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

Summary

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.

Abstract

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 however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Introduction

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 ^11–14.

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 & 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 ^17–19. 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.

Protocol

All samples were collected at the uranium liquid processing building of a uranium ISR facility in Wyoming.

1. Production Bleed Water (PBW)

1. 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.
2. 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.
3. Measure temperature and pH on-site and transport samples on ice to keep them cool.
4. Store PBW at 4 °C. Keep the PBW solution cool until after the concentrated Eagle’s minimum essential media (EMEM-10x) is added during media preparation as instructed in the following protocol.  
   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 °C before application to cells and during incubation.

2. Preparation of CuO Nanoparticles (CuO-NPs)

1. Combine a pure ethanolic solution containing 250 ml of 0.2 M CuCl₂ • 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.
2. 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).
3. Cool the solution to room temperature (20 °C), then decanted into 50 ml conical tubes, leaving the glass balls.
4. 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 °C), 100 ml ethanol, and 100 ml acetone.
5. Dry the CuO-NPs to room temperature (20 °C) in the 50 ml conical tubes.
6. 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 °C in an oven to remove the remaining liquid. Combine CuO-NPs into one batch and weigh the CuO-NPs.  
   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.

3. Treatment of PBW with CuO-NPs

1. 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.
2. Centrifuge sample tubes at 250 x g for 30 min and then filter the supernatant using a 0.45 μm syringe filter. Alter the centrifuge speed and time can depend on the nanoparticle to ensure the CuO-NPs become compact in the centrifuge tube.

4. Elemental Analysis

1. Prepare Untreated (control) and CuO-NP-treated PBW samples for elemental analysis as follows.
2. 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.
3. 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.  
   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.

5. Preparation of Cell Culture Media Using PBW

1. 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:
   1. For EMEM-1x control, purchase Eagle’s minimum essential media (EMEM-1x) with L-glutamine and sodium bicarbonate already added. Add fetal bovine serum (FBS) and antibiotics per manufacturer’s instructions.  
      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 µg/ml streptomycin). EMEM-1x is used as a control media because it is the manufacturer’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.
   2. 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.
   3. 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).
   4. 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).
2. 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.
3. Adjust the pH of each solution to 7.4 with NaOH or HCl.
4. 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 µg/ml streptomycin).
5. 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.
6. Filter each solution using a 0.22 μm vacuum filter unit, and store at 4 °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% & 17%.

6. Cell Viability  

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 ^24–26.

1. Prepare a culture of HEK and HEP cells 2-3 days before plating the 96-well plates used in the experiment per manufacturer’s instructions.
2. Measure cell viability using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay.  
   NOTE: The MTT assay protocol was modified from Meerloo et al. (2011) ²⁷.
   1. 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 μm syringe filter and aliquot into 1.5 ml freezer safe tubes. Protect tubes from light and store at 4 °C.
3. 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 μl 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 μl (100 μl/well).
4. Fill the perimeter wells of the plate with 200 μl PBS to control for evaporation.
5. Seed cells at a density of 500 cells/well adding 100 μl to each well, except for the perimeter wells (which are not plated with cells).  
   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.
6. Incubate cells for 24-hr at 37 °C allowing them to recover (form tight adhesions to the plate) before performing baseline MTT readings of cell density.
7. Perform baseline MTT readings of cell density by removing the seeding media from the first column (not including the perimeter) and adding 100 μl of MTT (5 mg/ml in media) to the wells for 1 hr.
8. After one hour, remove the MTT and add 100 μl of dimethyl sulfoxide (DMSO) to dissolve the MTT-formazan produced by viable cells (20 min).
9. Read the optical density (OD) of the first column at an absorption wavelength of 570 nm to obtain a baseline reading.
   1. 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.  
      NOTE: If DMSO is left in the plate overnight it pulls moisture from the adjacent column, causing a reduction in the media volume.
10. Warm the test solutions (i.e., the EMEM-1x, RO, untreated PBW and CuO-NP-treated PBW media solutions) to 37 °C in a water bath.
11. 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 µl 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).  
    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% & 17%) per experiment per cell line.
12. 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.
13. 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.
14. 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 μM of D-penicillamine to control and test solutions before adding the solutions to their respective plates. Perform data analysis using scientific graphing software.

7. Geochemical Modeling

1. Download Visual MINTEQ version 3.0/3.1 a freeware from the following website http://www2.lwr.kth.se/English/Oursoftware/vminteq/.
   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.
2. 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.

8. Inhibitory Concentration 50 (IC₅₀)

1. 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.
2. 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.
3. Subtract the percent inhibition from 100 (EMEM-1x viability) to get the percent viability for each untreated and CuO-NP-treated PBW+media concentration.
4. 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.

9. Data Analysis

1. Compare concentrations of elements in untreated and CuO-NP-treated PBW with a two-tailed, paired, Student T-test.
2. 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’s post hoc comparison between all groups (n = 3).
3. 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 <0.05 are considered significant.  
   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.

Results

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 ± 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...

Discussion

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...

Materials

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