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Fate and Toxicity of CuO Nanospheres and Nanorods used in Al/CuO Nanothermites Before and After Combustion Alan James Kennedy, Nicolas L Melby, Robert D Moser, Anthony J Bednar, Steven F. Son, Christopher D Lounds, Jennifer G Laird, Robert Nellums, David R. Johnson, and Jeffery A Steevens Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es401837f • Publication Date (Web): 23 Aug 2013 Downloaded from http://pubs.acs.org on August 25, 2013

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Environmental Science & Technology

254x190mm (96 x 96 DPI)

ACS Paragon Plus Environment


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Fate and Toxicity of CuO Nanospheres and Nanorods used in Al/CuO

2

Nanothermites Before and After Combustion

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Alan J. Kennedy†*, Nicolas L. Melby‡, Robert D. Moser†, Anthony J. Bednar†,

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Steven F. Son§, Christopher D. Lounds‡, Jennifer G. Laird†, Robert R.

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Nellums§, David R. Johnson†, Jeffery A. Steevens†

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† * U.S. Army Engineer Research and Development Center

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Environmental Laboratory, Building 3270, EP-R

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3909 Halls Ferry Rd

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Vicksburg, MS, 39180

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Voice: 601-634-3344

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Fax: 601-634-2263

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[email protected]

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‡ Badger Technical Services, 12500 San Pedro Ave., Suite 450, Vicksburg, MS,

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39180

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§ Purdue University, School of Mechanical Engineering, 500 Allison Rd., West

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Lafayette, IN 47907

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Word count

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Text: 7080 – 1702 (references) = 5378 (excludes references)

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Tables + Figures: (3 + 2) * 300 = 1500

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Total word equivalents: 6878


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While nanotechnology advancements should be fostered, the environmental health and

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safety (EHS) of nanoparticles used in technologies must be quantified simultaneously.

25

However, most EHS studies assess the potential implications of the free nanoparticles

26

which may not be directly applicable to the EHS of particles incorporated into in-use

27

technologies.

28

copper oxide (CuO) nanospheres relative to CuO nanorods used in nanoenergetic

29

applications to improve combustion. Particles were tested in both the as-received form

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and following combustion of a CuO/aluminum nanothermite.

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nanospheres were more stable and slowly released ions, while higher surface area

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nanorods initially released more ions and were more toxic but generally less stable.

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After combustion, particles sintered into larger, micron-scale aggregates, which may

34

lower toxicity potential to pelagic organisms due to deposition from water to sediment

35

and reduced bioavailability after complexation with sediment organic matter. While the

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larger nanothermite residues settled rapidly, implying lower persistence in water, their

37

potential to release dissolved Cu was higher which led to greater toxicity to

38

Ceriodaphnia dubia relative to parent CuO material (nanosphere or rod). This study

39

illustrates the importance of considering the fate and toxicology of nanoparticles in

40

context with their relevant in-use applications.

This investigation assessed the aquatic toxicological implications of

Results indicated

41 42

Introduction

43

Engineered metal oxide nanoparticles have potential to advance material applications,

44

yet may simultaneously pose potential environmental health and safety (EHS)

45

implications.

To sustain nanotechnologies using these particles, it is important to 2 ACS Paragon Plus Environment


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generate high quality data to expedite informed assessments of risk, including whether

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existing human and environmental health criteria for traditional metals are suitably

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protective of target or non-target releases of nanometals.

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Nanosilver may be the most studied nanometal in EHS assessments. It is generally

50

less toxic than dissolved silver (on a mass basis) in both pristine laboratory conditions

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[1,2,3] and in the presence of environmental ligands [4,5,6]. Less focus is given in the

52

literature to nanoscale copper (nano-Cu), which may have comparable binding sites and

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toxicity mechanisms (impact on Na+/K+ exchange and ATPase activity) to silver based

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on dissolved Cu studies [7,8,9,10]. Dissolved Cu is listed as a priority pollutant [11]. As

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discussed for nanosilver, nano-Cu is generally less toxic than the free Cu ion [2,10].

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While some research presents evidence that nano-Cu particles have a direct role in

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causing toxicity beyond ion release [2,10], other studies conclude that nano-Cu particles

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are more toxic than bulk particles due to their relatively greater release of ions [12,13,

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14].

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Applications of nano-Cu include electrodes, photovoltaic materials, antimicrobials,

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filtration, circuitry, sealants, wood preservation and catalysts [10,13,14,15]. Most EHS

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studies on nano-Cu consider only the parent nanomaterial, not the actual material

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incorporated into use applications.

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investigated the ecotoxicological implications of CuO based energetics, such as

65

nanothermites (we investigated a nano-aluminum (nano-Al) and CuO mixture).

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nanothermite is a physical mixture of an ultrafine (typically < 1 µm) metal fuel and metal

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oxide that offers advantages relative to micron-sized materials including decreased

68

reaction barriers (higher surface area and decreased diffusional scales), increased

We are unaware of published studies that

3 ACS Paragon Plus Environment

A

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mixture homogeneity, faster and more efficient combustion, increased ignition sensitivity

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and controllable energy release at high combustion temperatures [16,17,18,19,20].

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Typically, the metal fuel is nano-Al and the oxidizer is iron oxide, Fe2O3 [18,21].

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However, the utility of other metal oxides such as molybdenum oxide (MoO3), tungsten

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oxide (WO3), bismuth oxide (Bi2O3), and copper oxide (CuO) show promise [19].

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Recently the relative payoff of using CuO sphere vs. rod morphologies as oxidizing

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agents for nano-Al gained attention [16,17,19,22]. The shape and surface area of CuO

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particles may also be relevant to their environmental implications [15]. Nano-sized CuO

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may have greater environmental consequences due to the known high toxicity of the Cu

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ions (Cu+, Cu2+) in aqueous and sediment systems [11,23,24,25,26,27,28] relative to

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the other metal oxides used in nanothermites [29].

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vectors for environmental release on training ranges and in field deployment. Other

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applications (gun primers) are likely to disperse particles and runoff into aquatic

82

systems.

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submersed devices such as underwater cutting torches.

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Our objectives were to determine the effects of nano-CuO spheres and rods in aqueous

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suspension using a standard toxicological model (Ceriodaphnia dubia).

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information on the relative toxicity of the CuO nanoparticle ingredients used in

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nanothermites compared to the toxicity of the technology’s combustion residues was

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generated. We hypothesized that the aquatic toxicity of the nanorods would be greater

89

than the nanospheres due to their higher exposed surface area (and larger reactive

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surface for ion release); this hypothesis was based on Fan et al. [15], who concluded

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that higher surface area shapes of CuO micron-sized particles release more ions and

Energetics (explosives) have clear

Further, nanothermites require no external oxygen and are ideal for


Proactive


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are more toxic. We also hypothesized that the combustion residues would be less toxic

93

than the parent nano-CuO due to increases in particle size, based on previous reporting

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that nanothermite combustion leads to increased aggregate size due to sintering [29].

95

This is based on reporting by multiple authors [30,31,32] that smaller, unagglomerated

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particles are more toxic.

97

Methods

98

Test materials

99

Copper sulfate (CuSO4, CAS 7758-98-7, 98.2% purity, Fisher Chemical) was obtained

100

as a reference toxicant. Various CuO nanomaterials were obtained, including CuO

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nanospheres (NS), CuO nanorods (NR) and two associated Cu/Al2O3 nanothermite

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combustion residues (described below). Copper oxide NRs (diameter: 8-12nm; length:

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100-120nm; >97% purity; surface area = 100 – 120 m2/g) were obtained from

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NEMS/MEMS Work LLC (Columbia, MO). Aluminum nanopowder (average particle

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size: 80nm; 77% active aluminum content) was purchased from Novacentrix (Austin,

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TX).

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nanothermite application and Cu/Al2O3 residues were obtained by collecting combustion

108

products.

109

Required chemicals included N,N-Dimethylformamide (DMF; >99% purity, Sigma

110

Aldrich, St. Louis, MO), CuO NS powder (particle size < 50nm; surface area = 29 m2/g),

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and a nominally 8-9 micron ACS reagent grade CuO powder (>99% purity). The CuO

112

micro-powder and associated Cu/Al2O3 residue did not produce a stable water

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suspension (Table 1) and were not selected for aquatic toxicity testing.

The NS and NR represented oxidizing nanomaterials for the CuO/nano-Al


Nanothermite

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residues were synthesized by combusting stoichiometric (with respect to active nano-

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aluminum content) nanothermites inside a closed stainless steel combustion chamber.

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The combustion occurred in a millisecond to yield a calculated adiabatic temperature of

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3964K (upper limit) at 30% TMD using constant volume calculation at 7 atm. The ideal

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reaction is 2Al + 3CuO Al2O3 + 3Cu. Nanothermite batches were composed of 0.773

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g nano-CuO and 0.227 g nano-Al. Additional methodology on nanothermite residue

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synthesis is provided in the supporting information (SI). Residues were cooled and the

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resulting powder was collected from the combustion chamber using a stainless steel

122

spatula.

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Test media dosing

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Powders (CuO or combustion residues) were added to 20 mL ultrapure water (Milli-Q

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Plus ultrapure water system, 18.2 mΩ/cm, Billerica, MA) to create 100 mg/L working

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stocks.

127

PA,130W) for five minutes, followed by five minutes probe sonication (Branson Sonifier

128

450; 50 W, 40% duty cycle) to achieve dispersal. One hour was allowed for larger

129

aggregates to settle, based on preliminary observations and concentrations too low for

130

DLS measurement (see Figure S1, SI, for photographs and photospectrometric decay

131

curves). It was impractical to include rapidly settling agglomerates in water column

132

bioassays.

133

dissolved concentrations, and used in bioassays.

Stocks were bath sonicated (Fisher Scientific model FS-60, Pittsburgh,

Stocks were characterized for hydrodynamic diameter (HD), total and



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Particle characterization and analytical chemistry

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Particle size and morphology were determined by backscattered scanning electron

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microscopy (SEM; FEI Nova NanoSEM 630, FEI Company, Hillsboro, OR, USA).

137

Primary particle aspects were quantified from SEM images using ImagePro Plus

138

software (v7, Media Cybernetics Inc., Bethesda, MD), as in Poda et al [29].

139

conjunction with SEM imaging, energy-dispersive x-ray (EDX) microanalysis (Bruker

140

AXS, Fitchburg, WI, USA) was performed to generate elemental maps. X-ray diffraction

141

(XRD) measurements were conducted to determine alterations in phases present in the

142

nanothermites before and after combustion. Additional details on SEM, EDX and XRD

143

methodology are available in the SI. The HD of suspended particles in stocks was

144

determined by dynamic light scattering (DLS; 635 nm laser; 90 Plus/BI-MAS,

145

Brookhaven

146

autocorrelation function.

147

Total Cu concentrations were determined by Inductively Coupled Plasma Atomic

148

Emission Spectroscopy(Perkin Elmer Optima 5300DV ICP-AES), or inductively coupled

149

plasma mass spectrometry (Elan DRC-II ICP-MS, Waltham, MA, USA) as needed for

150

concentrations observed in and volumes available from test solutions.

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dissolved Cu concentrations were determined by ultracentrifugation of 10 mL samples

152

at 100,000 x G for 60 minutes (Beckman Optima XL-80K, Rotor 70.1 Ti, Brea, CA, USA)

153

and ICP-MS or GF-AAS as previously described for nanosilver [3,4].

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dissolved was used since this method theoretically removes Cu particles >3 nm (based

155

on calculations derived from Stoke’s Law and material density), which are not expected

156

based on SEM images (Figure 1; smallest measured particle was 12 nm). Aqueous

Instruments,

Holtsville,

NY,

USA)

using


an

In

intensity-weighted

Functionally

Functionally

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supernatant samples (3% nitric acid v/v) were analyzed using ICP following USEPA

158

Methods 6020 and 7010 [33]. The analytical detection limit was 0.5 µg/L and additional

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QA-QC information is supplied in the SI.

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Particle stability and dissolution

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Two mg of each power was sonicated at 100 mg/L (as described above) to determine

162

stability in ultrapure water. Following a one hour settling (discussed above), the HD

163

was determined for materials with adequate particle concentrations remaining in

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suspension. The total concentration was determined for all materials.

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The dissolution kinetics of CuO NSs and NRs were compared by spiking 10 mL of

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stock, prepared as described above, into 600 ml of 100 µS/cm (ionic strength = 1.45

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meq/L) at a nominal concentration of 1.67 mg/L. The 100 µS/cm water was used since

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higher ionic strength water was previously shown to increase agglomeration [3,34,35,

169

,36]. The 100 µS/cm water was prepared as hard reconstituted water recommended for

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bioassays [37] then diluted using ultrapure water (hardness = 27 mg/L as CaCO3, Ca2+

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= 4.7 mg/L; Mg2+ = 4.0 mg/L). The test medium (200 mL) was allocated to each of three

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replicate 250 mL glass beakers. Samples were taken 1 cm below the water surface

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from each replicate for total and functionally dissolved Cu at 2, 6, 24, and 48 h.

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Toxicity bioassays

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The toxicity of the CuO nanomaterials and Cu/Al2O3 nanothermite residues was

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determined using the standard 48-h Ceriodaphnia dubia test method [37].

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organism actively filter feeds from the water and is thus relevant to nanoparticle EHS


This


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research. Briefly, tests consisted of six concentrations obtained by 50% serial dilution,

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using 100 µS/cm water as diluent and control.

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replicate, 20 mL glass scintillation vials containing 15 mL water and five organisms.

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Organisms

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Environmental Testing and Consulting, Superior, WI, USA). Exposures were conducted

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in temperature and humidity controlled environmental rooms (Darwin, St. Louis, MO,

184

USA) at 25 ± 1 °C. Bioassays were terminated after 48-h and the number of surviving

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individuals was assessed.

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dissolved Cu at 1 and 48 h following dosing of the test medium.

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The contribution of the dissolved Cu fraction in causing the toxicity of NR and NS

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suspensions was assessed by both supernatant and ethylenediaminetetraacetic acid

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(EDTA) chelation experiments. For supernatant studies, working stocks (60 mL each)

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were ultracentrifuged as previously described, the supernatant was spiked into synthetic

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freshwater (resulting conductivity of 100 µS/cm) and a 50% serial dilution was created

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for bioassay testing.

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purity) experiments, a 17.5 mg/mL EDTA stock was spiked into the highest NS and NR

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test treatment at 35, 70 and 140 mg/L based on methods and toxicity reference values

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in standard guidance [38]. EDTA is known to chelate and thus reduce the bioavailability

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and toxicity of certain metals, including dissolved Cu [38,39]. The previously described

197

C. dubia test method was employed, with each treatment triplicated (n = 3).

were

acquired

from

in-house

Each concentration included four

cultures

(originally

obtained

from

Samples were collected for determination of total and

For EDTA (Sigma Chemical Co., CAS No. 6381-92-6, 99.5%


Page 11 of 33


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Data analysis

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All statistical comparisons, were performed using SigmaStat software (SPSS, Chicago,

200

IL; α = 0.05. Data normality (Kolmogorov−Smirnov test), homogeneity (Levene’s test),

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and treatment differences were determined by the Holm−Sidak test. Survival data were

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square-root transformed. For nonparametric data, the Kruskal−Wallis one-way analysis

203

of variance (ANOVA) on ranks was applied (Dunn’s test). Since some EDTA chelation

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bioassay treatments lacked survival variability (e.g., 100 ± 0, 0 ± 0%), t-tests were used

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to compare controls and individual treatments. Survival response curves were plotted

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using SigmaPlot (SPSS, Chicago, IL) on a log concentration scale. Fifty percent lethal

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concentration values (LC50s) and associated 95% confidence intervals (95% CIs) were

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determined by the trimmed Spearman-Karber method (ToxCalc 5.0, Tidepool Scientific

209

Software, McKinleyville, CA). Statistical significance was defined by non-overlapping

210

CIs. Separate LC50 values were determined as expressed by the original material

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mass added to stocks prior to settling, total measurable Cu and the dissolved fraction.

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The original material mass calculation was performed to consider the impacts of stability

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and bioavailability on the toxicity of each material.

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concentration during the 48 h bioassays was calculated as the average loss from the

215

highest four test concentrations.

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Results and Discussion

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Particle characterization and stability

218

SEM images (Figure 1) confirmed the size and shape of the CuO spheres (NS) and

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nanorods (NR), with comparable nominal and measured sizes reported in Table 1. The 10 ACS Paragon Plus Environment

Percent loss in total Cu


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original morphology of the CuO nanothermite reactant (NS or NR) had little impact on

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the residue aggregation. Residue particles varied from micron to nanometer in size with

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a generally circum-spherical morphology.

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observed in the NS/Al2O3 and NR/Al2O3 residues was 3.6 and 50.1 µm, respectively.

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EDX chemical mapping shows presence of Cu and Al in both Cu/Al2O3 nanothermite

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residues (Figure 1). XRD confirmed that CuO particles (NS, NR) were pure CuO and

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were transformed into elemental Cu in the Cu/Al2O3 nanothermite residue while the Al

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oxidized to Al2O3 (Figure S2, SI). The aquatic implications of aluminum are generally

228

considered low at circum-neutral pH (7 - 9), although acidic conditions that were not

229

present in this study can result in toxic forms [40]. Nano-Al2O3 is generally reported to

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have very low aquatic and terrestrial toxicity [2,41,42]. Thus, the nano-CuO and the

231

associated dissolved fraction in the Cu/Al2O3 nanothermite residues are likely of greater

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environmental concern and thus the focus of this investigation.

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After sonication and settling of the 100 mg/L (nominal) dispersions in ultrapure water,

234

only stocks containing the NS, NR, and the NS/Al2O3 residue remained brownish-gray in

235

color (Figure S1, SI) indicating high suspended particle concentrations.

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these stable materials, in addition to the NR/Al2O3 residue due to its relevance to the

237

study, were considered in further testing. The concentrations and HD of the selected

238

materials, where measurable, are summarized in Table 1. Total measurable Cu in

239

ultrapure water stocks (after 1 h settling) from highest to lowest were as follows; NS >

240

NR > NS/Al2O3 residue > NR/Al2O3 residue. The HD of the CuNS and the associated

241

residue were similar, although the primary particle size was much larger for the residue

242

(Table 1; Figure 1). Generally, the HD was larger than the primary particle size by

While rare, the maximum particle size


Thus, only

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SEM, which is expected due to bias toward the larger particles shown in the samples

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[35] and agglomeration in the test media; such a size increase in nano-CuO powders is

245

corroborated by previous study [2].

246

(Figure S1, SI) and the total Cu concentration was considerably lower than the other

247

materials; due to the relatively low concentration of particles, reliable HD data could not

248

be obtained (low particle counts, autocorrelation >1). Thus, it could not be determined

249

from these data if the measured Cu concentration of the NR/Al2O3 residue was

250

predominantly dissolved or if particles contributed to it.

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Dissolution kinetics

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The temporal assessment of total and dissolved Cu concentrations varied remarkably

253

between NS and NR suspensions (Figure 2). While the total suspended concentrations

254

of both materials decreased over a 48-h period, a result of particle settling, the decrease

255

was substantially faster for the NR (Figure 2a). Further, while both materials were

256

dosed nominally at 1.67 mg/L, the initial measured NR concentration was much lower

257

than the NS, suggesting a larger portion settled in the ultrapure water stock during the

258

one hour equilibration period; this is supported by results in Table 1. This is logical

259

provided higher aspect ratio particles agglomerate and settle rapidly [43]. The dissolved

260

concentration present immediately following interaction with water was substantially

261

higher for the NR (Figure 2c) relative to the NS (Figure 2b). This relates to the larger

262

surface area of the NRs (100-120 m2/g) relative to the NS (29 m2/g). While previous

263

reporting suggested sonication may promote dissolution [44,45], a linear, two-fold

264

increase in dissolved Cu was observed for the more stable NS suspension during the

265

48-h measurement period (Figure 2b). In contrast, the less stable NR suspension had a

The NR/Al2O3 residue suspension was colorless



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relatively higher dissolved concentration at experiment initiation, which subsequently

267

decreased as the particles settled from suspension (Figure 2c). Others [30,31,32,46]

268

have reported greater metal nanomaterial dissolution when relatively smaller particles

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are well dispersed and lower dissolution when particles are in a state of aggregation.

270

This is explained by the relatively lower exposed surface area of aggregated particles.

271

However, analysis of the dissolved fraction in water stocks (Table 1) indicated that while

272

some dissolution occurred in the ultrapure water, more occurred in the 100 µS/cm test

273

medium (Figure 2). This can occur through complexation with concomitant anions in the

274

higher ionic strength water (according to Le Chatelier's principle); increased ion disassociation

275

from particles at higher ionic strength was also previously described [46, 47]. Both the NS

276

and NR suspensions reached a similar dissolved concentration (≈35 µg/L) by the end of

277

the 48-h interaction with the test medium (Figure 2b,c). However, the higher initial

278

dissolved Cu fraction concentration for NR is likely to have aquatic toxicological

279

implications, as shown below, since mortality due to Cu exposure occurs rapidly.

280

Aquatic toxicity

281

Preparation of stocks for bioassays resulted in comparable total Cu concentrations to

282

data in Table 1.

283

ultrapure water stocks was for the larger-sized residues (87 to 95% loss), while the

284

smaller parent NR (60% loss) and NS (37% loss) were relatively more stable. The

285

greater loss of the combusted residues was related to the larger size of these particles,

286

based on SEM images (Figure 1; Table 1). The material remaining in suspension after

287

one hour was subsequently used to dose bioassays (Figure S1; SI). During the 48-h

288

bioassays, the greatest loss in total Cu concentration was generally observed for the

Generally the greatest concentration loss from suspension in the


Page 15 of 33


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NR (38 ± 9%) and residues (NS/Al2O3 residue: 42 ± 20%, NR/Al2O3 residue: 52 ± 2%),

290

with relatively low loss for the NS (24 ± 1%). While the residues initially settled more in

291

ultrapure water due to presence of larger particles, reduction in total Cu concentrations

292

was lower during bioassays since only smaller particles remained in suspension

293

following the initial stock settling period; further, the residues had a relatively higher

294

dissolved fraction. Loss in total concentration for the NS and NR was slightly lower in

295

the bioassays than reported above for the stocks since the starting concentration was

296

lower (Table 1), resulting in fewer particle-to-particle interactions, agglomeration and

297

associated settling [4].

298

previously reported (50-60%) for CuO nanopowder [2]. The percent dissolved Cu in the

299

100 µS/cm bioassay medium was considerably higher than in the ultrapure water stocks

300

(Table 1), with the highest fraction observed for the NS/Al2O3 residue, followed by the

301

NR, NR/Al2O3 residue and the NS.

302

The toxicity of dissolved Cu as CuSO4 was considerably greater than all nano-CuO

303

materials and Cu/Al2O3 residues when expressed on a Cu mass basis (Table 2; Figure

304

S3, SI).

305

nanoparticles are less toxic to fish and macroinvertebrates than the associated ion. In

306

this study, the 48-h LC50 values for Cu as CuSO4 ranged from 7.6 to 8.2 µg/L. These

307

values are within a range of previously reported dissolved Cu toxicity values (5.6 – 17.5

308

µg/L) in the literature for C. dubia [26,49]. That range is relatively wide since dissolved

309

Cu toxicity is controlled by water hardness [49].

310

strength and hardness water (27 mg/L as CaCO3) to increase particle stability as

311

previously recommended [35], dissolved Cu toxicity is expected to be relatively higher.

The loss in NS and NR concentrations is comparable to

Recent literature generally supports that Cu [2,10,48] and Ag [2,3,4]

Since we selected a lower ionic



Page 16 of 33

312

This implies that the toxicity of nano-CuO would also be relatively higher; particles in

313

lower ionic strength media with lower divalent cation concentrations [50] are expected to

314

be relatively more stable than in high ionic strength media, leading to greater direct

315

particle exposure and more exposed surface area for ion release (Figure 1) and toxicity.

316

However, as previously noted [47, Table 1, this study], dissolution from particles can

317

increase in higher ionic strength media.

318

On a total measurable mass basis (Table 1), the NS was the least toxic material tested

319

(LC50 = 115 µg/L).

320

relatively higher surface area of the NR implies greater surface reactivity and potential

321

for ion release. A median effects dose metric standardized by total exposed surface

322

area of the suspension, previously described in Kennedy et al [4], better expressed the

323

toxicity of NS (3,332 [2,812-3,947)] mm2/L) and NR (3714 [3560 – 3875] mm2/L) than

324

mass, since 95% CIs overlapped. Higher surface area was previously suggested to

325

relate to increased toxicity of micron-sized CuO particles [15]. Surface area based dose

326

metrics were reported to be more predictive of nanosilver toxicity than the traditional

327

mass basis [4].

328

Cladocerans (LC50: 60 – 2,600 µg/L) and rotifers (LC50: 240 - 390 µg/L appear to be

329

among the more sensitive species to nano-CuO, while algae (540 – 4,300 µg/L), fish

330

(710 – 1,780 µg/L) and shrimp (8,500 – 9,800 µg/L) are relatively more tolerant

331

[2,13,14,45,51]. Since these ranges were obtained from different studies using different

332

media and CuO suspensions, it is not possible to directly compare species sensitivity

333

distributions. The C. dubia sensitivity to CuO may be greater in the present study

334

partially due to the use of a lower ionic strength medium that promotes particle stability,

The NR was significantly more toxic (LC50 = 34 µg/L).


The

Page 17 of 33


335

dissolution and contains lower Ca2+ and Mg2+ to reduce Cu bioavailability. In addition,

336

C. dubia directly filters particulate matter leading to enhanced exposure.

337

The NS/Al2O3 residue (LC50 = 30 µg/L) and NR/Al2O3 residue (LC50 = 20 µg/L) were

338

significantly more toxic than their CuO reactant (NS, NR), based on total measured Cu

339

concentrations (Table 2, Figure S3a, SI). While the NR/Al2O3 residue was significantly

340

more toxic than the NS/Al2O3 residue, the greatest relative increase in toxicity (74%)

341

following combustion was observed for the NS/Al2O3 residue (compared to its reactant).

342

The increase in toxicity of the residues relative to the CuO reactant is likely related to

343

the increase in dissolved Cu in both stocks and during bioassays (Table 1).

344

Combustion resulted in a change from CuO to elemental Cu in the residues (Figure S2,

345

SI), related to the oxidation of Al to Al2O3. The Cu particles in the residue may re-

346

oxidized when exposed to test media and release a relatively larger amount of ions. In

347

fact, the more toxic NR/Al2O3 residue contained relatively more elemental Cu

348

(compared to CuO) than the NS/Al2O3 residue. While sonication may promote some

349

dissolution [52], Manusadzianas et al. [45] reported no significant difference in the

350

toxicity of vortexed versus sonicated CuO nanoparticles to several aquatic test species.

351

The relative toxicity of the CuO nanomaterials generally correlated well (R = -0.8) with

352

the dissolved fraction measured in the stocks and bioassays (Table 1; Figure S3, SI).

353

When LC50 values were expressed as the dissolved mass in each of the exposure

354

concentrations, toxicity was generally statistically similar to that of dissolved Cu as

355

CuSO4 (Table 2).

356

concentrations at test initiation were used for LC50 calculations; however, when the

357

dissolved concentrations at test termination were used (which increased during the

The one exception was the NR/Al2O3 residue when dissolved



Page 18 of 33

358

bioassay), toxicity was not significantly different than Cu as CuSO4.

The NR and NS

359

supernatant exposures also indicated that the dissolved fraction from the nano-Cu

360

dispersions induced very similar toxicity to Cu toxicity as CuSO4 (Table 2), although the

361

NS supernatant was slightly more toxic than expected based upon the dissolved fraction

362

in comparison to toxicity of Cu as CuSO4.

363

While the toxicity of the residues was greater than the parent CuO reactant when

364

expressed as the total measurable Cu in the exposures, the residues themselves were

365

generally much less stable in water (Table 1); thus, exposure of water column

366

organisms to residue particles is less likely.

367

residue was the most toxic material, its toxicity was actually the lowest when expressed

368

as the total amount of material originally spiked into the water (Table 2), since the

369

majority of the material settled from suspension prior to test organism exposure. Thus,

370

the relative risk of the Cu/Al2O3 residues should be considered in this context.

371

Toxicity reduction evaluation

372

An EDTA toxicity reduction evaluation was conducted using toxic concentrations of

373

CuSO4, NS and NR, across a gradient of EDTA concentrations (0, 35, 70, 140 mg/L).

374

As recommended by guidance [38], the low end of the EDTA gradient was intended to

375

be non-toxic but potentially too low to complex all Cu (or reduce toxicity), while the high

376

end was intended to be toxic by itself but be more than sufficient to complex all Cu.

377

However, if sufficiently high concentrations of Cu are present to complex with higher

378

levels of EDTA, this complexation is expected to reduce both EDTA and Cu toxicity [38].

379

The survival of C. dubia in 100 µS/cm water (no Cu) was relatively high at 0 and 35

To demonstrate, while the NR/Al2O3


Page 19 of 33


380

mg/L EDTA. However, EDTA alone was toxic at ≥ 70 mg/L (Table 3) which is in general

381

agreement with guidance values (24 h LC50: 40 – 120 mg/L; 48 h LC50: 30 – 110 mg/L

382

[38]) at similar conductivity and hardness. At 70 mg/L, the full lethal effects of EDTA

383

took 48 h to occur; this is relatively slower than Cu concentrations >200 µg/L (300 µg/L total Cu after 24 or 48 h. The toxicity of 209

400

µg/L NR was significantly reduced by 35 mg/L EDTA (Table 3a) after 24 h exposure but

401

not 48 h (Table 3b). While 35 mg/L EDTA should have capacity to complex 7,600 µg/L

402

Cu, which is in excess of the 209 – 488 µg/L total Cu used in the nano-CuO exposures, 18 ACS Paragon Plus Environment


Page 20 of 33

403

toxicity was not reduced after 48 h exposure. Further, while 70 and 140 mg/L EDTA

404

would ordinarily result in complete C. dubia lethality as observed in EDTA-only

405

exposures and dissolved Cu exposures up to 253 µg/L, C. dubia survival was relatively

406

high at these higher EDTA concentrations when exposed to normally toxic

407

concentrations of NS (after 24 and 48 h exposure) and NR (after 24 h exposure). Thus,

408

the co-occurrence of higher concentrations of CuO nanoparticles and EDTA resulted in

409

complexation and reduction of their respective toxicities. While this phenomenon was

410

observed for the NR after 24 h, substantial mortality occurred after 48 h. The reason for

411

this is unclear. However, we provided evidence that NRs are less stable and settle out

412

of suspension within 24 h while the NS remain stable in suspension (Figure 2). Thus,

413

once the NRs settled from suspension after 24h, there was less available particle

414

surface for complexation with EDTA and 70 to 140 mg/L EDTA became toxic to C.

415

dubia.

416

These results highlight substantial differences in the complexation of EDTA with

417

dissolved Cu compared to CuO nanoparticles, suggesting an inherent difference

418

between the nano-CuO exposures relative to exposure to dissolved Cu. While some

419

authors attributed all observed nano-CuO aquatic toxicity to the dissolved fraction

420

[14,53], others suggested particles played a role based on the dissolved fraction being

421

inadequate to explain all observed toxicity [2]. This may be indicative of a particle-

422

specific effect, such as direct contact or ingestion and oxidative stress [12]. However,

423

we provided supportive data that particles release ions over time (Figure 2a) and the

424

fraction of released ions present in the nano-CuO suspensions is directly relatable to

425

observed toxicity as related to dissolved Cu as CuSO4 (Table 2). Thus, the particles 19 ACS Paragon Plus Environment

Page 21 of 33


426

may act as unique ion delivery systems (Trojan horse effect; [12]) that overcome the

427

kinetics of complexation with lower concentrations of EDTA (35 mg/L), as previously

428

reported for nanosilver and environmental ligands [4,54,55,56] and dissolved Cu [57].

429

While more EDTA (≥ 70 mg/L) was required to reduce the toxicity of nano-CuO, relating

430

to a greater density of EDTA molecules that more rapidly complex the continuously

431

released ions from the particles, the presence of nano-CuO also significantly reduced

432

the toxicity of the higher concentrations of EDTA.

433

complexing with the high surface area CuO particles had a role in reducing EDTA

434

toxicity that was not observed for dissolved Cu as CuSO4. Environmental ligands such

435

as DOC are likely to similarly reduce nanometal toxicity [4].

436

Overall, this study provides further evidence that metallic nanoparticles are less toxic to

437

macro-organisms than their associated ion.

438

hypothesis that the relatively higher surface area CuO NR lead to greater toxicity

439

relative to the CuO NS, likely due to greater ion release from its larger reactive surface.

440

However, our hypothesis that the post-combusted Cu/Al2O3 nanothermite residue would

441

be less toxic than the parent CuO nanoparticles (used as reactants) due to their larger

442

size was not directly supported. While the residues were substantially less stable, and

443

thus would be expected to be less persistent in the water due to their larger size, they

444

tended to release more dissolved Cu ions as after combustion. The greater ion release

445

related to reduction of the CuO to elemental Cu in the residue, as confirmed by XRD.

446

The relatively higher ion fraction in the residue suspensions explains their relatively

447

higher mass-based toxicity. Despite this, it should be noted that much of the residue

448

material was relatively large and would be expected to be less mobile.

Thus, it is likely that EDTA

The data generated support our initial


Further,


Page 22 of 33

449

environmental ligands such as dissolved organic carbon would have a role in reducing

450

the toxicity of both ionic and nano Cu toxicity if released into the environment [53,57].

451

Acknowledgments. This research was funded by the US Army Environmental Quality

452

Technology Research Program (Dr. Elizabeth Ferguson, technical director). Permission

453

was granted by the Chief of Engineers to publish this information. The use of trade,

454

product, or firm names in this report is for descriptive purposes only and does not imply

455

endorsement by the U.S. Government. The findings of this report are not to be

456

construed as an official Department of the Army position unless so designated by other

457

authorized documents.

458

Supporting information available. Supportive text on synthesis of nanothermite

459

residues, particle characterization, particle stability (Figure S1), XRD spectra of the

460

nanothermite residues (Figure S2) and C. dubia dose response curves (Figure S3) are

461

available free of charge via the Internet at http://pubs.acs.org.

462

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Aruoja, V.; Dubourguier, H.C.; Kasemets, K.; Kahru, A. Toxicity of nanoparticles of CuO, ZnO, and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ. 2009, 407, 1461-1468.



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Malchi, J.Y.; Yetter, R.A.; Foley, T.J.; Son, S.F. The effect of added Al2O3 on the propagation behavior of an Al/CuO Nanoscale thermite. Combust. Sci. Technol. 2008, 180,1278-1294.

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Gan, Y.; Chen, Z.; Gangopadhyay, K.; Bezmelnitsyn, A.; Gangopadhyay, S. An equation of state for the detonation product of copper oxide/aluminum nanothermite composites. J. Nanoparticle Res. 2010, 12, 719-726.

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Sanders, V.E.; Asay, B.W.; Foley, T.J.; Tappan, B.C.; Pacheco, A.N.; Son, S.F. Reaction propagation of four nanoscale energetic composites (Al/MoO3, Al/WO3, Al/CuO, and Bi2O3). J. Propul. Power 2007, 23, 707-714.

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Yetter, G.A.; Rish, G.A.; Son, S.F. Metal Particle Combustion and Nanotechnology. Proc. Combust. Instit., 2009, 32, 1819-1838.

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Miziolek, A.W. Nanoenergetics: an emerging technology area of national importance. Advanced Materials and Process Technology Information Analysis Center (AMPTIAC) Quarterly Newsletter, 2002, 6, 43–48.

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Carlson, A.R.; Nelson, H.; Hammermeister, D. Development and Validation of SiteSpecific Water Quality Criteria for Copper 242. Environ. Toxicol. Chem. 1986, 5, 997-1012.

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Belanger, S.E.; Farris, J.L.; Cherry, D.S. Effects of diet, water hardness, and population source on acute and chronic copper toxicity to Ceriodaphnia dubia. Arch. Environ. Contam. Tox. 1989, 18, 601-611.

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Naddy, R.B.; Stubblefield, W.A.; May, J.R.; Tucker, S.A.; Hockett, J.R. The effect of calcium and magnesium ratios on the toxicity of copper to five aquatic species in freshwater. Environ. Toxicol. Chem. 2002, 21, 347-352.

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Bossuyt, B.T.A.; Janssen, C.R. Copper toxicity to different field-collected cladoceran species: intra- and inter-species sensitivity. Environ. Pollut. 2005, 136, 145-154.

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Kennedy, A.J.; Millward, R.N.; Steevens, J.A.; Lynn, J.W.; Perry, K.D. Relative Sensitivity of Zebra Mussel (Dreissena polymorpha) Life-stages to Two Copper Sources. J. Great Lakes Res. 2006, 32, 596-606.

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Poda, A.R.; Moser, R.D.; Cuddy, M.F.; Doorenbos, Z.; Lafferty, B.J.; Weiss, C.A.; Harmon, A.; Chappell, M.A.; Steevens, J.A. Nano-aluminum thermite formulations: characterizing the fate properties of a nanotechnology during use. J. Nanomaterials Molecular Nanotechnology 2013, 2, 1-9.

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Page 29 of 33

464 465 466 467 468


Table 1. Summary of test material characteristics. Measured primary and hydrodynamic diameters were based on SEM and DLS, respectively, with means and standard deviations presented (10th and 90th percentiles in parentheses). Numbers in parentheses under measured stock concentrations represent the percent remaining in suspension following 1-hour settling. Error bars were not available for the dissolved concentrations in stocks due to insufficient volume for replication. NA = not available or tested due to low particle stability and insufficient concentration for measurement.

Material

Nominal Primary particle diameter (nm)

Measured Primary particle diameter (nm)

Measured Hydrodynamic Polydispersity diameter (nm)

Mean Total measured Cu in stock (mg/L)

Dissolved Cu in stock (mg/L)

Dissolved fraction in bioassays (mg/L)

CuO-nanosphere (NS)

< 50

39 ± 8 (18 – 54)

320 ± 3

0.272 ± 0.003

57.6 ± 8.4 (58%)

0.03%

9 ± 4%

CuO-nanorod (NR)

Width: 8-12 Length: 100-120

Width: 16 ± 5 (11 – 23) Length: 136 ± 49 (68 – 200)

238 ± 2

0.185 ± 0.006

36.6 ± 4.9 (37%)

1.08%

38 ± 12%

CuO-NS / Al2O3 residue

NA

193 ± 323 (86 – 671)

355 ± 1

0.231 ± 0.019

12.5 ± 1.3 (12%)

6.71%

47 ± 17%

CuO-NR / Al2O3 residue

NA

937 ± 759 (231 – 958)

NA

NA

4.9 ± 0.3 (5%)

4.78%

18 ± 2%

CuO Micron

8000 - 9000

NA

NA

NA

5.2 (5%)

NA

NA

Micron-sized CuO / Al2O3 residue

NA

NA

NA

NA

1.8 (2%)

NA

NA



469 470 471 472 473 474 475 476 477

Page 30 of 33

Table 2. Lethal median concentration (LC50) for CuO test materials, measured as copper (Cu). Toxicity is expressed as the original amount of material spiked into stocks (prior to settling), total measurable Cu in suspension during the bioassays and dissolved fraction. The original mass of material accounts for all material originally spiked into the system, included that which settled from the water column. LC50 values are supplied using 0 and 48 h dissolved Cu data for the nanorod residue since its dissolved concentrations increased substantially during the bioassay. Asterisks denote toxicity that was significantly different than dissolved copper (Cu) toxicity as CuSO4. NA = not applicable.

Material

Original mass material spiked (nominal)

Total Measurable Cu (µg/L)

Fraction Dissolved Cu (µg/L)

Supernatant test, dissolved Cu (µg/L)

Cu as CuSO4 (test 1)

NA

NA

8.2 (7.6 – 8.8)

NA

Cu as CuSO4 (test 2)

NA

NA

7.6 (6.2 – 9.5)

NA

CuO-nanosphere (CuNS)

203.0 (171.4 – 240.6)

114.9* (97.0 – 136.1)

9.2 (8.3 – 10.2)

4.3* (3.7 – 5.1)

CuO-nanorod (CuNR)

29.5 (26.2 – 33.3)

33.8* (32.4 – 35.2)

9.1 (8.1 – 10.2)

6.9 (5.9 – 8.0)

CuO-nanosphere (CuNS)/Al2O3 nanothermite residue

42.5 (33.8 – 53.4)

29.8* (27.9 – 31.8)

11.5 (9.3 – 14.1)

NA

CuO-nanorod (CuNR)/Al2O3 nanothermite residue

0h 3.4 Chemistry: (3.0 – 3.8)* 424.7 (358.1 – 503.6)

19.7* (17.0 – 22.8)

NA 48 h Chemistry:

6.9 (5.8 – 9.2)

478 479


29

Page 31 of 33


480 481 482 483

Table 3. Ceriodaphnia dubia survival results from the ethylenediaminetetraacetic acid (EDTA) toxicity reduction evaluation (TRE) after 24-hours (a) and 48-hours exposure (b). All concentrations are based on total measurable copper. Asterisks denote statistical significance relative to the control.

484

(a) Material

Total Cu (µg/L)

0 mg/L EDTA

35 mg/L EDTA

70 mg/L EDTA

140 mg/L EDTA

Control (no copper)

< 1 µg/L

93 ± 12

100 ± 0

47 ± 12

0±0

Cu as CuSO4

43 µg/L

0 ± 0*

100 ± 0

73 ± 12

0±0

Cu as CuSO4

253 µg/L

0 ± 0*

100 ± 0

67 ± 23

0±0

CuO nanorod

209 µg/L

0 ± 0*

80 ± 0*

53 ± 12

0±0

CuO nanorod

317 µg/L

0 ± 0*

33 ± 23*

93 ± 12*

67 ± 12*

CuO nanosphere

488 µg/L

27 ± 23

0 ± 0*

87 ± 12*

100 ± 0*

485

(b)

486

Material

Total Cu (µg/L)

0 mg/L EDTA

35 mg/L EDTA

70 mg/L EDTA

140 mg/L EDTA

Control (no copper)

< 1 µg/L

93 ± 12

93 ± 12

0±0

0±0

Cu as CuSO4

43 µg/L

0 ± 0*

80 ± 20

13 ± 23

0±0

Cu as CuSO4

253 µg/L

0 ± 0*

93 ± 12

0±0

0±0

CuO nanorod

209 µg/L

0 ± 0*

0 ± 0*

7 ± 12

0±0

CuO nanorod

317 µg/L

0 ± 0*

0 ± 0*

0±0

20 ± 20

CuO nanosphere

488 µg/L

7 ± 12*

0 ± 0*

73 ± 12*

87 ± 12*

487


30


488 489 490 491 492

Page 32 of 33

Figure 1. Images of study materials. Scanning electron microscopy of the (a) copper oxide (CuO)-nanospheres (NS), (b) NS/Al2O3 nanothermite residues, (c) CuO-nanorods (NR) and (d) NR/Al2O3 residues. Note differences in scale. Energy dispersive x-ray mapping shows the elemental distribution within the (e) NS/Al2O3 residue and (f) NR/Al2O3 residue. Copper and Al are shown in red and blue, respectively.

493


31

Page 33 of 33

494 495 496 497 498 499 500


Figure 2. Total and dissolved concentrations, measured as copper, in suspensions of copper oxide (CuO) nanospheres (NS) and nanorods (NR) in the 100 µS/cm test medium as a function of time. Panel (a) provides a relative comparison of NS and NR total concentrations and settling rates. Panel (b) provides a scaled down comparison between total and dissolved NS concentrations. Panel (c) provides a scaled down comparison between total and dissolved NR concentrations. Data points not sharing the same letter designation are statistically significantly different.

501


32

CuO

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