in the gut at peak Au concentrations (10−15 days) may have been due to soil particles remaining within the gut. We allowed the earthworms to void their gut contents on wet paper towels for 24 h, but this may not have been sufficient to clear all soil particles. The data were also characterized by high variance during that time period and all but one point was not significantly different from the lower concentration on day 25. This variation may also suggest that there were variable amounts of Au remaining within the gut. It is also possible that due to aging effects the bioavailability of the Au NPs decreased over time. This makes it difficult to determine actual uptake rates using these initial data points. Despite the fact that some Au remained within the gut, our previous research demonstrated that Au NPs are taken up intact by the earthworms and biodistributed throughout the body,9 so some proportion of the Au NPs were internalized within the tissues. Our previous studies investigating uptake of Ag NPs into earthworms also demonstrated very slow accumulation over time characterized by a rapid initial increase in apparent tissue concentrations over the first 7 days.12 After transfer of earthworms to clean media, very small declines in apparent tissue concentrations were observed over the first 3 days, but no decrease in tissue concentration was observed for the next 57 days of elimination time. We assume elimination for the first three days corresponds to clearance of any Au NP containing soil particles from the gut. The beginning earthworm fresh mass at the start of exposure was 0.73 ± 0.09 g; it was 0.66 ± 0.10 g at the beginning of the elimination period and 0.85 ± 0.17 g at the end of the elimination period. Therefore, there was no increase in tissue concentrations due to weight loss that would compensate for decreased body burden due to elimination and concentrations can be directly compared. This indicates that the earthworms have no efficient means of eliminating Au NPs. Previous studies in rats have suggested that insoluble CeO2 NPs are eliminated extremely
slowly.23 It is possible that the 15 nm particles, roughly the size of a very large protein molecule, are too large to be excreted from the nephridia. Elimination of CeO2 NPs in rats is mostly via the feces and little elimination was detected via the urine.23 The estimated uptake rate of 0.035 ± 0.006 mg Au kg−1 wet mass d−1 is based on the concentration observed after 3 days of gut clearance in clean media, which is the most accurate estimate of Au concentration accumulated within the tissues. This estimate assumes first-order kinetics. The estimate for a first-order elimination rate constant is 0 mg Au kg−1 wet mass d−1 based on the fact that apparent tissue Au concentrations were not significantly different for any measurement period from day 3 to day 60 of the elimination phase. Trophic Transfer of Au NPs to Rana catesbeiana. The cumulative administered dose over the 14 day exposure for bullfrogs undergoing trophic exposure was 27.5 ± 1.6 μg of Au per animal, while the administered gavage dose was 25 ± 0.75 μg per animal (no significant difference between treatments). There was no significant difference in the beginning body mass between the trophic and direct exposure treatments (9.29 ± 1.50 g and 9.48 ± 0.78 g, respectively), nor was there a significant difference in weight change over the 14 day exposure period among the three treatments (Figure 2). Given the
Figure 2. Change in body mass of Rana catesbeiana exposed to Au NPs either through gavage (Gavage Au, n = 7) or through feeding the worms previously exposed to Au NPs (Worm Au, n = 8) during the study. Error bars indicate standard deviation (control, n = 3) . 9756
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Figure 3. Au concentrations in organs from Rana catesbeiana exposed to Au nanoparticles either through exposure from enriched earthworms (Worm Au, n = 8) or oral gavage (Gavage Au, n = 7). Note the differences in scale in each panel. Asterisks indicates statistical significance at α = 0.05 (*) or 0.01 (**).
similarity in body mass and growth, the organ and carcass Au concentrations can be directly compared between trophic and direct exposure to test for differences in uptake and accumulation. The data also suggest that there were no effects of Au NP exposure on growth as measured by change in body mass. We detected Au in all organs tested for bullfrogs exposed via trophic and direct exposures (Figure 3). Au concentrations in control tissues were below the method detection limits. The majority of Au was detected in the liver, spleen, and kidney. This pattern was observed in a previous study where polyethylene glycol (PEG) coated Au NPs were injected into the tail vein of rats;27 however, we observed a much greater proportion of the Au body burden in the spleen. For rats exposed to10 nm Au NPs, only 2.2% of the applied dose was found in the spleen and 36.3% was found in the blood after 24 h. We found ∼57% and 25% of the body burden in the spleen for the gavage and trophic exposures, respectively. The particles in the rat study were diluted in phosphate buffered saline, which caused them to aggregate.27 A previous study also indicated that small (5−10 nm) CeO2 NPs are cleared from the blood relatively slowly compared to larger particles when administered intravenously in rats.28 CeO2NPs have also been found to have a similar biodistribution pattern after IV injection in rats. 23 This was attributed to the action of the reticuloendothelial system clearing exogenous particles from the blood. We observed significantly higher Au concentrations in liver and kidney in animals experiencing trophic compared to direct exposure (Figure 3), as well as a higher overall body burden (Figure 4). The percentage of the applied dose that was assimilated averaged 0.12% for trophic and 0.09% for direct exposure. High concentrations of Au NPs were observed in the spleen, with a higher mean concentration observed in the direct
Figure 4. Reconstructed body burden in Rana catesbeiana exposed to Au nanoparticles either through exposure from enriched earthworms (Worm Au, n = 8) or oral gavage (Gavage Au, n = 7).
exposure treatment; however, the concentrations were not significantly different between treatments due to high variance in the trophic exposure treatment (Figure 3). This variability in the spleen burden also made the differences in total body burden between treatments statistically nonsignificant; however, there is a consistent trend between the liver, kidney, muscle, carcass, and whole body burdens indicating that the Au NPs were more bioavailable through trophic compared to direct exposure. The differences in liver and kidney concentrations, where most of the body burden resided, were statistically significant. There were nonsignificant differences between treatments in the stomach and intestine Au concentrations; however, these were not included in the reconstruction of the whole body burden, because it is possible that a large proportion of the Au detected had not been absorbed. 9757
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demonstrates uptake and biodistribution, since the Au NPs could not be present in those organs unless they were absorbed from the GI tract and transported in the blood. Other studies examining microbial food webs involving protozoans have demonstrated trophic transfer, but in this case, the entire prey item is engulfed by the predator.2,31 Another study investigated uptake of TiO2 NPs by zebrafish (Danio rerio) by cladocerans (Daphnia magna) which had been pre-exposed to TiO2.14 Again, this study did not analyze internal organs or provide evidence for actual uptake and biodistribution of the TiO2, although elimination (gut clearance) experiments provided strong indirect support for uptake. In some cases, an increase in concentration from one trophic level to the next (biomagnification) has been observed for one trophic transfer step,1,2 while in other cases, including the present study, there was a decrease in concentration, or trophic dilution.2 Because we only exposed the bullfrogs for 14 days and elimination was likely slow, it is not likely that steady state was achieved. However, our modified biodynamic model suggests that a tropic transfer factor (TTF; Concentration in trophic level n + 1/concentration in trophic level n) greater than 1 is unlikely, indicating no possibility of biomagification (Figure 6). In the case of microbe-protozoan trophic transfers2 where AE is theoretically 100%, since the entire microbe is engulfed by the protozoan, it is fairly clear that biomagnifcation could occur, particularly if elimination mechanisms are limited. In the present study AE was only 0.001, so even if ke is zero, biomagnification is unlikely, particularly given that the animal is growing during the exposure. In our previous study, where biomagnificaiton from plants to invertebrates1 was observed, we did not quantify AE; however, we do know that the IR was high. The study involved transfer from tobacco (Nicotiana xanthi) to tobacco hornworms (Manduca sexta). The larvae started with a small body mass and derived all of their biomass from the Au NP-containing tobacco during the exposure. It is possible that the accumulation was enhanced by their relatively high IR, which would have a similar effect on the accumulated concentration as a high assimilation efficiency (eq 3). Also, hornworm caterpillars are known to have a spatially variable gut pH with regions having pH values up to 12; while the bullfrogs have an acidic gut environment. High pH would tend to electrostatically stabilize the negatively charged Au NPs,
Although there was a logarithmic decrease in Au concentration with each trophic step from soil to earthworm to frog (Figure 5), this study provides, to our knowledge, the
Figure 5. Concentrations of Au in each trophic level from the study (soil and earthworms n = 5; whole frogs n = 8). Error bars indicate standard deviations. Please note the logarithmic scale.
first definitive evidence that manufactured NPs can be transmitted from soil through a terrestrial food web consisting of two trophic steps. In our previous study, which demonstrated trophic transfer and biomagnification of Au NPs from plants to herbivores, the Au NPs were taken up by the plants via hydroponic exposure.1 Our previous study demonstrated unequivocally that earthworms absorb Au NPs intact with no dissolution from soil and distribute them among their tissues.9 While organ concentrations were generally too low to determine speciation of the Au in frog tissue, Au is insoluble under physiological conditions, and we have shown in a variety of species that Au NPs are taken up intact, with no dissolution.1,4,9,13,29 A previous study investigated trophic transfer of Au nanorods in estuarine mesocosms; however, no Au was found within the internal organs of fish or spatially localized or chemically speciated within the tissues of other species.30 Because of this, it is difficult to know if the Au NPs were truly absorbed or simply bound to the exterior surfaces of the organisms, including the gut. Since we were able to dissect individual internal organs from the bullfrogs, the present study
Figure 6. Change in apparent trophic transfer factor (TTF) as a function of body mass and days post metamorphosis modeled using a modified biodynamic approach. 9758
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Notes
perhaps preventing aggregation with food particles within the gut. Note that Au is not likely to dissolve in either environment. Environmental Implications. We have demonstrated that manufactured Au NPs applied to soil may ultimately be transferred to higher order consumers through detrital based food webs in terrestrial environments. This is the first study that has tracked the transmission of manufactured NPs from soil through food webs. Taken together with our previous study that showed uptake in herbivores feeding on plants, this indicates that trophic transfer of MNMs accumulating in terrestrial ecosystems, for example from sewage sludge biosolids application, is likely. As discussed, there are conflicting results regarding the possibility of biomagnificaiton. The biology, physiology, and life history traits of the organisms involved at each trophic step are likely to determine whether trophic transfer is efficient. Thus, even for MNMs with similar physicochemical properties, prevailing ecological conditions and community composition at a given site will have a major impact on the nature and extent of trophic transfer. Finally, we obtained what we believe is the first evidence to suggest that Au NPs are more bioavailable through trophic compared to direct exposure. While there are several possible explanations for this, one that must be considered is the difference in the residence time of NPs in the intestines between the trophic exposure and direct exposure, which could account for differences in bioavailability. The Au concentrations in stomach and intestine (Figure 3) were similar between the two treatments, which does not support this explanation. Another explanation is that the food web acts as a “trophic filter”. The nanoparticles occur as a population with variability in properties such as particle size. Since only the most bioavailable fraction of NPs is absorbed at one trophic level, the available pool of particles for the next trophic step would have greater bioavailability than the original pool. This is analogous to trophic transfer of Hg, where more bioavailable methylated form is present at each trophic level, resulting in increases in the trophic transfer efficiency with each subsequent level.32 The importance of this may be limited in this case since the particles were fairly monodisperse, although our previous study demonstrated that some aggregation of Au NPs does occur in the soil pore water.9 Finally, it is possible that the surfaces of the NPs are modified in the soil and perhaps biologically within earthworm tissues, such as the formation of a protein corona, and that this modification may enhance uptake.19,33 Based on the importance of surface chemistry in determining fate34 and uptake19,35 of NPs, it is perhaps the most likely explanation. Regardless of the actual mechanism(s) involved, it is clear that dietary uptake and trophic transfer of NPs is a route of exposure that should be considered in risk assessments and that some form of transformation that enhances bioavailability possibly occurs in the soil or during trophic transfer.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This material is based upon work supported by the National Science Foundation and the Environmental Protection Agency under EPA Science to Achieve Results (STAR) grant numbers 833335 and 834574 and NSF Cooperative Agreement EF0830093, Center for the Environmental Implications of NanoTechnology. Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF or the EPA. The authors acknowledge the assistance of J. Ye at the University of Kentucky Electron Microscopy Center and the helpful comments of three anonymous reviewers.
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ASSOCIATED CONTENT
S Supporting Information *
TEM images, histograms of the particle size distribution and an example EDS spectrum from a particle. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
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