Influence of Carbon Nanotubes on Pyrene Bioaccumulation from

Apr 27, 2009 - Kennedy , A. J; H , M. S.; Steevens , J. A.; Dontsova , K. M.; Chappell , M. A.; Gunter , J. C.; Weiss , C. A. , Jr. Factors influencin...
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Environ. Sci. Technol. 2009, 43, 4181–4187

Influence of Carbon Nanotubes on Pyrene Bioaccumulation from Contaminated Soils by Earthworms E L I J A H J . P E T E R S E N , * ,† ROGER A. PINTO,† PETER F. LANDRUM,‡ A N D W A L T E R J . W E B E R , J R . * ,† Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, and Great Lakes Environmental Research Laboratory, National Oceanic and Atmospheric Administration, 4840 State Road, Ann Arbor, Michigan 48108

Received October 27, 2008. Revised manuscript received March 21, 2009. Accepted March 26, 2009.

Increasing production of and application potentials for carbon nanotubes (CNTs) suggest these materials will enter soil and sediment ecosystems in significant masses in upcoming years. This may result in ecological risks, either from the presence of the CNTs themselves or, given their exceptional sorption capacities, from their effects on the fate and accumulation of concurrently present hydrophobic organic chemicals (HOCs). Here we test the influence of additions of singlewalled CNTs (SWNTs) and multi-walled CNTs (MWNTs) to two different pyrene-contaminated soils on uptake of this HOC by earthworms (Eisenia foetida). The effects of nanotube additions to the soils were observed to be CNT concentration dependent, with 0.3 mg nanotubes per gram of soil having no impact, while 3.0 mg/g of SWNTs or MWNTs substantially decreased pyrene bioaccumulation from both contaminated soils. The presence of CNTs also affected pyrene elimination rates. After a 14-day exposure to pyrene-spiked soils, earthworms showed enhanced elimination rates in soils amended with 3.0 mg CNT/g but not 0.3 mg CNT/g. These results suggest that the presence of SWNTs or MWNTs in terrestrial ecosystems will have concentration-dependent effects on decreasing HOC accumulation by earthworms in a manner similar to that expected of most “hard” carbons.

Introduction The unique characteristics of carbon nanotubes (CNTs) promise numerous applications thereof in areas such as medicine (1), hydrogen storage (2), sensors (3), and environmental applications (4). CNTs are now being synthesized in significant quantities on a worldwide scale, with an estimated 350 tons having been produced in the year 2007/ 2008 (5) and increasing future production being expected. As such, CNTs will be released increasingly into terrestrial and aquatic ecosystems, either intentionally or unintentionally. Despite intensive foci on applications of CNTs, knowledge regarding their environmental behaviors and ecological and human health risks is limited (6, 7). Research on ecological risks has focused primarily on risks posed to organisms by * Address correspondence to either author. Phone: (734)-485-7955 (W. J. W. Jr.); (734)-936-3069 (E. J. P). E-mail: [email protected] (W. J. W. Jr.); [email protected] (E. J. P.). † University of Michigan. ‡ National Oceanic and Atmospheric Administration. 10.1021/es803023a CCC: $40.75

Published on Web 04/27/2009

 2009 American Chemical Society

exposure to CNTs themselves (8-13) and on their potentials for bioaccumulation (8, 12, 14-16). Given the strong sorption capacity of CNTs for a broad range of hydrophobic organic compounds (HOCs) (17-20), these materials may pose additional risks by influencing the biological uptake of and toxic effects caused by other environmental contaminants. It is possible that nanotubes would act similarly to other “hard” carbon materials (21) and decrease HOC bioaccumulation by organisms (22). Soil and sediment organic carbons and their associated sorption properties vary with their diagenetic aging, younger carbons exhibiting weak, linear sorption patterns and older, “hard” carbons showing substantially stronger nonlinear sorption (21, 23). The typical nonlinear adsorption behaviors of hard carbons are well documented, and recognition of the effects of these nonlinear behaviors in risk assessment represents a significant advance over the use of commonly assumed linear sorption models (22). It is also possible that CNTs may well concentrate HOCs, causing increased accumulation of HOCs via passage of chemical-laden nanotubes into and through organisms (15). A recent study by Ferguson and co-workers showed that addition of single-walled carbon nanotubes (SWNTs) to sediments decreased the availability of a range of HOCs to Streblospio benedicti but did not significantly affect HOC accumulation by Amphiascus tenuiremis, and that the addition of soot, another modestly hard carbon, actually increased HOC uptake by S. benedicti (16). These results provide additional evidence that organism physiology and feeding behavior can substantially influence uptake of HOCs in sediment ecosystems containing CNTs or other hard carbons, and that various forms of hard carbon may have quite different effects on bioaccumulation by organisms (24-26). It was recently estimated that concentrations of nanotubes in soils would be a factor of 20 higher than that in water (5), a result underscoring the importance of determining ecological implications of nanotubes in terrestrial ecosystems. Earthworms are one of the most commonly used receptors for testing ecological uptake of chemicals in soils because of their continual processing of soil and frequent consumption by vertebrate organisms (27, 28). Uptake of SWNTs and multi-walled carbon nanotubes (MWNTs) by Eisenia foetida was recently tested using carbon-14 labeled nanotubes (14). Despite the lack of longterm nanotube accumulation for either type of nanotubes, significant concentrations were found to pass through organism guts, a finding that may strongly affect the impact of nanotubes on HOC bioaccumulation. Here, we investigated the effects of spiking pyrenecontaminated soils with CNTs on bioaccumulation of this polycyclic aromatic hydrocarbon and representative HOC by Eisenia foetida. Two concentrations of SWNTs and MWNTs (0.3 and 3 mg/g) were spiked to two soils. These concentrations are smaller than those used previously by Ferguson et al. (16) (5 mg/g) to study the effects of nanotubes on HOC accumulation in aquatic ecosystems, but they are larger than would typically be expected in most environmental settings. Nevertheless, if nanotube amendments are found not to have a significant impact at these higher concentrations, it can be reasonably expected that they would not have an impact at lower levels. Three different pyrene spiking conditions were evaluated to fully understand the effects of HOC adsorption by nanotubes on bioaccumulation: (i) pyrene spiked to soils without the addition of nanotubes, (ii) pyrene spiked to soils followed by the addition of nanotubes, and (iii) adsorption of pyrene to nanotubes followed by nanotube amendment VOL. 43, NO. 11, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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to soils. To assess the impact of the presence of nanotubes on pyrene elimination rates, earthworms that had accumulated pyrene in pyrene-only spiked soils were added to unmodified soils or soils amended with different nanotube concentrations of 0.3 or 3 mg/g.

Experimental Methods Chemicals. Nickel nitrate hexahydrate (99%), magnesium nitrate hexahydrate (reagent grade), ferric nitrate (reagent grade), citric acid (99%), methanol (HPLC grade), and acetone (HPLC grade) were purchased from Fisher. Alkaline magnesium carbonate, 14C-pyrene (g95% purity; positions 4, 5, 9, 10), and nonlabeled pyrene (98%) were purchased from Sigma Aldrich. Helium gas (99.95%), argon gas (99.998%), and methane gas (99.97%) were purchased from Cryogenic Gases. Carbon Nanotube Synthesis and Purification. MWNTs were synthesized via chemical vapor deposition with a nickel magnesium oxide catalyst, and SWNTs were synthesized using iron on a magnesium oxide support material, both using methane as the feedstock gas (14, 15). The nanotubes were purified by bath sonication in full-strength hydrochloric acid (100 mL/g nanotubes) for 1 h. Carbon nanotube characterization is described in full elsewhere (14, 15). In brief, thermal gravimetric analysis (TGA) revealed almost complete removal of the catalyst impurities by the purification process with carbon percentages of 91.1% ( 0.2 and 99% ( 1 for the SWNTs and MWNTs, respectively. TGA did not reveal amorphous carbon impurities for purified SWNTs or MWNTs, and Raman spectroscopy also revealed high carbon purity with respect to amorphous carbon for the SWNTs. Transmission electron microscopy revealed diameters of 1-2 nanometers for the SWNTs and mainly from 30 to 70 nanometers for the MWNTs. Surface area measurements were taken for MWNTs and SWNTs using a Micromeritics ASAP 2020. Uptake Experiments. Pyrene uptake by Eisenia foetida was determined using modified standard procedures (29). The soils, collected from Chelsea, Michigan and the North Campus of the University of Michigan (Ann Arbor, MI) are referred to here accordingly as “Chelsea” and “NC” soils. Each was air-dried and passed through a 2 mm mesh sieve, then the soils analyzed by HPLC to ensure that pyrene concentrations were less than the detection limit for the instrument (1.37 ppb). The organic carbon fractions for the Chelsea and NC soils were 2.45 ( 0.04% and 2.2 ( 0.2% (n ) 4), respectively. Hard carbon percentages were measured using method CT0375 and found to be 0.04 ( 0.02% (n ) 4) and 0.03 ( 0.01% (n ) 3) for the Chelsea and NC soils, respectively (30). Pyrene only spiked soils were prepared using the following procedure. Nonradiolabeled pyrene at a concentration of 37.5 µg per gram of soil was dissolved in a serum bottle containing a mixture of 5 mL methanol and 1 mL acetone. After complete dissolution of pyrene, 14C-pyrene (58.7 mCi per mM) was added via microsyringe to yield a radioactivity of 0.0014 µCi per gram of soil. After thorough mixing, this solution was transferred dropwise to the soil while rotating the soil container, which was then sealed and tumbled overnight at 60 rpm. After soil homogenization, the solvents were allowed to evaporate in a Labconco freeze-dryer for two hours; preliminary tests indicated that 3.6 ( 0.1% (n ) 3) of the initial pyrene is lost during this step. To test for homogeneity of the pyrene soil distribution, at least three one-gram soil samples were combusted in a biological oxidizer (R.J. Harvey OX500), and the radioactivity assessed using liquid scintillation counting. Upon verification of proper pyrene distribution within the soil (standard deviation was less than 7% of the mean), deionized water was added to the soil at a ratio of 22.5-25% w/w and the wetted soil was 4182

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allowed to sit for at least one hour before addition of earthworms. For uptake experiments of pyrene preadsorbed to carbon nanotubes, CNTs were measured at ratios of 0.3 or 3 mg/g of soil and added to borosilicate centrifuge vials containing 20 mL of deionized water. The amendments of 0.3 or 3 mg/g nanotubes were designed to yield approximately a 1:1 or 10:1 ratio of the masses of added nanotubes and native hard carbons, respectively. A solution with labeled and nonradioactive pyrene was prepared as described above in a serum bottle containing 5 mL of methanol. This solution was transferred completely to the centrifuge vial containing the nanotubes and water, and was mixed for 48 h under constant horizontal shaking (100 rpm). After this period, the vial was centrifuged and a 0.5-mL aliquot of the supernatant was transferred to a scintillation vial for 14C radioactivity counting to verify that a minimum of 98% of the pyrene load had been adsorbed to the nanotubes. Then, the supernatant was withdrawn and the CNTs transferred to a volume of water designed to yield a soil moisture content of 22.5-25%. This mixture was then homogenized using a Cole Parmer ultrasonic processor for 30 min in an ice-water bath. Preliminary tests showed that ultrasonication did not cause desorption of pyrene from the nanotubes but did cause pyrene losses of 4.8 ( 0.1% and 4.3 ( 0.3% (n ) 3) for pyrene preadsorbed to SWNTs and MWNTs, respectively. Additional byproducts were not detected by HPLC analyses, and the decrease is thus postulated to result from volatilization losses. The nanotube and pyrene solution was subsequently completely transferred to soil by addition of small volumes using a manual pipet while rotating the soil container as it received the solution. The container was then sealed and tumbled overnight at 60 rpm. A drying step was not included in this procedure. After soil and moisture homogenization, the consistency of the pyrene distribution in soil was confirmed as described above. Uptake experiments in which pyrene was spiked to soils followed by nanotube addition were also conducted. A mixture of radioactively labeled and nonlabeled pyrene was prepared and spiked to soil as describe above, the soils freezedried, and an adequate distribution of pyrene in the soils determined. The nanotubes were then sonicated in water and added to the soils. To initiate the uptake experiments, three adult worms with combined masses between 1.2 and 2.0 g were transferred to moist soil (30 g dry mass) in 100-mL glass jars, the jars were loosely closed with a cap to prevent worms from escaping and to allow air exchange, and then kept in the dark at 21 ( 2 °C. Three worms were added to each of triplicate containers for each data point. Worms were removed after 1, 7, 14, or 28 days, washed with deionized water, and transferred to wet paper towels in Petri dishes for 24 h in the dark to allow purging of gut contents. The worms were rinsed with clean deionized water before being transferred to glass centrifuge tubes, freeze-dried overnight, weighed, combusted in a biological oxidizer, and the radioactivity determined using liquid scintillation counting. Bioaccumulation factors (BAFs) were calculated as ratios of the pyrene concentrations in the earthworms divided by that in the soils. Pyrene concentrations in the soils after 28 days of exposure were also measured using biological oxidation and liquid scintillation counting. After 28 days of earthworm exposure, samples of NC soils were extracted with hexane and the solvent liquidphase filtered to a borosilicate vial to test for pyrene biotransformation and biodegration. After evaporation under forced nitrogen flow, the residues from the hexane extraction were redissolved in acetonitrile for HPLC analysis (1100 Agilent HPLC). The fluorescence spectrum profile for triplicate samples from samples aged 28 days were compared with those of the time zero controls. Lipid contents were

measured gravimetrically using a 1:1 chloroform:methanol extraction for earthworms added to pyrene-spiked NC soil, and NC soil spiked separately with pyrene and 3.0 mg/g of SWNT or MWNTs. Depuration Experiment. After exposure for 14 days to pyrene-spiked NC or Chelsea soil, earthworms were transferred to containers with unmodified soil or soil amended with MWNTs or SWNTs at concentrations of 0.3 or 3 mg CNT per gram of soil. The soils were spiked with CNTs by dispersing them in water as described above, adding them to the soil, and tumbling the soil mixture for at least 24 h. After 2, 6, and 9 days, the radioactivity in the earthworms was measured as described above. Three worms were removed from each of triplicate containers for each data point, except for the data point for the Chelsea depuration experiment after nine days for which the worms in two of the containers unexpectedly died and only one replicate was used. Modeling. Data from uptake experiments was fit to the following first-order accumulation rate model given in eq 1 using nonlinear curve fitting (SAS Institute). Ct )

Csks (1 - e-ket) ke

(1)

In this equation, Ct is the concentration of the compound in the organism at time t (dpm g-1 dry weight), Cs is the initial concentration in the soil (dpm g-1 dry weight), ks is the uptake coefficient of the compound from the soil (g dry soil g-1 dry organism d-1), ke is the elimination rate constant of the compound by earthworms in soil (d-1), and t is the time (days). This model assumes relatively constant bioavailability of pyrene, limited biotransformation, that uptake and depuration mass transfer processes involved can be reasonably described by first-order uptake and depuration rate models, and a simple exponential decay between the initial and equilibrium bioaccumulation factors. A first-order exponential decay model was fit to the depuration data. Experimental details for measuring the sorption of pyrene to SWNTs, MWNTs, and NC soil, and modeling to estimate changes to the aqueous-phase pyrene concentration after nanotube amendment are included in the Supporting Information.

FIGURE 1. Bioaccumulation factors (BAFs) for pyrene in the absence and presence of carbon nanotubes in North Campus soil. “NCS” and “NCS2” indicates uptake in pyrene-only soil, “Pyr+SWNT” indicates uptake in SWNT-spiked soils, ”Pyr+MWNT” indicates uptake in MWNT-spiked soils, “SWNTPyr” indicates preadsorption of pyrene to SWNTs, “MWNTPyr” indicates preadsorption of pyrene to MWNTs, and “0.3” or “3.0” indicate spiking the soils with 0.3 or 3.0 mg nanotubes per gram soil. Mean and standard deviation values were calculated from triplicate samples.

Results and Discussion Pyrene concentrations in soil measured by biological oxidation and liquid scintillation counting after 28d of exposure with earthworms ranged from 99-102% of the initial radioactivity at the beginning of the accumulation experiments. Differences observed using HPLC analysis of pyrene in the NC soil before and after the 28 day exposures were lower than 1.7%. These results indicate minimal losses due to biodegradation, biotransformation, or volatilization during the course of the experiment. The lack of pyrene biotransformation accords with earlier studies (28, 31) and supports one of the assumptions of our model. Pyrene Bioaccumulation. Lipid concentrations did not vary among earthworms exposed to pyrene-only spiked soil and to soils amended separately with pyrene and 3.0 mg /g of SWNTs or MWNTs, and lipid concentrations did not change during a set of time points covering the duration of the experiment. The average lipid value measured was 2.0 ( 0.3% (n ) 36). While dry earthworm masses decreased on average only from 14 to 28 days for the Chelsea soils, there was not a significant mass change on average for the earthworms exposed to NC soil. The addition of 3.0 mg/g of CNTs did not appear to impact the earthworms’ lipid concentrations or masses, a result that accords with previous results obtained for earthworms in soils spiked with doublewalled carbon nanotubes (32). It is important to note though that the initial earthworm masses of the three adult worms

FIGURE 2. Bioaccumulation factors (BAFs) for pyrene in the absence and presence of carbon nanotubes in Chelsea soil. “Chelsea” indicates uptake in pyrene-only soil, “Pyr+SWNT” indicates uptake in SWNT-spiked soils, ”Pyr+MWNT” indicates uptake in MWNT-spiked soils, and “0.3” or “3.0” indicate spiking the soils with 0.3 or 3.0 mg nanotubes per gram soil. Mean and standard deviation values were calculated from triplicate samples. in our experiments spanned a fairly large range from 1.2 to 2 g, and this experimental approach was not specifically designed to test for chronic effects. Biota-soil accumulation factors (BSAF) can be estimated from the BAF values in Figures 1 and 2 using the provided organic carbon and lipid content values. BAF values after 28 days of exposure were almost twice as high for the NC soil as for the Chelsea soil even though the organic carbon fraction of the Chelsea soil was only 14% larger (see Figures 1 and 2). This difference in BAF values between the NC and Chelsea soils cannot be explained by VOL. 43, NO. 11, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Uptake Coefficients (ks) and Elimination Rate Constants (ke)a

NC soil NCS NCS2 Pyr+SWNT 0.3 Pyr+MWNT 0.3 SWNTPyr 0.3 MWNTPyr 0.3 Pyr+SWNT 3.0 Pyr+MWNT 3.0 SWNTPyr 3.0 MWNTPyr 3.0 Chelsea Soil Chelsea Pyr+SWNT 0.3 Pyr+MWNT 0.3 Pyr+SWNT 3.0 Pyr+MWNT 3.0

ks

ks 95% confidence interval

ke (uptake)

ke 95% confidence interval

ke (depuration)

R2

3.1 ( 0.7 2.8 ( 0.3 2.2 ( 0.4 2.6 ( 0.6 3.5 ( 0.5 4.5 ( 1.0 0.83 ( 0.1 0.75 ( 0.1 1.0 ( 0.1 0.73 ( 0.1

1.6, 4.6 2.1, 3.4 1.4, 3.0 1.6, 3.9 2.4, 4.7 2.3, 6.6 0.57, 1.1 0.49, 1.0 0.81, 1.2 0.51, 0.95

0.45 ( 0.11 0.46 ( 0.05 0.31 ( 0.06 0.39 ( 0.10 0.50 ( 0.08 0.61 ( 0.14 0.24 ( 0.04 0.23 ( 0.04 0.29 ( 0.03 0.22 ( 0.04

0.21, 0.68 0.34, 0.58 0.18, 0.44 0.17, 0.60 0.33, 0.66 0.30, 0.91 0.15, 0.33 0.14, 0.32 0.24, 0.35 0.14, 0.30

0.27 0.17 0.26 0.30 0.26 0.30 0.23 0.29 0.23 0.29

0.94 0.98 0.98 0.94 0.98 0.94 0.93 0.96 0.93 0.96

2.3 ( 0.3 2.3 ( 0.4 1.8 ( 0.2 0.86 ( 0.16 0.70 ( 0.10

1.7, 2.9 1.3, 3.3 1.3, 2.3 0.50, 1.2 0.48, 0.92

0.56 ( 0.07 0.54 ( 0.11 0.45 ( 0.06 0.24 ( 0.05 0.21 ( 0.03

0.41, 0.70 0.30, 0.78 0.32, 0.58 0.12, 0.36 0.13, 0.29

0.16 0.15 0.15 0.18 0.22

0.82 0.84 0.74 0.80 0.87

a “NCS” and “NCS2” indicates uptake or depuration in NC soil without nanotubes, “Chelsea” indicates uptake or depuration in Chelsea soil without nanotubes, “Pyr+SWNT” indicates uptake in SWNT-spiked soils, ”Pyr+MWNT” indicates uptake in MWNT-spiked soils, “SWNTPyr” indicates preadsorption of pyrene to SWNTs, “MWNTPyr” indicates preadsorption of pyrene to MWNTs, and “0.3” or “3.0” indicate spiking with 0.3 or 3.0 mg nanotubes per gram of soil. ke values determined from depuration experiments for the NC soils do not directly relate to the ke and ks values on that row; the ke value of 0.17 was determined using data shown in Figure 3b while the value of 0.27 was determined from that shown in Figure 3c. R2 values are for the depuration data.

the differences in their respective hard carbon concentrations. Rather, differences in the BAF values more likely result from Chelsea organic carbon having an older diagenetic age and thus a “harder” chemical structure (21), but diagenetic ages were not determined in this study. The data generally fit the assumption for the model. There was minimal biotransformation or change in the earthworm lipid concentrations, and bioavailability typically appeared to remain constant from 14 to 28 days, as indicated by the similar BAF values. For one treatment though, 0.3 mg/g of MWNTs spiked separately from pyrene to NC soil, there was a statistically significant decrease in the BAF values from 14 to 28 days (t test, R < 0.05). This result is not readily explainable by changes in the earthworm masses and thus suggests a decrease in pyrene bioavailable over time. Fitting an additional parameter to our model is possible, but the assumption of relatively constant pyrene availability appears reasonable for all other treatments. Nanotube amendments had a concentration-dependent effect on the BAF values. For both the NC and the Chelsea soils, the addition of 0.3 mg/g of SWNTs or MWNTs did not have a significant effect on the bioaccumulation factors, whereas 3.0 mg/g CNTs did have an impact. Given that the sorption capacities of the nanotubes were approximately 103 greater than those for NC soil (see Supporting Information Figure S1 and Table S1), it was expected that preadsorbing the pyrene to the nanotubes would lead to substantially decreased accumulation. In experiments conducted with NC soil, however, preadsorption of pyrene to the nanotubes or the direct addition of nanotubes to the soils after pyrene spiking did not affect the BAF values for nanotube additions of 0.3 or 3.0 mg/g (see Figure 1 and Table 1). This suggests that nanotube-associated pyrene was redistributed among the soil, nanotube, and pore water sufficiently rapidly that it did not affect the accumulation rates. Otherwise, lower BAF values would be expected given the greater sorption coefficients for pyrene adsorbed to the nanotubes compared to that sorbed to soil organic carbon. This apparently rapid redistribution is similar to the 5 day equilibration period for nanotube desorption to water described in a previous study (18). The addition of 3.0 mg nanotubes per gram of soil significantly decreased pyrene uptake for both Chelsea and 4184

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NC soils. As shown in Table 1, nanotube addition of 3.0 mg/ g, but not 0.3 mg/g, caused a statistically significant decrease in uptake coefficients for both soils. There was also a substantial decrease in BAF values with a decline of approximately 25 and 50% for the Chelsea and NC soils, respectively. Although the addition of the nanotubes increased the fraction of hard carbon by roughly an order of magnitude for both soils, the apparent stronger sorption capacity of the organic carbon in the Chelsea soil caused this addition to have a lower impact on pyrene accumulation. Sorption isotherms were determined to compare the BAF values obtained experimentally with predicted changes in soil pore water concentrations. MWNTs had a higher sorption capacity compared to SWNTs and both were approximately 103 higher than NC soil (see Supporting Information Table S1). The cause of this difference in sorption capacity between the two types of nanotubes is unclear, but given the much higher surface area of the SWNTs (763 m2/g) compared to the MWNTs (118 m2/g), cannot be explained by the surface areas. According to the equilibrium modeling, the addition of 0.3 mg/g and 3.0 mg/g SWNTs should decrease the aqueous phase pyrene compared to the pyrene-only treatment by factors of 1.3 and 3.7, respectively, while those same concentrations of MWNTs would lead to respective decreases by factors of 1.4 and 5.3. The large difference between the predicted decreases in bioavailability and measured changes in BAF values was likely not a result of changes in the earthworm lipid concentration or dry masses. Instead, it is expected that equilibrium between pyrene and the soils and nanotubes was not complete. It is well-known that sequestration for nonpolar compounds can take months to years to come to true equilibrium (22). Additionally, the rapidly desorbing contaminant fraction from sediments, measured using a 6 h Tenax extraction, can be used to predict HOC bioavailability for a variety of aquatic organisms (33). Given that nanotubes are known to fully desorb pyrene relatively quickly (18), it is possible that their presence may not decrease HOC accumulation to the extent that would be expected based on their high sorption coefficients. It is also likely that CNTs will undergo extensive sorption interactions with soils after nanotube spiking, a process which may decrease the number and quality of pyrene sorption sites on the nanotubes and the soil. Interactions of dissolved organic matter with

FIGURE 3. Depuration of pyrene in Chelsea soil (a) and NC soil (b and c) with and without carbon nanotubes. “NCS” indicates North Campus soil, “MWNT” indicates multiwalled carbon nanotubes, “SWNT” indicates single-walled carbon nanotubes, “0.3” indicates a nanotube concentration of 0.3 mg/g, and “3.0” indicates a nanotube concentration of 3.0 mg/g. Mean and standard deviation values were calculated from triplicate samples. nanotubes has previously been shown to decrease their sorption capacity for PAHs to varying extents (17). Thus, calculations based on equilibrium modeling provide the

maximum decrease in bioaccumulation expected, but many additional factors such as those described above may cause the actual decreases to be much lower. For each of these experiments, the presence of carbon nanotubes, possibly loaded with pyrene, remaining in the guts of the organisms after the 24 h purging interval is not expected to have affected the bioaccumulation factor values. Bioaccumulation factors for radioactively labeled nanotubes after 14 days of exposure remaining in the organisms’ guts after the gut purging interval were previously shown not to exceed 0.03 (14), while pyrene BAF values measured here were typically 2 orders of magnitude larger than that value. Overall, it appears that the presence of nanotubes in soils influences pyrene bioaccumulation by earthworms similarly to that typical for black carbons. This impact is similar to that observed for SWNT addition to sediment and uptake by S. benedicti, but not by A. tenuiremis (16). At the estimated average nanotube soil concentration of 0.01 µg kg-1 (5), the presence of nanotubes in the environment is not expected to influence the fate and distribution of HOCs. It is likely, however, that there will be “hot spots” with elevated nanotube concentrations and in these locations, the presence of nanotubes could have a significant impact. Given the relatively high costs of CNTs and the current lack of comprehensive data regarding their toxicity and fate in environmental systems, the findings described here should not be construed as endorsing the use of CNTs as soil amendments for reducing HOC uptake. Other more suitable materials have been developed by our group and others for such purposes (34-36). Pyrene Depuration. The presence of carbon nanotubes in soils at concentrations of 3.0 mg/g but not 0.3 mg/g hastened pyrene elimination (Figure 3) and increased the ke values (see Table 1). Given the higher sorption coefficients of the nanotubes (Supporting Information Table S1), this result likely stems from the higher sorption capacity of the soil with the addition of 3.0 mg/g nanotubes, which is expected to enhance sorption interactions between the pyrene eliminated from the worm and the soil that passes through the earthworm guts. This result agrees with previous findings for the amphipod Diporeia spp. which excreted polycyclic aromatic hydrocarbons more rapidly in the presence of solid substrates, and the degree of increased depuration rate was found to direct relate to the sorption capacity of the substrates (37). The addition of 3.0 mg/g of MWNTs increased the elimination rate more quickly than the addition of 3.0 mg/g SWNTs, a result attributed to the higher sorption capacity of the MWNTs. The ke values determined here are close to a pyrene value previously determined of roughly 0.2 d-1 using OECD soils (27). It was unexpected that two different depuration experiments in unamended NC soil yielded substantially different ke values. This difference likely stems from the fact that earthworms lost or gained mass on average for the data shown in Figure 3b and c, respectively. The cause of these mass changes is unclear, and values obtained from these two experiments should not be directly compared. The ke values determined from fitting the two-compartment model to the data from the uptake experiments were generally close to or slightly higher than those determined during the depuration experiments. This result indicates that the accumulation modeling worked sufficiently well for making comparisons among the effects of the various CNT amendments and spiking procedures. Overall, CNTs act to sorb nonpolar contaminants, and as such they will enhance elimination when contaminant concentrations on the CNTs are below the equilibrium condition between the organisms and the CNTs, and restrict uptake when the condition is reversed. Thus, manufactured VOL. 43, NO. 11, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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CNTs will be expected to act largely as a new source of black carbon in soils.

Acknowledgments We thank Kyle Roebuck, Hayley Smithkort, and Jesse Tzeng for their experimental assistance. We thank Marja Noponen and Dr. Jussi Kukkonen for making the hard carbon measurements and Drs. Xiaoyin Chen and Johannes Schwank for assistance with surface area measurements. This work was supported by awards from the University of Michigan Graham Environmental Sustainability Institute and from U.S. EPA grant RD833321. This is Great Lakes Environmental Research Laboratory contribution 1513.

Note Added after ASAP Publication There was an error in Table S1 of the Supporting Information file in the version of this paper published ASAP April 27, 2009; the corrected version published ASAP April 30, 2009.

Supporting Information Available Detailed experimental methods for earthworm culturing, sorption of pyrene to nanotubes and NC soil, and equilibrium modeling to assess the impact of nanotube additions on pyrene availability; sorption isotherms of pyrene with nanotubes and NC soil; and a table summarizing the sorption coefficients. This material is available free of charge via the Internet at http://pubs.acs.org.

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