We thank Dr. Roger Hopper and the Wyoming Department of Agriculture, Analytical Services Lab for the mass spectroscopy analysis of our samples. We would like to express our gratitude to the University of Wyoming, School of Pharmacy for allowing us to video this protocol in their laboratories. We would also like to thank the Theodore O. and Dorothy S. King Endowed Professorship Agreement for their graduate assistantship (SC), the University of Wyoming for the Graduate Assistantship support (JRS), and the Science Posse (NSF GK-12 Project # 084129) for the teaching fellowship (JRS). We would also like to thank Uranium One for allowing us to obtain samples and assisting us with questions. This work was supported by the School of Energy Resources, University of Wyoming.

All samples were collected at the uranium liquid processing building of a uranium ISR facility in Wyoming.
1. Production Bleed Water (PBW)
<ol>
	<li>Collect two types of water samples from an ISR uranium facility: PBW and reverse osmosis (RO) water. Collect PBW from a monitoring tap after the ion exchange process but before reverse osmosis decontamination. Collect RO samples after the PBW is decontaminated by reverse osmosis treatment. 
	NOTE: Lixiviant is transported in pipelines from multiple well fields to the uranium liquid processing building, where it is collected in a column and prepared for ion exchange. Approximately 1-3% of the lixiviant after ion exchange is removed from the circuit and termed production bleed water (PBW). PBW is re-used in the mining processes or decontaminated/demineralized with RO filtration.</li>
	<li>Collect water samples in high density polyethylene (HDPE) bottles with zero head space according to standard operating procedures for sample collection and analysis of the Wyoming Department of Environmental Quality (WYDEQ) 23.</li>
	<li>Measure temperature and pH on-site and transport samples on ice to keep them cool.</li>
	<li>Store PBW at 4 &#176;C. Keep the PBW solution cool until after the concentrated Eagle&#8217;s minimum essential media (EMEM-10x) is added during media preparation as instructed in the following protocol. &#160; 
	NOTE: PBW is an oxidized solution that will precipitate if allowed to freeze or warmed to room temperature. After dilution the PBW solution is sufficiently dilute that it will not precipitate when heated to 37 &#176;C before application to cells and during incubation.</li>
</ol>
2. Preparation of CuO Nanoparticles (CuO-NPs)
<ol>
	<li>Combine a pure ethanolic solution containing 250 ml of 0.2 M CuCl2 &#8226; 2H2O, 250 ml of 0.4 M sodium hydroxide (NaOH), and 5 g polyethylene glycol (PEG) in a round-bottom flask with six mm borosilicate glass balls.</li>
	<li>Place the solution in a modified microwave oven and allow it to react under reflux at ambient air pressure for 10 min at 20% power (intervals of 6 sec on, 24 sec off).</li>
	<li>Cool the solution to room temperature (20 &#176;C), then decanted into 50 ml conical tubes, leaving the glass balls.</li>
	<li>Centrifuge the solution in the 50 ml conical tubes at 1,000 x g for 30 min, decanted, and then wash the CuO-NPs with a sequence of 300 ml hot water (60-65 &#176;C), 100 ml ethanol, and 100 ml acetone.</li>
	<li>Dry the CuO-NPs to room temperature (20 &#176;C) in the 50 ml conical tubes.</li>
	<li>Scrape the CuO-NPs out of their tubes into a mortar. Cover the CuO-NPs with tin foil and heat the CuO-NPs to 110 &#176;C in an oven to remove the remaining liquid. Combine CuO-NPs into one batch and weigh the CuO-NPs. &#160; 
	NOTE: The preparation of CuO-NPs and CuO-NP treatment of PBW were conducted in Water Quality Laboratory of Ecosystem Science and Management, University of Wyoming. CuO-NP synthesis followed the procedure of Martinson and Reddy (2009) 11.</li>
</ol>
3. Treatment of PBW with CuO-NPs
<ol>
	<li>Add 50 mg (1 mg/ml) of CuO-NP to a 50 ml conical tube followed by 50 ml of PBW. Seal the tube and reacted for 30 min on a bench top orbital shaker at 250 rpm.</li>
	<li>Centrifuge sample tubes at 250 x g for 30 min and then filter the supernatant using a 0.45 &#956;m syringe filter. Alter the centrifuge speed and time can depend on the nanoparticle to ensure the CuO-NPs become compact in the centrifuge tube.</li>
</ol>
4. Elemental Analysis
<ol>
	<li>Prepare Untreated (control) and CuO-NP-treated PBW samples for elemental analysis as follows.</li>
	<li>Acidify aliquots (40 ml) of CuO-NP-treated and untreated PBW with trace metal grade nitric acid to a pH of 2.0. Analyze acidified PBW aliquots for cations by inductively coupled plasma-mass spectroscopy (ICP-MS) as described in Reddy and Roth (2012) 13.</li>
	<li>Prepare unacidified aliquots (20 ml) of CuO-NP-treated and untreated PBW and analyze the unacidified aliquots for anions by ion chromatography (IC) as described in Reddy and Roth (2012) 13. &#160; 
	NOTE: Aliquots were analyzed by the Wyoming Department of Agriculture Analytical Services, Laramie WY 82070. A description of the IC and ICPMS procedure can be found in Reddy and Roth, (2012) 13.</li>
</ol>
5. Preparation of Cell Culture Media Using PBW
<ol>
	<li>Use two control (EMEM-1x and RO+media) and eight PBW test media solutions (four concentrations each of untreated PBW and CuO-NP-treated media) in the viability studies. Overviews of the solutions are as follows:
	<ol>
		<li>For EMEM-1x control, purchase Eagle&#8217;s minimum essential media (EMEM-1x) with L-glutamine and sodium bicarbonate already added. Add fetal bovine serum (FBS) and antibiotics per manufacturer&#8217;s instructions. &#160; 
		NOTE: EMEM-1x is purchased diluted to the proper concentration for cell growth and containing L-glutamine and sodium bicarbonate. EMEM-1x requires the addition of fetal bovine serum (FBS) and an antibiotic mix of penicillin and streptomycin (50 I.U./ml penicillin and 50 &#181;g/ml streptomycin). EMEM-1x is used as a control media because it is the manufacturer&#8217;s recommended growth media for both cell types used in this study. Concentrated EMEM-10x is diluted with RO water from the facility or untreated or CuO-NP-treated PBW to produce the test solutions. Concentrated EMEM-10x when purchased does not contain L-glutamine or sodium bicarbonate so these are added in addition to the fetal bovine serum (FBS) and an antibiotic mix of penicillin and streptomycin.</li>
		<li>For the RO control solution use RO water collected from the ISR facility. Use the same protocol as the PBW test media only substitute 100% RO water from the ISR facility in place of PBW. To dilute the untreated and CuO-NP-treated solution use RO or ultrapure water from the laboratory.</li>
		<li>Dilute untreated PBW into four test concentrations before mixing with the cell culture media components. Prepare the four different concentrations of untreated PBW solutions by mixing untreated PBW with RO (from the laboratory) in the following combinations: 100% (pure PBW + no RO water), 75% (187.5 ml of PBW + 62.5 ml RO water), 50% (125 ml of PBW + 125 ml of RO water) or 25% (62.5 ml of PBW + 187.5 ml of RO water).</li>
		<li>Dilute CuO-NP-treated PBW into four test concentrations before mixing with the cell culture media components. Prepare the four different concentrations of CuO-NP-treated PBW solutions by mixing PBW (pre-treated with 1 mg/ml CuO-NP for 30 min) with RO (from the laboratory) in the following combinations: 100% (pure CuO-NP-treated PBW + no RO water), 75% (187.5 ml of CuO-NP-treated PBW + 62.5 ml RO water), 50% (125 ml of CuO-NP-treated PBW + 125 ml of RO water) or 25% (62.5 ml of CuO-NP-treated PBW + 187.5 ml of RO water).</li>
	</ol>
	</li>
	<li>Prepare 250 ml of RO+media, untreated PBW+media and CuO-NP-treated PBW+media concentration by adding 25 ml of concentrated EMEM-10x to 190 ml of the 100% RO and the 100%, 75%, 50% or 25% of the premade untreated or CuO-NP-treated PBW concentrations created in step 6.1.3 and 6.1.4.</li>
	<li>Adjust the pH of each solution to 7.4 with NaOH or HCl.</li>
	<li>Supplement each concentration of untreated and CuO-NP-treated PBW as well as RO+media with the following standard components: 25ml (10%) fetal bovine serum (FBS), 2.5 ml L-glutamine, 0.55 g NaHCO3 and 1.25 ml Pen/Strep (50 I.U./ml penicillin and 50 &#181;g/ml streptomycin).</li>
	<li>Adjust the osmolality of each concentration of untreated PBW+media, CuO-NP-treated PBW+media and RO+media to 290-310 mOSM/kg by adding RO water and measure using an osmometer.</li>
	<li>Filter each solution using a 0.22 &#956;m vacuum filter unit, and store at 4 &#176;C. 
	NOTE: Due to slight variations in the amount of RO water used to adjust osmolality, vary final media concentrations within a 5% range, with untreated PBW+media concentrations at 56%, 44%, 29% and 16.5% and CuO-NP-treated PBW+media concentrations at 53%, 45%, 30% &#38; 17%.</li>
</ol>
6. Cell Viability &#160;
NOTE: Given that kidney and liver are target organs of heavy metal toxicity, employ cultured human embryonic kidney (HEK293) cells (HEK) and human hepatocellular carcinoma (HepG2) cells (HEP) testing methods 24&#8211;26.
<ol>
	<li>Prepare a culture of HEK and HEP cells 2-3 days before plating the 96-well plates used in the experiment per manufacturer&#8217;s instructions.</li>
	<li>Measure cell viability using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. &#160; 
	NOTE: The MTT assay protocol was modified from Meerloo et al. (2011) 27.
	<ol>
		<li>Obtain MTT in powder form. Add phosphate buffered saline (PBS) to make up a stock concentration of 50 mg/ml. Agitate the solution for 2 hr and then filter with a 0.45 &#956;m syringe filter and aliquot into 1.5 ml freezer safe tubes. Protect tubes from light and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Remove HEK and HEP cells from their culture dishes using trypsin, centrifuge at 1,000 x g for 5 min and decant the trypsin. Add 5 ml of PBS and mix cells to obtain a single cell solution. Then, apply 20 &#956;l of the single cell solution to a hemocytometer to obtain a cell count per milliliter of solution. Centrifuge the cells again at 1,000 x g for 5 min and decant the PBS used to rinse the cells. Add the appropriate amount of EMEM-1x to adjust the concentration of cells to 500 cell/100 &#956;l (100 &#956;l/well).</li>
	<li>Fill the perimeter wells of the plate with 200 &#956;l PBS to control for evaporation.</li>
	<li>Seed cells at a density of 500 cells/well adding 100 &#956;l to each well, except for the perimeter wells (which are not plated with cells). &#160; 
	NOTE: Seeding density for HEK and HEP cells is based on experimental growth curves that allow the peak of growth to occur around days 4-5. Prepare growth curves for all cell lines to estimate seeding density.</li>
	<li>Incubate cells for 24-hr at 37 &#176;C allowing them to recover (form tight adhesions to the plate) before performing baseline MTT readings of cell density.</li>
	<li>Perform baseline MTT readings of cell density by removing the seeding media from the first column (not including the perimeter) and adding 100 &#956;l of MTT (5 mg/ml in media) to the wells for 1 hr.</li>
	<li>After one hour, remove the MTT and add 100 &#956;l of dimethyl sulfoxide (DMSO) to dissolve the MTT-formazan produced by viable cells (20 min).</li>
	<li>Read the optical density (OD) of the first column at an absorption wavelength of 570 nm to obtain a baseline reading.
	<ol>
		<li>Use baseline readings to ensure all plates were seeded correctly and that cells are growing consistently between plates. Remove the DMSO from the column being tested before incubating for the next 24 hr. &#160; 
		NOTE: If DMSO is left in the plate overnight it pulls moisture from the adjacent column, causing a reduction in the media volume.</li>
	</ol>
	</li>
	<li>Warm the test solutions (i.e., the EMEM-1x, RO, untreated PBW and CuO-NP-treated PBW media solutions) to 37 &#176;C in a water bath.</li>
	<li>Remove the seeding media from the rest of the plate (not including the perimeter or the first column which was used for the baseline reading) and replaced with 100 &#181;l of EMEM-1x, RO+media, untreated PBW+media concentrations or CuO-NP-treated PBW+media concentrations (one solution per plate). Incubate cells in their test concentrations or control solutions for a total of seven days (Days 2-8). &#160; 
	NOTE: There 10 plates total: 1 EMEM-1x, 1 RO+media, 1 of each untreated PBW+media concentration (56%, 44%, 29% and 16.5%) and one plate of each CuO-NP-treated PBW+media concentration (53%, 45%, 30% &#38; 17%) per experiment per cell line.</li>
	<li>Each day following baseline MTT reading, remove the control and test solutions (listed in the note under 6.11) from the next column of their respective plate (e.g. Day 2 test and control media are removed from row 3, wells B-G; Day 3: row 4, wells B-G etc.) and repeat the MTT protocol as described in steps 6.7-6.9 above.</li>
	<li>Repeat the protocol every day for seven days. Average the OD results for each row (6 wells) and reported against time to generate a seven-day growth curve.</li>
	<li>To assess the effect of copper chelation on cell viability in CuO-NP-treated PBW+media follow the same procedure as above, except add 100 &#956;M of D-penicillamine to control and test solutions before adding the solutions to their respective plates. Perform data analysis using scientific graphing software.</li>
</ol>
7. Geochemical Modeling
<ol>
	<li>Download Visual MINTEQ version 3.0/3.1 a freeware from the following website <a href="http://www2.lwr.kth.se/English/Oursoftware/vminteq/">http://www2.lwr.kth.se/English/Oursoftware/vminteq/</a>. 
	NOTE: Visual MINTEQ is a freeware chemical equilibrium model for the calculation of metal speciation, solubility equilibria, sorption etc. for natural waters. In addition it is used to predict ion speciation, ion activities, ion complexes and saturation indices which is compared to the concentration of elements before and after treatment (mass spectroscopy results) to examine possible mechanisms of element removal 28.</li>
	<li>Open the program and input the mass spectroscopy data from step 4, including pH, alkalinity and the concentrations of different elements, into the program. 
	NOTE: Given that groundwater is oxidized during in situ uranium extraction process, use oxidized species of arsenic, vanadium, and uranium for input.</li>
</ol>
8. Inhibitory Concentration 50 (IC50)
<ol>
	<li>Calculate the IC50 for the untreated and CuO-NP-treated PBW+media concentrations by first averaging the viability (OD averages) on day 5 of three separate runs.</li>
	<li>Subtract day five viability averages of the untreated and CuO-NP-treated PBW+media concentrations from day five viability averages of EMEM-1x to calculate viability differences. Then divide the viability differences by the average viability on Day 5 in EMEM, and multiply by 100 to get percent inhibition.</li>
	<li>Subtract the percent inhibition from 100 (EMEM-1x viability) to get the percent viability for each untreated and CuO-NP-treated PBW+media concentration.</li>
	<li>Input into scientific graphing software by setting EMEM-1x at a concentration of one and a percent viability of 100; transform all concentrations into log scale (X = Log (X)) and perform nonlinear regression with least square fit analysis.</li>
</ol>
9. Data Analysis
<ol>
	<li>Compare concentrations of elements in untreated and CuO-NP-treated PBW with a two-tailed, paired, Student T-test.</li>
	<li>Calculate the areas under the curve (AUC) by using the growth curve data collected over seven days and analyze the variance with repeated measures analysis of variance (ANOVA), followed by Tukey&#8217;s post hoc comparison between all groups (n = 3).</li>
	<li>Compute the IC50 by using data from day five of the growth curve for both untreated and CuO-NP-treated PBW+media solutions (described above). P values of &#60;0.05 are considered significant. &#160; 
	NOTE: For the purpose of statistical analysis, mass spectroscopy values of half the detection limit was assigned to ions concentrations levels below that limit 29.</li>
</ol>

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Physiology. 99 (4), 105-110 (2005).</li><li>Zheng, X., Watts, G., Vaught, S., Gandolfi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-level+arsenite+induced+gene+expression+in+HEK293+cells.">Low-level arsenite induced gene expression in HEK293 cells.</a> Toxicology. 187 (1), 39-48 (2003).</li><li>Li, Z., Piao, F., Liu, S., Wang, Y., Qu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subchronic+exposure+to+arsenic+trioxide-induced+oxidative+DNA+damage+in+kidney+tissue+of+mice.">Subchronic exposure to arsenic trioxide-induced oxidative DNA damage in kidney tissue of mice.</a> Experimental and Toxicologic Pathology. 62 (5), 543-547 (2010).</li><li>Farombi, E., Akintunde, J., Nzute, N., Adedara, I., Arojojoye, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Municipal+landfill+leachate+induces+hepatotoxicity+and+oxidative+stress+in+rats.">Municipal landfill leachate induces hepatotoxicity and oxidative stress in rats.</a> Toxicology and Industrial Health. 28 (6), 532-541 (2011).</li><li>Das, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic+exposure+through+drinking+water+increases+the+risk+of+liver+and+cardiovascular+diseases+in+the+population+of+West+Bengal.">Arsenic exposure through drinking water increases the risk of liver and cardiovascular diseases in the population of West Bengal.</a> India. BMC public health. 12 (1), 639-648 (2012).</li><li>Valko, M., Morris, H., Cronin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metals,+toxicity+and+oxidative+stress.">Metals, toxicity and oxidative stress.</a> Current Medicinal Chemistry. 12 (10), 1161-1208 (2005).</li><li>Horie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Adsorption+of+Ultrafine+Metal+Oxide+and+Its+Influence+on+Cytotoxicity+toward+Cultured+Cells.">Protein Adsorption of Ultrafine Metal Oxide and Its Influence on Cytotoxicity toward Cultured Cells.</a> Chemical Research in Toxicology. 22 (3), 543-553 (2009).</li></ol>

The authors have nothing to disclose.

Previous studies reported that CuO-NPs removed arsenic from groundwater 11,13,30,31. This study supports these previous findings and also reports that CuO-NPs remove additional contaminants from PBW. This study also confirms previous reports that CuO-NPs are effective at arsenic removal, despite the presence of other contaminants and potential competing ions 11. Speciation modeling predicted that 97% of vanadium species in PBW are negatively charged, allowing for adsorption to CuO-NPs, and batch tre...

Approximately 20% of the US electrical supply is provided by nuclear energy and, based in part on national incentives to increase energy independence, US nuclear capacity is expected to increase 1. Worldwide growth of nuclear energy also is expected to continue, with much of the growth occurring outside the US 2. As of 2013, 83% of US uranium was imported, but 952,544 metric tons of reserves exist in the US 3,4. In 2013 there were 7 new facility applications and 14 restart/expansion applications between Wyoming, New Mexico, and Nebraska 5. In the US, uranium is predominately extracted through in situ recovery (ISR) processes 6. ISR causes less land disruption and avoids creating tailing piles that can release environmental contaminants 7. ISR uses water-based oxidizing solutions to leach uranium from the underground ore body, after which the uranium is extracted from the leachate through an ion exchange process 8. To maintain a negative water balance in the ore body, a portion of the leachate, called production bleed water (PBW), is bled off. A portion of the PBW is decontaminated using reverse osmosis (RO) and re-introduced into the mining process, but PBW also could have beneficial industrial or agricultural uses, if toxic contaminants can be reduced to acceptable levels determined by state regulatory agencies for surface and groundwater 9. Currently, most ISR uranium facilities use RO to remove contaminants from PBW. However, RO processing is energy intensive and produces toxic waste brine, which requires regulated disposal.
Many water decontamination methods exist, including adsorbents, membranes, and ion exchange. Of these, adsorption is the most commonly used, and recent developments in nanoparticle synthesis has enhanced the capabilities of adsorbent-based water decontamination processes 10. Cupric oxide nanoparticles (CuO-NPs) previously had not been extensively studied on uranium ISR PBW, but in recent studies of contaminant removal from groundwater, CuO-NPs were found to have unique properties, including not requiring pre- or post-water treatment steps (e.g., adjusting pH or redox potential) and performing well in different water compositions (e.g., in different pHs, salt concentrations, or competing ions) 11. In addition, CuO-NPs are easily regenerated by leaching with sodium hydroxide (NaOH), after which the regenerated CuO-NPs can be reused. Details of CuO-NP trace metal filtering capabilities from natural waters have been previously published 11&#8211;14.
Although useful for water treatment, metal oxide nanoparticles can be toxic to living organisms, but the extent of the toxicity depends, in part, on nanoparticle characteristics and constituents 10,15,16. Therefore, it is important to study simultaneous contaminant removal and nanoparticle toxicities before field applications. The current study determined the capability of CuO-NPs to remove PBW priority contaminants (including arsenic, selenium, vanadium and uranium), and assessed the effect of CuO-NP treatment on PBW cytotoxicity.
PBW was collected from an active ISR uranium facility and utilized to determine the efficacy of CuO-NP treatment in priority contaminant removal. PBW cytotoxicity before and after CuO-NP treatment also was assessed. PBW is a complex geological (industrial/environmental) mixture and both the National Institute of Environmental Health and Science (NIEHS) and the Agency for Toxic Substances &#38; Disease Registry (ASTDR) are placing emphasis on studying the toxicity of environmentally relevant mixtures, including mixtures as they exist in nature or industrial settings, as well as promoting in vitro testing to prioritize chemicals for further in vivo testing 17&#8211;19. Studies of chronic, low-dose mixture exposures are challenging because chronic exposure to a low dose mixture not produce obvious effects, at least not in the short time frame of most laboratory studies. Similarly, most in vitro studies of chemical mixtures expose cells to a defined lab-made mixture of 2 or more metals 20,21. These studies provide baseline information, but simplified mixtures do not replicate the complex antagonistic and synergistic interactions that may occur in a native, environmental sample, where the full range of mixture components are present.
The goals of this study were to examine alternate contaminant removal processes for PBW and to evaluate the effect of (CuO-NP) treatment on PBW cytotoxicity using cultured human cells. The results could benefit the uranium industry through the development of more efficient or environmentally friendly methods for contaminant removal. This study provides the first evidence that reduction of priority contaminants in PBW by CuO-NPs reduces cytotoxicity in mammalian cells 22.

<table><tbody><tr><td>CuCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>203149</td><td></td></tr><tr><td>Borosilicate glass balls</td><td>VWR</td><td>26396-639</td><td>6 mm</td></tr><tr><td>Nitric Acid</td><td>Fisher</td><td>A509-P500</td><td>Trace metal grade</td></tr><tr><td>0.45 &amp;#956;m syringe filter</td><td>Fisher</td><td>SLHA 033S S</td><td></td></tr><tr><td>10x EMEM</td><td>Fisher</td><td>BW12-684F</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>L-glutamine</td><td>Fisher</td><td>BP379-100</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>ATCC</td><td>30-2300</td><td></td></tr><tr><td>0.22 &amp;#956;m vacuum filter unit</td><td>Fisher</td><td>09-740-28C</td><td></td></tr><tr><td>HEK293</td><td>ATCC</td><td>CRL-1573</td><td></td></tr><tr><td>HEPG2</td><td>ATCC</td><td>HB-8065</td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>SV3003101</td><td></td></tr><tr><td>MTT</td><td>Sigma</td><td>M2128</td><td></td></tr><tr><td>D-penicillamine</td><td>Fisher</td><td>ICN15180680</td><td></td></tr><tr><td>96-well plates</td><td>Fisher</td><td>07-200-92</td><td></td></tr><tr><td>DMSO</td><td>Fisher</td><td>D12814</td><td></td></tr><tr><td>Spectra Max 190</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Visual MINTEQ version 3.0</td><td>KTH Royal Institute of Technology</td><td></td><td></td></tr><tr><td>ICP-MS</td><td>Agilent</td><td></td><td>Details of instruments, models and detection limits were published in Reddy &lt;em&gt;et al.&lt;/em&gt;, 2013.</td></tr><tr><td>IC DIONEX DX 500</td><td>Dionex</td><td></td><td>Details of instruments, models and detection limits were published in Reddy &lt;em&gt;et al.&lt;/em&gt;, 2013.</td></tr><tr><td>VWR Incubator</td><td>VWR</td><td></td><td></td></tr></tbody></table>

removal of trace elements by cupric oxide nanoparticles from uranium in situ recovery bleed water and its effect on cell viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

PBW component concentrations and pH in untreated and CuO-NP-treated PBW are reported in Table 1. Martinson and Reddy (2009), reported that the point of zero charge of the CuO-NP is estimated at 9.4 &#177; 0.4. Given that the pH of PBW was 7.2-7.4, in these conditions, water donates protons to CuO-NPs, causing the nanoparticle surface to be positively charged allowing for the adsorption of negatively charged species. CuO-NP treatment removed priority contaminants from PBW, including arsenic, selenium, ura...

Watch this Scientific Journal Video about Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability at JoVE.com

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

removal-trace-elements-cupric-oxide-nanoparticles-from-uranium-situ

Research

JoVE Journal

Environment

9.7K Views.  University of New Mexico. Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

Video: Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in industrialized countries. Here, the development of optical mesosensors/adsorbents (MSAs) for efficient recognition and selective recovery of Pd(II), Au(III), and Co(II) from urban mine was achieved. A simple, general method for preparing MSAs based on using high-order mesoporous monolithic scaffolds was described. Hierarchical cubic Ia3d wagon-wheel-shaped MSAs were fabricated by anchoring chelating agents (colorants) into three-dimensional pores and micrometric particle surfaces of the mesoporous monolithic scaffolds. Findings show, for the first time, evidence of controlled optical recognition of Pd(II), Au(III), and Co(II) ions and a highly selective system for recovery of Pd(II) ions (up to ~95%) in ores and industrial wastes. Furthermore, the controlled assessment processes described herein involve evaluation of intrinsic properties (e.g., visual signal change, long-term stability, adsorption efficiency, extraordinary sensitivity, selectivity, and reusability); thus, expensive, sophisticated instruments are not required. Results show evidence that MSAs will attract worldwide attention as a promising technological means of recovering and recycling palladium, gold and cobalt metals.

Because of the importance and extensive use of palladium, gold and cobalt metals in high-technology equipment, their recovery and recycling constitute an important industrial challenge. The metal recovery system described herein is a simple, low-cost means for the effective detection, removal, and recovery of these metals from the urban mine.

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Engineering

Speciation and Bioavailability Measurements of Environmental Plutonium Using Diffusion in Thin Films

The biological uptake of plutonium (Pu) in aquatic ecosystems is of particular concern since it is an alpha-particle emitter with long half-life which can potentially contribute to the exposure of biota and humans. The diffusive gradients in thin films technique is introduced here for in-situ measurements of Pu bioavailability and speciation. A diffusion cell constructed for laboratory experiments with Pu and the newly developed protocol make it possible to simulate the environmental behavior of Pu in model solutions of various chemical compositions. Adjustment of the oxidation states to Pu(IV) and Pu(V) described in this protocol is essential in order to investigate the complex redox chemistry of plutonium in the environment. The calibration of this technique and the results obtained in the laboratory experiments enable to develop a specific DGT device for in-situ Pu measurements in freshwaters. Accelerator-based mass-spectrometry measurements of Pu accumulated by DGTs in a karst spring allowed determining the bioavailability of Pu in a mineral freshwater environment. Application of this protocol for Pu measurements using DGT devices has a large potential to improve our understanding of the speciation and the biological transfer of Pu in aquatic ecosystems.

The technique of diffusive gradients in thin films (DGT) is proposed for speciation studies of plutonium. This protocol describes diffusion experiments probing the behavior of Pu(IV) and Pu(V) in presence of organic matter. DGTs deployed in a karstic spring allow assessment of the bioavailability of Pu.

The biological uptake of plutonium (Pu) in aquatic ecosystems is of particular concern since it is an alpha-particle emitter with long half-life which can ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Chemistry

Two-way Valorization of Blast Furnace Slag: Synthesis of Precipitated Calcium Carbonate and Zeolitic Heavy Metal Adsorbent

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with sufficient heavy metal adsorption capacity. To this end, blast furnace slag, a residue from iron-making, is utilized as the starting material. Calcium is selectively extracted from the slag by leaching with acetic acid (2 M CH3COOH) as the extraction agent. The filtered leachate is subsequently physico-chemically purified and then carbonated to form precipitated calcium carbonate (PCC) of high purity (&lt;2 wt% non-calcium impurities, according to ICP-MS analysis). Sodium hydroxide is added to neutralize the regenerated acetate. The morphological properties of the resulting calcitic PCC are tuned for its potential application as a filler in papermaking. In parallel, the residual solids from the extraction stage are subjected to hydrothermal conversion in a caustic solution (2 M NaOH) that leads to the predominant formation of a particular zeolitic mineral phase (detected by XRD), namely analcime (NaAlSi2O6&#8729;H2O). Based on its ability to adsorb Ni2+, as reported from batch adsorption experiments and ICP-OES analysis, this product can potentially be used in wastewater treatment or for environmental remediation applications.

A protocol for the parallel production of precipitated calcium carbonate and zeolitic material from blast furnace slag via mineral carbonation and alkaline hydrothermal conversion, respectively, is presented. The performance of the zeolitic material towards nickel adsorption is tested.

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with ...

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uranium is known to affect bone metabolism. Here, we present a protocol aimed at investigating the effect of natural uranium exposure on the viability, the differentiation, and the function of osteoclasts, the cells in charge of bone resorption.

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about ...

Developmental Biology

U2O5 Film Preparation via UO2 Deposition by Direct Current Sputtering and Successive Oxidation and Reduction with Atomic Oxygen and Atomic Hydrogen

We describe a method to produce U2O5 films in situ using the Labstation, a modular machine developed at JRC Karlsruhe. The Labstation, an essential part of the Properties of Actinides under Extreme Conditions laboratory (PAMEC), allows the preparation of films and studies of sample surfaces using surface analytical techniques such as X-ray and ultra-violet photoemission spectroscopy (XPS and UPS, respectively). All studies are made in situ, and the films, transferred under ultra-high vacuum from their preparation to an analyses chamber, are never in contact with the atmosphere. Initially, a film of UO2 is prepared by direct current (DC) sputter deposition on a gold (Au) foil then oxidized by atomic oxygen to produce a UO3 film. This latter is then reduced with atomic hydrogen to U2O5. Analyses are performed after each step involving oxidation and reduction, using high-resolution photoelectron spectroscopy to examine the oxidation state of uranium. Indeed, the oxidation and reduction times and corresponding temperature of the substrate during this process have severe effects on the resulting oxidation state of the uranium. Stopping the reduction of UO3 to U2O5 with single U(V) is quite challenging; first, uranium-oxygen systems exist in numerous intermediate phases. Second, differentiation of the oxidation states of uranium is mainly based on satellite peaks, whose intensity peaks are weak. Also, experimenters should be aware that X-ray spectroscopy (XPS) is a technique with an atomic sensitivity of 1% to 5%. Thus, it is important to obtain a complete picture of the uranium oxidation state with the entire spectra obtained on U4f, O1s, and the valence band (VB). Programs used in the Labstation include a linear transfer program developed by an outside company (see Table of Materials) as well as data acquisition and sputter source programs, both developed in-house.

U2O5 Film Preparation via UO2 Deposition by Direct Current Sputtering and Successive Oxidation and Reduction with Atomic Oxygen and Atomic Hydrogen

This protocol presents the preparation of U2O5 thin films obtained in situ under ultra-high vacuum. The process involves oxidation and reduction of UO2 films with atomic oxygen and atomic hydrogen, respectively.

We describe a method to produce U2O5 films in situ using the Labstation, a modular machine developed at JRC Karlsruhe. The Labstation, an essential part ...

Separation of Uranium and Thorium for 230Th-U Dating of Submarine Hydrothermal Sulfides

The age of a submarine hydrothermal sulfide is a significant index for estimating the size of hydrothermal ore deposits. Uranium and thorium isotopes in the samples can be separated for 230Th-U dating. This article presents a method to purify and separate U and Th isotopes in submarine hydrothermal sulfide samples. Following this technique, the separated U and Th fractions can meet measuring requirements by multi-collector inductively coupled plasma mass spectrometry (MC-ICPMS). The age of the hydrothermal sulfide sample can be calculated by measuring the present-day activity ratios of 230Th/238U and 234U/238U. A super clean room is necessary for this experiment. Cleaned regents and supplies are used to reduce the contamination during the sample processes. Balance, hotplate, and centrifuge are also used. The sulfide sample is powdered for analysis and less than 0.2 g sample is used. Briefly, the sample is weighed, dissolved, added to 229Th-233U-236U double spike solution, Fe co-precipitated, and separated on an anion-exchange resin extraction column. Approximately 50 ng U is consumed for 230Th-U dating of sulfides sample by MC-ICPMS.

Separation of Uranium and Thorium for 230Th-U Dating of Submarine Hydrothermal Sulfides

The protocol describes a method to purify and separate the U and Th nuclide in submarine hydrothermal sulfide sample with Fe co-precipitation and extraction chromatography for 230Th-U disequilibrium dating.

The age of a submarine hydrothermal sulfide is a significant index for estimating the size of hydrothermal ore deposits. Uranium and thorium isotopes in ...

Accumulation and Analysis of Cuprous Ions in a Copper Sulfate Plating Solution

Knowledge of the behavior of cuprous ions (monovalent copper ion: Cu(I)) in a copper sulfate plating bath is important for improving the plating process. We successfully developed a method to quantitatively and easily measure Cu(I) in a plating solution and used it for evaluation of the solution. In this paper, a quantitative absorption spectrum measurement and a time-resolved injection measurement of Cu(I) concentrations by a color reaction are described. This procedure is effective as a method to reproduce and elucidate the phenomenon occurring in the plating bath in the laboratory. First, the formation and accumulation process of Cu(I) in solution by electrolysis of a plating solution is shown. The amount of Cu(I) in the solution is increased by electrolysis at higher current values &#8203;&#8203;than the usual plating process. For the determination of Cu(I), BCS (bathocuproinedisulfonic acid, disodium salt), a reagent that selectively reacts with Cu(I), is used. The concentration of Cu(I) can be calculated from the absorbance of the Cu(I)-BCS complex. Next, the time measurement of the color reaction is described. The color reaction curve of Cu(I) and BCS measured by the injection method can be decomposed into an instantaneous component and a delay component. By analysis of these components, the holding structure of Cu(I) can be clarified, and this information is important when predicting the quality of the plating film to be produced. This method is used to facilitate the evaluation of the plating bath in the production line.

Here, accumulation of cuprous ions in a copper sulfate plating solution in a model experiment and an analysis based on quantitative measurements are described. This experiment reproduces the accumulation process of cuprous ions in the plating bath.

Knowledge of the behavior of cuprous ions (monovalent copper ion: Cu(I)) in a copper sulfate plating bath is important for improving the plating process. ...

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsr&#248;d et al. designed for rat liver cell isolation and provides high yield (up to 14 x 106 cells per mouse) and high purity (&#62;95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

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