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 ...

<ol>
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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+arsenic+removal+process+for+water+using+cupric+oxide+nanoparticles.">A novel arsenic removal process for water using cupric oxide nanoparticles.</a> Journal of Colloid and Interface Science. 397, 96-102 (2013).</li><li>Reddy, K., Roth, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic+Removal+from+Natural+Groundwater+Using+Cupric+Oxide.">Arsenic Removal from Natural Groundwater Using Cupric Oxide.</a> Ground Water. 51 (1), 83-91 (2012).</li><li>Zhang, G., Ren, Z., Zhang, X., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+iron(III)-copper(II)+binary+oxide:+a+novel+adsorbent+for+enhanced+arsenic+removal+from+aqueous+solutions.">Nanostructured 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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 border="0" cellpadding="0" cellspacing="0" width="892">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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...

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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 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.