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.

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			<td height="21" style="height:21px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:243px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:315px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CuCl2</td>
			<td style="width:243px;">Sigma</td>
			<td align="right" style="width:119px;">203149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Borosilicate glass balls</td>
			<td style="width:243px;">VWR</td>
			<td>26396-639</td>
			<td>6 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nitric Acid</td>
			<td style="width:243px;">Fisher</td>
			<td>A509-P500</td>
			<td>Trace metal grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.45 mm syringe filter</td>
			<td style="width:243px;">Fisher</td>
			<td style="width:119px;">SLHA 033S S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10X EMEM</td>
			<td style="width:243px;">Fisher</td>
			<td style="width:119px;">BW12-684F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:243px;">ATCC</td>
			<td>30-2020</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-glutamine</td>
			<td style="width:243px;">Fisher</td>
			<td style="width:119px;">BP379-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:243px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:243px;">ATCC</td>
			<td style="width:119px;">30-2300</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.22 mm vacuum filter unit</td>
			<td style="width:243px;">Fisher</td>
			<td style="width:119px;">09-740-28C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEK293</td>
			<td style="width:243px;">ATCC</td>
			<td style="width:119px;">CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPG2</td>
			<td style="width:243px;">ATCC</td>
			<td style="width:119px;">HB-8065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:243px;">Sigma</td>
			<td style="width:119px;">SV3003101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MTT</td>
			<td style="width:243px;">Sigma</td>
			<td style="width:119px;">M2128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-penicillamine</td>
			<td style="width:243px;">Fisher</td>
			<td style="width:119px;">ICN15180680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well plates</td>
			<td style="width:243px;">Fisher</td>
			<td style="width:119px;">07-200-92</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:243px;">Fisher</td>
			<td style="width:119px;">D12814</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Spectra Max 190</td>
			<td style="width:243px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Visual MINTEQ version 3.0</td>
			<td style="width:243px;">KTH Royal Institute of Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ICP-MS&#160;</td>
			<td style="width:243px;">Agilent</td>
			<td style="width:119px;"></td>
			<td>Details of instruments, models and detection limits were published in Reddy et al., 2013.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IC DIONEX DX 500</td>
			<td style="width:243px;">Dionex</td>
			<td style="width:119px;"></td>
			<td>Details of instruments, models and detection limits were published in Reddy et al., 2013.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">VWR Incubator</td>
			<td style="width:243px;">VWR</td>
			<td style="width:119px;"></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 ...

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9.7K vistas.  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

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, &#947; was calculated for each of 75 PCB congeners in each of the eight tanks. Mean &#947; was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean &#947; could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

Determination of Inorganic Arsenic in a Wide Range of Food Matrices using Hydride Generation - Atomic Absorption Spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to inorganic arsenic through diet, information about distribution of arsenic species in various food types must be generated. A method, previously validated in a collaborative trial, has been applied to determine inorganic arsenic in a wide variety of food matrices, covering grains, mushrooms and food of marine origin (31 samples in total). The method is based on detection by flow injection-hydride generation-atomic absorption spectrometry of the iAs selectively extracted into chloroform after digestion of the proteins with concentrated HCl. The method is characterized by a limit of quantification of 10 &#181;g/kg dry weight, which allowed quantification of inorganic arsenic in a large amount of food matrices. Information is provided about performance scores given to results obtained with this method and which were reported by different laboratories in several proficiency tests. The percentage of satisfactory results obtained with the discussed method is higher than that of the results obtained with other analytical approaches.

The usefulness of an analytical method to determine inorganic arsenic in a wide range of food matrices is demonstrated. The method consists of selective extraction of inorganic arsenic into chloroform with a final determination by hydride generation-atomic absorption spectrometry.

The European Food Safety Authority (EFSA) underlined in its Scientific Opinion on Arsenic in Food that in order to support a sound exposure assessment to ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...