Activity Inhibition on Municipal Activated Sludge by Single-Walled Carbon Nanotubes

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1 Activity Inhibition on Municipal Activated Sludge by Single-Walled Carbon Nanotubes Alex Parise 1 and Jackie Zhang 2 Department of Civil and Environmental Engineering University of Massachusetts Lowell Lowell, MA Abstract: Nanotechnology is a rapidly expanding field of study that shows promising use in sectors such as medical, industrial, and residential applications. As one of the many nanomaterials being developed, carbon nanotubes have many unique physical and electrical properties that make them ideal candidates for plastics reinforcement, electrical components, and drug delivery systems. However, as research and development push these materials into everyday use, the risk of environmental contamination grows more and more real. This study aimed to compare the possible reduction in respiratory activity of the activated sludge used in a typical wastewater treatment plant through contamination by four types of singlewalled carbon nanotubes: short, functionalized short, long, and functionalized long. Based on the effective concentration (EC50) values obtained, we found that functionalized single-walled nanotubes resulted in a higher microbial inhibition than the non-functionalized nanotubes and long single-walled nanotubes gave a higher inhibition than their short counterparts. SEM imaging indicates that the long nanotubes dispersed more favorably after sonication than the short variety, which likely had an impact on the experimental results. Keywords: CNTs, activated sludge, respiratory activity, respiratory inhibition Introduction Discovered in the early 1990 s, carbon nanotubes (CNTs) are essentially single sheets of graphite rolled into tubes (Qian, 2002) with diameters no larger than low nanometers (Bastus, 2008). Many companies are attempting to mass produce different varieties of nanotubes for industrial applications (Baughman, 2002). As industrial use rises, the probability of a spill involving these new nanomaterials rises as well, leading to the question of what will happen. Carbon nanotubes are known to have a negative impact on cell viability (Pogodin, 2010). Bottini et al. (2006) reported that oxidized CNTs were more toxic than the un-oxidized, pristine CNTs. However, most studies were conducted in a pure-culture microbial system, only limited information is available for a mixed-cultured microbial system. In this study, we focused on examining the impact of the functionalization of CNTs on the microbial activity of activated sludge used in wastewater treatment systems.

2 Experimental Setup Materials and Methods One day prior to experimentation, liters of activated sludge was collected from the Lowell Regional Wastewater Utility in Lowell Massachusetts. The sample was immediately returned to the laboratory and aerated. An initial chemical oxygen demand (COD) and mixed liquor suspended solids (MLSS) reading was taken following procedures dictated by the Standard Methods for the Examination of Water and Wastewater (American Public Health Association, 1998). The sludge was then decanted and washed three times with tap water and allowed to settle. A second MLSS measurement was taken, and the sludge concentrated to 4000 mg/l. After concentration, a second COD measurement was taken. Using the obtained data, synthetic feed was added to the sample as food, with the volume varying to ensure the food/microorganism ratio was similar to the initial sample. On the day of the experiment, two six-jar beaker tests were run simultaneously to accommodate the eleven beakers needed for the test run. Each beaker was prepared according to Table *, and allowed to run for three hours each. Conditions for each beaker were kept identical by using air flow meters to hold aeration at 1 L/min, and mixed at 90 RPM. The beakers were staggered 15 minutes apart to allow for preparation and dissolved oxygen (DO) readings. At the end of each beaker s three hour run, the DO levels were recorded over a 10 minute span at 30 second intervals using a YSI Model 52CE meter and probe. Nanotube Preparation Four types of single-walled carbon nanotubes were purchased from Cheaptubes, located in Brattleboro, VT. They were long, carboxylic functionalized long, short, and carboxylic functionalized short. (Their characteristics are shown in Table 1). Table 1 Single-walled carbon nanotube characterization Outer Diameter (nm) Length (nm) Specific Surface Area (m^2/g) Purity (%) COOH Functional Content (%) Ash (%) Additional MWNT content (%) Short >407 >90 0 <1.5 5 Functionalized short >407 > <1.5 0 Long >407 >90 0 <1.5 0 Functionalized long >407 > <1.5 0

3 Into a 100 ml beaker, 75 ml of D.I. water and 5 ml of synthetic feed were added to a measured amount of nanotubes (Table 2). Each beaker was stirred by hand using a glass stir rod prior to sonication. A Misonex Sonicator 3000 with cup horn was used to sonicate the samples to achieve better dispersion of the nanotubes. The mixture was sonicated at a dial setting of 7.5 for 2 hours, pulsing on and off for 30 second intervals. Water was pumped through the system to ensure steady temperatures. The setup for the cooling water can be seen in Figure 1. Following sonication, the beakers were placed into a refrigerator for storage. Prior to use in the experiment, the beakers were stirred by hand to eliminate any nanotubes that had caked onto the bottom of the beaker and sonicated for 10 minutes straight at a dial setting of 7.5. Table 2 Composition of samples for sonication Sample Number Nanotubes (g) Synthetic Feed (ml) D.I. water (ml) Resulting concentration in the activated sludge beaker (mg/l) Soundproof box 1:00:00 80 ml Sample beaker Peristaltic pump Cooling water flow 7.5 Misonex Sonicator 3000 To sink 20 L D.I. water Cup-horn sonicator Figure 1. Sonication setup.

4 Respiration Inhibition test Respiratory inhibition of the bacteria by the nanotubes was examined using a slightly modified OPPTS Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals (EPA, 1996). Synthetic feed was used as a food source for the bacteria to ensure the respiration results were not affected by starvation. The composition of the synthetic feed is as follows: 16 g peptone, 11 g meat extract, 3g urea, 0.7 g NaCl, 0.4 g CaCl 2 2 O, 0.2 g MgSO 4 2 O, and 2.8 g K 2 HPO 4 mixed into 1L of D.I. water. This feed was added to the sludge for overnight storage, as well as in each beaker during experimentation. A reference chemical, 3,5-dichlorophenol, was used to ensure the bacteria in the sludge were not already stressed or damaged. Three beakers containing 5, 10, and 25 mg/l of the reference were run along with the samples. The EC50 of the three references must fall within 5-30 mg/l. EC50 being the effective concentration of toxins that produce a 50% inhibition rate. Two controls were run as well, one at the beginning of the experiment and the other at the end. The respiration rates from the two controls must fall within 15% of each other (Equation 1). If either the reference or the controls were out of range, the experimental results were considered invalid. RC1 RC 2 % difference of controls = *100% ( R R 2 ) / 2 C1 C (1) Respiration inhibition values of the activated sludge were determined by comparing the D.O. usage rates of the samples to the two controls, and were calculated by the use of equation 2. 2RS % inhibition of samples = 1 *100% RC1 RC 2 (2) where: R C1 = Respiration rate of control 1 R C2 = Respiration rate of control 2 R S = Respiration rate of the sample

5 From the respiration inhibition values, the potential toxicity for the different nanotubes can be compared by examining the Effective Concentrations that will give the activated sludge a 50% respiration inhibition (EC50 values). SEM imaging A scanning electron microscope (SEM) was used to take before and after images of the sludge sample mixed with carbon nanotubes. The machine used was a JEPL JSM-6390 using a tungsten electrode. Samples were prepared a day in advance for imaging to ensure proper dehydration. One ml of sample was placed on a filter, and washed with 1 ml 2.5% glutaraldehyde and allowed to set in a refrigerator for two hours. Following that, the sample was washed with a 0.1M phosphate buffer, and immersed in increasing concentrations of ethanol to remove any moisture. The dehydrated sample was then stored in a desiccator until use. Prior to imaging, the sample was gold-coated using a Denton Vacuum Desk IV Sputter Coater at 25% power for 2 minutes to provide a better imaging surface. Results and Discussion Inhibition results for all four nanotubes are listed below in Table 3. These EC50 values indicate the concentrations that would produce a 50% reduction in oxygen use by the microorganisms in the activated sludge. Lower EC50 values show that less of the nanotubes are required to produce the inhibitory effect, meaning that smaller EC50 values indicate a higher inhibition. Analysis of this data leads to two conclusions. First, long single-walled carbon nanotubes are more inhibitory than short single-walled carbon nanotubes. Secondly, functionalized carbon nanotubes are more inhibitory than non-functionalized. Table 3 - EC50 results SWCNTs Studied EC50 (mg/l) (1) Functionalized long 1656 (2) Long 2907 (3) Functionalized short 4902 (4) Short 7939

6 A scanning electron microscope (SEM) image was taken of each 300 mg/l nanotube sample after sonication to demonstrate the dispersion of CNTs (see Figures 2, 3 and 4). The images are of the nanotubes having been sonicated in a mixture of D.I. water and the synthetic feed used to provide nutrients to the bacteria. From examining the images, it can be seen that the short single-walled nanotubes did not disperse as well as the long single-walled nanotubes. This conclusion was arrived on by noting the discrete nanotube fibers of the long tubes, as compared to the more aggregated short tubes. The level of dispersion impacts the contact between bacteria and nanotubes, which could have had an effect on the results of the experiment. Figure 2. SEM of Functionalized Long Single-Walled CNTs (300 mg/l)

7 Figure 3. SEM of Long Single-Walled CNTs (300 mg/l) Figure 4. SEM of Short Single-Walled CNTs (300 mg/l) This finding demonstrates that the toxicity of CNTs (exhibited by respiration inhibition) is related to their physical properties; the carboxylic functionalization made SWCNTs more toxic towards the activated sludge. In addition, we observed that the longer SWCNTs were more readily dispersed during sonication, providing more contact between CNTs and the

8 microorganisms in the activated sludge, therefore more respiration inhibition. The hydrophobicity of the CNTs might also play a role in their toxicity. Henriques and Love (2007) reported that the penetration of the toxin into the floc matrix is reduced if the toxin is hydrophobic, suggesting that more hydrophilic toxins (e.g., functionalized SWCNTs) would penetrate deeper into the flocs and impose higher toxicity towards the microorganisms present in the flocs. These findings are important because the environmental impact of carbon nanotube contamination is an under-studied area of research, and could help treatment plant operators and engineers devise contingency plans in the event of a spill of nanotubes into the sewer systems. References Baughman, R., Zakhidov, A., and Heer, W. (2002) Carbon Nanotubes The Route Towards Applications. Science 297, Bastús, N. G., Casals, E., Vázquez-Campos, S. and Puntes, Victor (2008) Reactivity of engineered inorganic nanoparticles and carbon nanostructures in biological media. Nanotoxicology 2, Bottini, M., Bruckner, S., Nika, K., Bottini, N., Bellucci, S., Magrini, A., Bergamaschi, A. and Mustelin, T. (2006) Multi-walled carbon nanotubes induce T lymphocyte apoptosis. Toxicology Letters 160, Henriques, I.D.S. and Love, N. (2007) The role of extracellular polymeric substances in the toxicity response of activated sludge bacteria to chemical toxins. Wat. Res. 41, Luongo, L. and Zhang, X. (2010) Toxicity of carbon nanotubes to the activated sludge process. Journal of Hazardous Materials 178, Pogodin, S., and Baulin, V. (2010) Can a Carbon Nanotube Pierce Through a Phospholipid Bilayer? ACSNano 4, Qian, D., Wagner, G., and Liu, W. (2002) Mechanics of Carbon Nanotubes. Applied Mechanics Reviews. 55, Name of the First Author Mr. Alex Parise is a Master s student in the Department of Civil & Environmental Engineering at the University of Massachusetts Lowell. He received his BS degree in Environmental Engineering from the University of Maine in His research project is funded through the the NSF-supported Center for High-rate Nanomanufacturing at UML. In addition to devoting time and energy on his research project, he has also assisted with several undergraduate courses and laboratory sessions. Name of the Second Author Dr. Xiaoqi (Jackie) Zhang is a Professor in the Department of Civil & Environmental Engineering at the University of Massachusetts Lowell. She has been working on wastewater treatment for over 15 years. Her research interests include biological wastewater treatment, nutrients removal, environmental remediation using innovative nanoparticles and environmental impact assessment of nanomaterials.