Article pubs.acs.org/est
Unique Toxicological Behavior from Single-Wall Carbon Nanotubes Separated via Selective Adsorption on Hydrogels Justin G. Clar,*,†,§ Sarah A. Gustitus,† Sejin Youn,†,∥ Carlos A. Silvera Batista,‡,⊥ Kirk. J. Ziegler,‡ and Jean Claude J. Bonzongo*,† †
Engineering School of Sustainable Infrastructure and Environment, Dept. of Environmental Engineering Sciences, University of Florida, Gainesville, Florida 32611, United States ‡ Department of Chemical Engineering, University of Florida, Gainesville, Florida 32611, United States S Supporting Information *
ABSTRACT: Over the past decade, extensive research has been completed on the potential threats of single-wall carbon nanotubes (SWCNTs) to living organisms upon release to aquatic systems. However, these studies have focused primarily on the link between adverse biological effects in exposed test organisms on the length, diameter, and metallic impurity content of SWCNTs. In contrast, few studies have focused on the bioeffects of the different SWCNTs in the as-produced mixture, which contain both metallic (m-SWCNT) and semiconducting (s-SWCNT) species. Using selective adsorption onto hydrogels, high purity m-SWCNT and s-SWCNT fractions were produced and their biological impacts determined in dose−response studies with Pseudokirchneriella subcapitata as test organism. The results show significant differences in the biological responses of P. subcapitata exposed to high purity m- and s-SWCNT fractions. Contrary to the biological response observed using SWCNTs separated by density gradient ultracentrifugation, it is found that the high-pressure CO conversion (HiPco) s-SWCNT fraction separated by selective adsorption causes increased biological impact. These findings suggest that s-SWCNTs are the primary factor driving the adverse biological responses observed from P. subcapitata cells exposed to our as-produced suspensions. Finally, the toxicity of the s-SWCNT fraction is mitigated by increasing the concentration of biocompatible surfactant in the suspensions, likely altering the nature of surfactant coverage along SWCNT sidewalls, thereby reducing potential physical interaction with algal cells. These findings highlight the need to couple sample processing and toxicity response studies.
■
INTRODUCTION Since their discovery, single-wall carbon nanotubes (SWCNTs) have been extensively researched by both scientists and engineers due to their unique properties (e.g., strength, high adsorption capacity, controllable conductivity, etc.) and their potential to be used in both industrial applications and consumer products.1 However, a common concern for all emerging technologies is whether increased production and subsequent inclusion in consumer products would likely lead to increased environmental and human exposure. Upon introduction to waste streams, SWCNTs could reach natural systems and potentially interact with ecological receptors and impact the biosphere. In fact, special attention must be paid to aquatic systems since they function as environmental sinks by integrating pollutants from atmospheric deposition, terrestrial surface runoffs, and groundwater discharges.2,3 Over the past decade, significant research has been conducted to evaluate the potential toxicity of SWCNTs.4−10 However, published results on the toxic effects of SWCNTs have been rather controversial since studies using the same test © 2015 American Chemical Society
organism point to either severe acute toxicity or little to no toxic effect.11−13 With no standardized methods on how to evaluate the potential consequences of an organism’s exposure to the varying types of engineered nanomaterials, different research groups are left to determine which traditional tests and additional modifications best fit their needs. Assessing the potential toxicity of SWCNTs is further complicated when one considers the wide variety of surface alterations used to aid SWCNT dispersion in aqueous media.14 For example, researchers have used a large number of amphiphilic dispersants for SWCNT stabilization, including traditional surfactants like sodium dodecyl sulfate (SDS), bile salts, such as sodium cholate (SC), or nonionic surfactants and polymers, such as pluronic acids. While the adverse biological impacts exhibited by model organisms exposed to SWCNTs could come from the Received: Revised: Accepted: Published: 3913
December 5, 2014 February 18, 2015 February 24, 2015 February 24, 2015 DOI: 10.1021/es505925m Environ. Sci. Technol. 2015, 49, 3913−3921
Article
Environmental Science & Technology
The supernatants containing well-suspended SWCNTs were separated from the pellets and characterized using the spectroscopic methods described below. Additional analysis of the dispersion is included in the Supporting Information (See Figure S1). SWCNT Separation into m-SWCNTs and s-SWCNTs. All column experiments were completed using a method adapted from Kataura and co-workers,19,20 as described in our previous publications.21−23 Glass columns purchased from Bio-Rad with an inner diameter of 2.5 cm were used in all studies. The columns were connected to a Bio-Rad Econo gradient pump using a flow adaptor. Each column was packed with approximately 40 mL of the desired medium. The column length was approximately 8 cm in height, depending on the rigidity of the medium used in separation. After packing, columns were stabilized with at least 4 column volumes (CV), ∼160 mL, of Nanopure water to remove the preservative and potential contaminants not covalently bonded to the matrix. The medium was then flushed with 4 CV of 1 wt % SDS to equilibrate the column prior to separation. SWCNT suspensions were then injected into the column at a volume of 8 mL (20% CV). After the injection of the SDS-SWCNT suspension into the column, 1 CV of 1 wt % SDS was passed through the column at a constant flow rate of 1 mL/min. Using this eluent, a highly enriched m-SWCNT fraction is eluted from the column and collected. The remaining adsorbed s-SWCNTs were eluted from the column by changing the eluent to a 2 wt % SC solution. This portion of collected nanotubes is highly enriched in the s-SWCNT species. After each run, the column was restabilized with at least 3 CV of 1 wt % SDS in preparation for subsequent separations. All collected fractions containing either m- or s-SWCNTs were combined and concentrated using the Amicon ultrafiltration cell described below to produce fractions with the appropriate concentration and surfactant identity for toxicity studies. Surfactant Exchange on SWCNT Surfaces. To produce large quantities of separated SWCNT fractions using hydrogelpacked columns, nanotubes had to be suspended in SDS surfactant.20,25 However, our previous study showed that SDS alone is highly toxic to several aquatic organisms, including P. subcapitata, the freshwater algae used in this study.3 Therefore, following the separation of the SWCNT fractions, an extra step was used to remove the toxic SDS surfactant from both m- and s-SWCNT surfaces/suspensions and replace it with sodium cholate (SC), which is less toxic to P. subcapitata. Surfactant exchange was achieved using an Amicon 8200 ultrafiltration cell equipped with a regenerated cellulose membrane (MW cutoff 30 kDa). This system allows for SDS molecules to pass through the membrane under a pressure head of ultrahigh purity nitrogen gas. After an initial concentration step, the suspension was mixed with approximately 150 mL of a 1 wt % SC solution and stirred for at least 20 min to allow for equilibration. Introduction of a 1 wt % SC solution to the previously concentrated SWCNT suspension creates a concentration gradient that results in an energetic driving force to remove SDS from the SWCNT sidewalls in order to equilibrate with the surrounding solution. Simultaneously, the SC in solution was driven to equilibrate with the SWCNT sidewalls. Several ultrafiltration passes eliminated SDS from the SWCNT sidewalls, resulting ultimately in SWCNTs well-dispersed in a 1 wt % SC solution as evident by the strong fluorescence intensity and lack of peak broadening (See Supporting Information Figure S3). This method of sample concentration
nanotubes themselves, published data do suggest that many dispersants commonly used in the stabilization of SWCNTs are toxic to several organisms used in bioassays.3 Therefore, any attempt to elucidate the toxicity of SWCNTs must first evaluate the potential for the dispersants to contribute to the toxic response. In addition to the different dispersants used in the stabilization process, variability in physical SWCNT parameters, such as length, diameter, and concentration or identity of residual metal catalysts, adds another level of complexity to the observed toxic response. A summary of these findings has been extensively reviewed elsewhere.15−17 In contrast, little research has been conducted on the role of SWCNT electronic type, that is, whether the potential toxic response is associated with the metallic (m-) or semiconducting (s-) nature of the nanotubes. To our knowledge, the toxicity of m- and sSWCNT species has only been investigated by one study, which used Escherichia coli as the test organism.18 This study used SWCNTs separated by density gradient ultracentrifugation (DGU) and found that m-SWCNTs were significantly more toxic than s-SWCNTs. The lack of studies has been hampered by the inability to obtain significant quantities of high-purity fractions needed for toxicity analysis. One widely researched strategy for the large-scale separation of SWCNTs into m- and s-SWCNT fraction utilizes the selective adsorption of SDS-coated SWCNTs onto hydrogel surfaces.19−22 This work uses a comparative approach to investigate the adverse biological effects of the as-prepared SWCNT mixture to that of SWCNT suspensions fractionated by their electronic character through selective adsorption onto hydrogels using the freshwater green algae, Pseudokirchneriella subcapitata, as the test organism. To our knowledge, this is the first study that combines downstream toxicity screening after separation of SWCNTs into s- and m- fractions using selective adsorption onto hydrogels.
■
MATERIALS AND METHODS Materials. Nanopure water (18 mΩ) was used in all experiments. High-pressure CO conversion (HiPco) SWCNTs were obtained from Rice University (HPR 164.1) and used as received. The surfactants, sodium dodecyl sulfate (SDS) and sodium cholate (SC), were purchased from Sigma-Aldrich. Aqueous suspensions of type-separated SWCNTs produced through Arc-discharge were supplied by NanoIntegris and purified by ultrafiltration as described below. Finally, two types of hydrogels (Sepharose 6 FF and Sephacryl 200 HR), which are manufactured by GE, were used for the separation of SDSSWCNT suspensions into m- and s-SWCNTs fractions. These gels were selected for their benefits of both high-throughput and high-purity separations.23 Preparation of the Aqueous Suspensions of SWCNTs. Aqueous suspensions were prepared as described previously.21,22,24 Briefly, approximately 40 mg of raw SWCNT powder were added to 100 mL of a 1 wt % desired aqueous surfactant solution. The suspension was homogenized at 8 000 rpm (IKA T-25 Ultra-Turrax) for 30 min. Following the mixing step, the suspension was ultrasonicated in a cup-horn for 10 min (120 W, Misonix S3000). This ultrasonication step was repeated three times to ensure a high degree of SWCNT dispersion. Next, SWCNT bundles, amorphous carbon, and residual iron catalysts (4 mM (Figure 1b). Accordingly, the presence of SC in the growth 3915
DOI: 10.1021/es505925m Environ. Sci. Technol. 2015, 49, 3913−3921
Article
Environmental Science & Technology
Figure 2. Characterization data of the initial SWCNT suspension and type-separated SWCNTs used in this study. The initial suspension was prepared in 1 wt % SDS aqueous solution, resulting in a dark black color. Isolated m-SWCNT fractions are red in color and show strong absorbance in the M11 optical transitions and a lack of distinct fluorescence peaks compared to the original suspension. Isolated s-SWCNTs are green with strong absorbance increases in the S11 and S22 regions and increased fluorescence intensity when compared to the initial suspension.
medium of P. subcapitata at concentrations 98% m-SWCNTs. Alternatively, the s-SWCNT fraction shows large increases in fluorescence intensity coupled with absorbance increases in the S11 and S22 ranges as well as decreases in the M11 range that collectively indicate a high level of purity, which is estimated to be greater than 95%. 3916
DOI: 10.1021/es505925m Environ. Sci. Technol. 2015, 49, 3913−3921
Article
Environmental Science & Technology Biological Responses of P. subcapitata to SWCNT Mixtures and m-SWCNT and s-SWCNT Fractions. Following the determination of the adequate background concentrations of SC for toxicity studies using P. subcapitata as the organism, bioassays were first conducted to determine the effect of as-prepared SWCNT suspensions (i.e., mixtures containing both m- and s-SWCNT fractions) on P. subcapitata. Figure 3 shows a decrease in algal growth with increasing
Figure 4. Effect of increasing concentrations of m- and s-SWCNT species on the growth of P. subcapitata in a standard 96 h chronic algal assay. The final concentration of sodium cholate (SC) in all treatments was identical and held under the threshold concentration of 4 mM. Changes in biomass measured by chlorophyll-a. Doses of 0.75 ppm sSWCNTs resulted in complete growth inhibition. The dotted horizontal line represents the growth of P. subcapitata in the control culture media containing only SC and no SWCNTs. Standard error bars are calculated based on four replicates. (*) Indicates a statistical difference from the control (α = 0.05).
media. Increasing the concentration of s-SWCNTs to 0.75 mg/ L resulted in total algal growth inhibition. These results suggest that the observed toxicity of the nonseparated and SCsuspended SWCNTs seen in Figure 3 can be attributed primarily to the s-SWCNT species in the mixture. Mitigation of s-SWCNT Toxicity. Previous studies have indicated that the concentration of a nontoxic surfactant can have a significant impact on the observed biological response.7,24 To investigate the effect of SC concentrations to the observed s-SWCNT toxicity, P. subcapitata cultures were exposed to identical concentrations of s-SWCNTs (0.2 mg/L) while increasing the SC concentration in growth culture media. The results presented in Figure 5 show significant increases in algal viability as the SC concentration is increased. Increasing the background concentrations to 2 mM of SC effectively eliminates the toxic response observed in Figure 4. It is important to note that optical spectroscopy shows no drastic changes to dispersion quality over this SC concentration range (not shown). This mitigation of toxicity is evidence that dispersant concentration and its effects on SWCNT coverage and stabilization have a major impact on the potential for SWCNTs to cause toxicity. Biological Response of P. subcapitata to m- and sSWCNT Fractions Produced by Density Gradient Ultracentrifugation (DGU). As previously discussed, only one prior study has systematically examined the biological response of test organisms to electronically sorted SWCNTs.18 In the study, SWCNTs produced through Arc-discharge synthesis and separated via DGU were exposed to E. coli. To ensure that our biological response results were not organism dependent, NanoIntegris SWCNTs, similar to those used in the previous study, were used in P. subcapitata exposure studies. The results of this analysis are presented in Figure 6. At an identical dose of 0.25 mg/L, the NanoIntegris m-SWCNT fraction separated via DGU completely inhibit the growth of P. subcapitata when compared to control growth media. Alternatively, the identical concentration of s-SWCNTs separated via DGU only results in
Figure 3. Effect of increasing concentrations of nonseparated SWCNTs (i.e., mixture of m- and s-SWCNT species) on the growth of P. subcapitata in a standard 96 h chronic algal assay as measured by chlorophyll-a. The final concentration of sodium cholate (SC) in all treatments was adjusted to 1.0 mM. The dotted horizontal line represents the growth of P. subcapitata in the control culture media containing SC but no SWCNTs added. Standard error bars are calculated based on four replicates. (*) Indicates a statistical difference from the control (α = 0.05).
SWCNT concentrations. At SWCNT concentrations ≥0.25 mg/L, a significant adverse biological impact was observed, reaching a 50% algal growth inhibition in culture media containing a final concentration of 0.75 mg/L of SWCNTs. In these experiments, the background SC concentration in all treatments was 1 mM, which is well below the previously identified threshold concentration for normal growth of P. subcapitata in the presence of SC. Overall, these results are consistent with previously published data using P. subcapitata and SWCNTs dispersed in either SC or other nontoxic surfactants.7,24 The results of dose−response assays using the purified SWCNT fractions are presented in Figure 4. Prior to these dose−response studies, both m- and s-SWCNT fractions were washed by identical procedures (see Materials and Methods). At a concentration ≤0.5 mg/L, m-SWCNTs do not induce a statistically significant loss in algal viability when compared to control samples. In fact, the m-SWCNT fraction at a dose of 0.5 mg/L induces less biological impact compared to asprepared suspensions at the same concentration. In contrast, exposure to s-SWCNTs resulted in significant adverse impacts on algal growth, regardless of the concentration tested. At the lowest tested concentration of 0.25 mg/L s-SWCNTs, the algal growth was only ∼46% of what was observed in control culture 3917
DOI: 10.1021/es505925m Environ. Sci. Technol. 2015, 49, 3913−3921
Article
Environmental Science & Technology
Specifically, the purified s-SWCNT fraction produced via selective adsorption resulted in a significant growth inhibition of the green algae as compared to cell growth recorded from control media (i.e., no s-SWCNTs added). In contrast, the purified m-SWCNT fraction produced by selective adsorption resulted in a less pronounced but noticeable adverse biological impact only to cells exposed to the highest concentrations. Interestingly, the results presented in Figure 4 show toxicity trends that are the opposite of those previously reported by Vecitis and co-workers when exposing E. coli to Arc-discharge SWCNTs separated by DGU (NanoIntegris).18 They linked the adverse biological impact on E. coli to m-SWCNTs rather than the s-SWCNTS found in this study.18 While the biochemical differences between algal and bacterial cells may be a likely candidate for the varied observations in biological response, the data in Figure 6 confirmed the response trend previously reported by Vecitis and co-workers.18 This similarity using different test organisms is compelling evidence that the variability in biological response are not driven by the type of test organism, but rather some inherent differences in the produced SWCNT fractions. A closer look at the potential impacts of the production/ speciation methods on the inherent properties of SWCNTs requires a direct comparison of the same SWCNT sample separated by different methods. However, major roadblocks exist in separating Arc-discharge SWCNTs using selective adsorption onto hydrogels. To our knowledge, only one published study has type-separated high-purity fractions of SWCNTs of larger average diameter (>1 nm) than the HiPco SWCNTs used here through selective adsorption column systems. A critical step in their study was preprocessing the SWCNT suspension through DGU.31 Although good separation was achieved, the throughput is low due to the extensive preprocessing. This low throughput makes it very difficult to obtain sufficient material to conduct adequate toxicity assays. Collected SWCNTs samples would require additional post separation processing to remove the density gradient medium and cosurfactant solution, reducing the yield of separated nanotubes further. Importantly, Figure 1a indicates that any residual SDS on the separated nanotubes can have a substantial toxic effect to test organisms. Vecitis and co-workers removed these impurities using combinations of precipitation in methanol, vacuum filtration, and acid washing prior to conducting their dose−response studies.18 In similar fashion, it is equally difficult to obtain enough separated materials using HiPco SWCNTs through the DGU separation method. While the needed comparison remains elusive, our results and those published by Vecitis and co-workers18 indicate that important differences could exist between the biological responses of organisms when exposed to SWCNTs produced by different manufacturing methods. On a positive note, one could take advantage of these two production/separation techniques to produce nontoxic fractions of either m- or s-SWCNTs. It is interesting to note that differences in the length distribution of type-separated SWCNTs are unlikely to be the driver of the observed difference in toxicity response. Vecitis and co-workers report similar length distributions of both their m- and s-SWCNT fractions.18 Critically, the lengths reported are indicated as heavily aggregated SWCNTs due to the substantial preprocessing. Previous analysis has also shown that imaging strategies to determine SWCNT length are unreliable; it was found that optical spectroscopy was a reliable characterization tool.32 With regards to the SWCNT separated
Figure 5. Effect of increasing concentrations of sodium cholate (SC) at a fixed concentration of s-SWCNTs on the growth of P. subcapitata in a standard 96 h chronic algal assay. The final concentration of sSWCNTs in all treatments was 0.2 mg/L. Changes in biomass measured by chlorophyll-a. The dotted horizontal line represents the growth of P. subcapitata in the control culture media containing only SC and no SWCNTs. Standard error bars are calculated based on four replicates. (*) Indicates a statistical difference from the control (α = 0.05).
Figure 6. Toxic effect of Arc-discharge SWCNTs separated by DGU from NanoIntegris on the growth of P. subcapitata in standard 96 h chronic algal assay. The final concentration of SWCNTs in both treatments was 0.25 mg/L. The concentration of sodium cholate (SC) was constant at 1 mM in all treatments. The dotted horizontal line represents the growth of P. subcapitata in a control culture media containing only SC and no SWCNTs. Columns show changes in the algal biomass measured by chlorophyll-a. Culture media treated with m-SWCNT resulted in total algal growth inhibition, while those containing s-SWCNTs were more favorable to algal growth with an inhibition
