NOTE: End date changed to 9/30/2011 per B. Woolford/JSC (01/2011)

NOTE: Start/end dates changed to 10/2/2006-12/31/2010 (previously 4/30/2006-1/31/2011) per B. Woolford/JSC via S. Steinberg-Wright/JSC (9/2009)

Our research will focus on several related areas of research regarding lunar soil: 1) understanding the activation and deactivation processes of lunar soil, as well as how to monitor these processes, 2) understanding the properties of lunar soil in solution (dissolution), and 3) understanding the effects of lunar soil on cellular systems. Initial studies will be carried out using several different materials. Due to the scarcity of pristine lunar soil, tests will be conducted with lunar simulant, JSC-1A-vf, and quartz and titania, which have been used as positive and negative controls, respectively, in toxicological studies. Knowledge of the activation and deactivation processes is important due to the likely passivation of the active surfaces of lunar soils prior to their transfer to long-term storage. In order to determine methods for dust mitigation on the lunar surface, we must first activate the materials and determine the best methods for deactivation. Additionally, the particles themselves may not require activation in order to be toxic. Therefore, dissolution and cellular toxicity studies will be performed to determine if any toxic properties of lunar soil are due simply to their chemical makeup.

There is no standard protocol for experiments aimed at understanding the solubility and dissolution characteristics of mineral dusts. We have previously developed a protocol using lunar dust simulant (JSC-1A-vf). In this protocol, 0.5 mg/mL mixtures of dust and the solution of interest were placed in sealed containers and rotated for 72 hours in order to ensure consistent mixing. At the completion of this time period, the mixtures were filtered, and the resulting solution was tested using Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) for the presence of metals. These results were compared to tests run concurrently on solutions containing no dust. The current work used jet-milled lunar dust, Apollo sample 14003, and 3 different solutions: distilled water, pH 4.0 buffer (citrate-phosphate), and pH 7.14 buffer (phosphate-buffered saline).

After only 2 hours in distilled water, the pH of the lunar dust mixture was significantly higher than that of the control. Over the 30-day testing period, both the control solution and dust mixtures showed increases in pH with respect to the starting pH. While a number of species were measured using ICP-MS after 3, 14, and 30 days, only silicon, calcium, and magnesium were present in quantities above the detection limit of the instrument.

While the effects of water on lunar dust are of interest for its possible use as a plant growth medium, studies of dust in the body become more complicated. Fluids in the body are buffered and much more complex in their composition. In an attempt to understand the effects of pH on lunar dust, we performed 3-day dissolution experiments at pH 4.0 and pH 7.14. In contrast to the distilled water testing, many more ions are released into solution in the buffered solutions, especially at lower pH.

NOTE: End date changed to 9/30/2011 per B. Woolford/JSC (01/2011)

NOTE: Start/end dates changed to 10/2/2006-12/31/2010 (previously 4/30/2006-1/31/2011) per B. Woolford/JSC via S. Steinberg-Wright/JSC (9/2009)

Our research will focus on several related areas of research regarding lunar soil: 1) understanding the activation and deactivation processes of lunar soil, as well as how to monitor these processes, 2) understanding the properties of lunar soil in solution (dissolution), and 3) understanding the effects of lunar soil on cellular systems. Initial studies will be carried out using several different materials. Due to the scarcity of pristine lunar soil, tests will be conducted with lunar simulant, JSC-1A-vf, and quartz and titania, which have been used as positive and negative controls, respectively, in toxicological studies. Knowledge of the activation and deactivation processes is important due to the likely passivation of the active surfaces of lunar soils prior to their transfer to long-term storage. In order to determine methods for dust mitigation on the lunar surface, we must first activate the materials and determine the best methods for deactivation. Additionally, the particles themselves may not require activation in order to be toxic. Therefore, dissolution and cellular toxicity studies will be performed to determine if any toxic properties of lunar soil are due simply to their chemical makeup.

Aim 2: Solution properties

Tests aimed at determining the behavior of lunar dust simulant in solution provided a protocol for dissolution studies that was determined to be sufficient for testing of lunar soil. Previous tests on lunar dust simulant showed an increase in solution pH when the dust was added to water. Therefore, it was decided to use buffer solutions in order to remove a variable in the testing process. A citrate-phosphate buffer (pH 4.0) and phosphate-buffered saline (pH 7.14) were prepared for testing. Additionally, distilled water was used in order to determine the behavior of the dust in an unbuffered solution for comparison to the results of lunar simulant dissolution. The lunar dust used for the testing was prepared from a larger sample of Apollo 14 dust, 14003, by a jet-milling process. Dust concentrations of 0.5 mg/mL were added to the solutions and agitated at regular intervals for 3-30 days to ensure consistent mixing. After filtering the mixtures, Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) was used to determine the concentration of various elements leached into the solution during the dissolution tests (aluminum, antimony, barium, beryllium, cadmium, chromium, cobalt, copper, iron, lead, manganese, molybdenum, nickel, selenium, silicon, silver, titanium, and zinc, as well sodium, potassium, magnesium, and calcium). A lower pH was found to lead to higher levels of dissolution in the buffer solutions, as seen previously in the tests using lunar dust simulant. However, there were significantly lower levels of dissolution seen in the distilled water, even though its pH (6.7) was somewhat lower than that of the phosphate-buffered saline (7.14). This indicates that the solubilization of many of these elements requires the presence of Na+, which is indicative of cation exchange phenomena. For each of the tests, the major constituent released into solution was silicon, followed by aluminum (low pH) and iron (high pH).

The testing of the dust in distilled water also included the measurement of pH and oxidation-reduction potential. Addition of the dust to water led to an immediate increase in the pH compared to the control solution. Throughout the testing period, a gradual increase in the pH was seen for both the dust sample and control. The behavior of the oxidation-reduction potential was not as clear. Addition of the dust to the water led to an immediate decrease in the potential. However, over the testing period, this potential oscillated, though, once again, this oscillation mirrored the control solution.

Future work will include testing of the jet-milled lunar dust in solutions used for intratracheal instillation of the dust in rats. This will help to determine if there is any significant change in the dust after it is placed in solution but before it is instilled in the animals.

NOTE: Start/end dates changed to 10/2/2006-12/31/2010 (previously 4/30/2006-1/31/2011) per B. Woolford/JSC via S. Steinberg-Wright/JSC (9/2009)

Our research will focus on several related areas of research regarding lunar soil: 1) understanding the activation and deactivation processes of lunar soil, as well as how to monitor these processes, 2) understanding the properties of lunar soil in solution (dissolution), and 3) understanding the effects of lunar soil on cellular systems. Initial studies will be carried out using several different materials. Due to the scarcity of pristine lunar soil, tests will be conducted with lunar simulant, JSC-1A-vf, and quartz and titania, which have been used as positive and negative controls, respectively, in toxicological studies. Knowledge of the activation and deactivation processes is important due to the likely passivation of the active surfaces of lunar soils prior to their transfer to long-term storage. In order to determine methods for dust mitigation on the lunar surface, we must first activate the materials and determine the best methods for deactivation. Additionally, the particles themselves may not require activation in order to be toxic. Therefore, dissolution and cellular toxicity studies will be performed to determine if any toxic properties of lunar soil are due simply to their chemical makeup.

Aim 1: Activation and Monitoring

The lunar soil is subjected to constant bombardment by galactic/cosmic-ray particles, solar ultraviolet rays and energetic ions, and solar-wind protons and alpha particles, in addition to micrometeorite impacts, and all of this occurs in the vacuum of the Moon (10-12 torr). These forces affect the soil to different degrees, with crushing and grinding by micrometeorites being the most energetic in creating a highly reactive product. The production of a multitude of ‘dangling’ bonds, mostly situated at oxygen atoms in the 40-50 wt% oxygen soil, gives the potential for chemical reactivity with respiratory tissue in humans. The loss of vacuum integrity in the lunar sample containers during the Apollo era ensured that the present lunar samples are not in the same condition as they were on the Moon; they have been passivated by oxygen and water vapor. Hence, there is a need to study the effects of grinding and crushing for the production of chemical reactivity in lunar soil. We have studied these effects in quartz, lunar dust simulant (JSC-1A-vf), and a variety of lunar soils using grinding by mortar and pestle, as this can serve as a first approximation for meteorite bombardment.

In order to compare the reactivity of lunar dust to terrestrial materials such as lunar dust simulant and quartz, we used the ability of the different dusts to produce hydroxyl radicals in solution as a marker. Due to the transient nature of the hydroxyl radical, we used the terephthalate molecule as a probe. This molecule is non-fluorescent, but will become highly fluorescent when upon reaction with a hydroxyl radical. Additionally, the fluorescence intensity of this product molecule increases linearly with concentration, allowing a determination of the number of radicals produced. Using this technique, we monitored the production of hydroxyl radicals in aqueous solution by ground and unground lunar soil (Apollo 16 soil, 62241). We determined that grinding produced approximately a 10-fold increase in radical production over the unground soil. Grinding of quartz and lunar dust simulant also produced an increase in radical production, but much less than that of the lunar soil. The radical production of the ground lunar soil was approximately 10-fold and 3-fold greater than ground quartz and ground JSC-1A-vf, respectively. The increased reactivity produced for the quartz by grinding was attributed to the presence of silicon- or oxygen-based radicals (“dangling bonds”) on the surface. These “dangling bonds” may also play a part in the reactivity of the lunar soil and lunar simulant. However, other factors would seem to be required to account for the greatly increased reactivity of the lunar soil. In order to determine the source of the increased reactivity of ground lunar soil, we ground 8 lunar soils of varying maturity and source (highland or mare) and measured the hydroxyl-radical production. It was determined that there is a direct correlation between the reactivity and the amount of nanophase (3-33 nm) metallic iron particles (as a function of soil maturity) in the samples, both highland and mare. Additionally, the highland soils were found to be generally less reactive than the mare soils, a fact explained by the lesser amounts of total iron (and therefore less nanophase iron) in highland samples. As these nanophase iron particles are formed under the unique conditions on the Moon, they are not present in quartz or lunar simulant, thereby explaining the decreased reactivity in those samples.

Aliquots of the different lunar soils, as well as the lunar simulant and quartz samples, were exposed to humidified air (50% relative humidity, 25 oC) for set time points in order to understand the kinetics of deactivation. After a time exposure, their fluorescence was measured using the terephthalate method. It was found that lunar simulant reached 50% of its initial reactivity in approximately 3 hours, with quartz reaching 50% reactivity in approximately 2 hours. The lunar samples had quite a bit of scatter, with an approximate time to reach 50% reactivity of 3.5 hours with a spread of 1.5 hours.

Aim 2: Solution Properties

There is no standard protocol for experiments aimed at understanding the solubility and dissolution characteristics of mineral dusts. We developed a protocol using lunar dust simulant (JSC-1A-vf). Initial tests showed that an increase in pH occurred when simulant was placed in water. Therefore, it was determined that buffer solutions must be used in order to remove one variable in the dissolution experiment. Buffer solutions (citrate or citrate-phosphate) were prepared at physiologically-relevant pHs of 4.0, 5.3, and 6.7. Dust concentrations of 0.5 mg/mL were added to the solutions and rotated for 3 days to ensure constant suspension. After filtering the solutions, concentrations of silicon, aluminum, titanium, iron, copper, nickel, zinc, sodium, magnesium, calcium, and potassium were measured using Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) and compared to concentrations from solutions containing no dust. At all pHs, the concentrations of nickel, copper, zinc, and sodium were not significantly higher than the controls. However, increased amounts of the other elements were measured in the filtered solutions, with the more acidic buffer solutions being more effective at leaching the measured species from the dust. Grinding of the dust prior to placement in the buffer solutions led to consistently higher concentrations of the species of interest.

In order to determine the effects of lung fluid on dissolution kinetics, lunar dust simulant was placed in a modified Gamble’s solution (used in the literature to mimic lung fluid) of pH 7.38 under the conditions described above. After measurement using ICP-MS, it was found there was a significant change in the concentrations of silicon, aluminum, titanium, and iron when dust was added to the lung fluid simulant. Additionally, there was a considerable increase in the concentrations found in the lung fluid simulant/lunar dust simulant mixture (pH 7.38) in comparison to those in the phosphate buffer/lunar dust simulant mixture (pH 7.4).

Future work will include a study of the dissolution properties of a lunar dust sample in buffer solutions of selected pH as well as a time-course study of the dissolution of lunar dust simulant in selected buffers.

Aim 3: Cell culture

It has been shown previously that freshly ground quartz is more harmful to cellular systems than aged quartz. The ability of ground quartz to oxidize lipids is directly correlated to the number of radicals produced in solution and to the freshness of the silica. We aimed to compare the ability of lunar dust simulant to produce inflammatory mediators in cellular systems, as well as effects on viability, with that of quartz.

For viability testing, A549 alveolar epithelial cells and BEAS-2B bronchial epithelial cells were grown and exposed to ground and unground quartz and lunar simulant for 24 hours at concentrations = 1000 µg/cm2. Viability was tested using the trypan blue exclusion method. In both cell lines, exposure to unground quartz led to a significant loss in viability at concentrations = 200 µg/cm2. In contrast, concentrations of at least 500 µg/cm2 of unground lunar dust simulant were required to lead to any noticeable loss in viability. When exposed to ground quartz and lunar dust simulant, A549 cells began to show a decrease in viability at lower concentrations of dust, but the loss in viability at higher concentrations was less than that of the unground samples. Similar results were also seen for BEAS-2B cells.

The production of the inflammatory mediators IL-6 and IL-8 was used as a marker of cellular stress. Similar to the viability testing, cells were plated and allowed to grow for 72 hours prior to exposure to quartz and lunar dust simulant. After 72 hours, dust concentrations = 1000 µg/cm2 were added to the cells for 6, 24, 48, and 72 hours. The supernatants were removed and tested for the presence of IL-6 and IL-8. For both cell lines, some increase in the cytokine concentration was seen after addition of both ground and unground dust. The cytokine concentrations found for the controls were often high, and the cytokine concentrations found at longer time points and dust concentrations did not follow any expected trends. In general, these cells seem to be quite robust with regards to exposure to quartz and lunar dust simulant at the physiologically relevant concentrations used in our studies.

Our research will focus on several related areas of research regarding lunar soil: 1) understanding the activation and deactivation processes of lunar soil, as well as how to monitor these processes, 2) understanding the properties of lunar soil in solution (dissolution), and 3) understanding the effects of lunar soil on cellular systems. Initial studies will be carried out using several different materials. Due to the scarcity of pristine lunar soil, tests will be conducted with lunar simulant, JSC-1A-vf, and quartz and titania, which have been used as positive and negative controls, respectively, in toxicological studies. Knowledge of the activation and deactivation processes is important due to the likely passivation of the active surfaces of lunar soils prior to their transfer to long-term storage. In order to determine methods for dust mitigation on the lunar surface, we must first activate the materials and determine the best methods for deactivation. Additionally, the particles themselves may not require activation in order to be toxic. Therefore, dissolution and cellular toxicity studies will be carried out to determine if any toxic properties of lunar soil are due simply to their chemical makeup.